21st Annual Stem Cell Symposium Program

21ST ANNUAL

STEM CELL SYMPOSIUM

STEMCELL.UCLA.EDU

A stem cell-derived brain organoid created from a cell line mutant for autism risk gene CHD8 Credit: George Chen/Daniel Geschwind Lab

THE SYMPOSIUM IS SUPPORTED BY A GENEROUS ENDOWMENT FROM THE MARGARET M. BLOOMFIELD FAMILY FOUNDATION.

INDEX OF PAGES WELCOME LETTER FROM THE DIRECTOR

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SCHEDULE

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ABOUT THE BSCRC

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SPEAKER BIOGRAPHIES

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APARNA BHADURI, PH.D.

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SHUIBING CHEN, PH.D.

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GEORGE DALEY, M.D., PH.D.

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MARK KRASNOW, M.D., PH.D.

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TIPPI MACKENZIE, M.D.

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CHARLES MURRY, M.D., PH.D.

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SAMIRA MUSAH, PH.D.

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DAVID V. SCHAFFER, PH.D.

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POSTER ABSTRACTS

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Welcome to the 21st Annual Stem Cell Symposium!

Dear Colleagues and Friends: On behalf of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, it is my privilege to welcome you to the 21st Annual Stem Cell Symposium. This year’s gathering carries particular significance as it coincides with the 20th anniversary of our center’s founding — two decades of groundbreaking research, transformative innovation, and a shared commitment to improving human health.

We come together in the aftermath of the recent wildfires, which have had a profound impact on our Los Angeles community. Many have faced devastating losses — of homes, cherished possessions, and stability — and we extend our heartfelt support to all who are rebuilding in the wake of these events. In such trying times, the power of community becomes a vital force, reminding us of the resilience and compassion that define our shared humanity.

This symposium has always been more than an annual event. Over the past 21 years, it has been a touchpoint — a place for collaboration, conversation, and connection. It has brought together a cadre of researchers who understand that stem cell science is not just about discovery but about addressing the most urgent health challenges of our time.

This year, we are thrilled to showcase an exceptional lineup of speakers who will share cutting-edge advancements in stem cell research, along with nearly 60 early-career scientists presenting their work in our poster session. These scholars, trained through our center’s interdisciplinary mentoring programs, represent the future of regenerative medicine and the hope it brings to patients worldwide.

Looking back, our 20-year journey has been nothing short of extraordinary. But what excites me most is what lies ahead. From paradigm-shifting technologies to life-changing therapies, the field of stem cell science continues to transform possibilities into reality. At the Broad Stem Cell Research Center, we remain committed to driving this progress, guided by the power of collaboration and the shared vision of what is possible.

Of course, none of this progress happens in isolation. It is your collective efforts as researchers, clinicians, educators, and philanthropists that propel our field forward. As we celebrate this milestone, we do so with immense gratitude for your dedication and partnership. You are an integral part of our journey, and together we will continue to forge new paths in stem cell research and regenerative medicine.

Thank you for being here and for being part of this vibrant and enduring community.

Sincerely, Dr. Thomas A. Rando Director, UCLA Broad Stem Cell Research Center 3

21ST ANNUAL

STEM CELL SYMPOSIUM

FRIDAY, FEBRUARY 7, 2025 COVEL COMMONS 8:00AM-4:35PM

8:00-8:30am

Check-in Begins

8:30-8:45am

Welcome Remarks by Thomas Rando, M.D., Ph.D. Director, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA

8:45-9:25am

Mark Krasnow, M.D., Ph.D. (Stanford University) Lung Stem Cells, their Niches, and the Initiation of Lung Cancer Moderator: Brigitte Gomperts, M.D.

9:25-10:05am

Aparna Bhaduri, Ph.D. (UCLA) Understanding Cell Types in the Developing Human Brain and Glioblastoma Moderator: Lili Yang, Ph.D.

10:05-10:30am

Coffee Break

10:30-11:10am

Samira Musah, Ph.D. (Duke University) Advancing Stem Cell Differentiation and Human Disease Modeling with Organ Chips Moderator: Melody Li, Ph.D.

11:10-11:50am

David V. Schaffer, Ph.D. (University of California, Berkeley) Engineering Biomaterial Microenvironments to Scale Stem Cell Expansion and Differentiation for Clinical Translation Moderator: April Pyle, Ph.D.

11:50am-12:30pm

Lunch

12:30-1:30pm

Poster Session

1:30-2:10pm

Charles Murry, M.D., Ph.D. (University of Southern California) Heart Regeneration: Peeling an Onion?

Moderator: Arjun Deb, M.D.

2:10-2:50pm

Shuibing Chen, Ph.D. (Cornell University) Human Pluripotent Stem Cells, Organoids and Drug Screening Moderator: Song Li, Ph.D.

2:50-3:15pm

Coffee Break

3:15-3:55pm

Tippi MacKenzie, M.D. (University of California, San Francisco) Prenatal Therapies for Single Gene Disorders Moderator: Amander Clark, Ph.D.

3:55-4:35pm

George Daley, M.D., Ph.D. (Harvard University) Hematopoietic Lineages from Pluripotent Stem Cells Moderator: Thomas Rando, M.D., Ph.D.

4:35-6:00pm

Reception

THE SYMPOSIUM IS SUPPORTED BY A GENEROUS ENDOWMENT FROM THE MARGARET M. BLOOMFIELD FAMILY FOUNDATION.

ABOUT THE BROAD STEM CELL RESEARCH CENTER History In 2005, UCLA launched the Institute for Stem Cell Biology and Medicine, now the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, to bring together scientific, legal, and ethical experts from across the university to focus on fulfilling the great promise of stem cell research.

Since then, our scientists have advanced the field in meaningful ways—initiating cell and gene therapy clinical trials for patients with deadly immune diseases, late-stage cancers, blinding eye diseases and more.

Mission Our center supports innovation, excellence, and the highest ethical standards focused on taking groundbreaking stem cell research from the laboratory to the patient. We're committed to a multidisciplinary and integrated collaboration of scientific, academic, and medical disciplines for the purpose of revolutionizing the treatment of disease through personalized cellular therapies and regenerative medicine.

Funding Our research is made possible by granting agencies such as the California Institute for Regenerative Medicine (CIRM) and the National Institutes of Health (NIH). Many of our hallmark programs are generously supported by philanthropic organizations including The Eli and Edythe Broad Foundation and The Rose Hills Foundation, as well as by individual contributors.

2025 Stem Cell Symposium Organizing Committee Members: Thomas Rando, M.D., Ph.D. Director, UCLA Broad Stem Cell Research Center Professor, Neurology and Molecular, Cell and Developmental Biology

Melody Man Hing Li, Ph.D. Assistant Professor, Microbiology, Immunology and Molecular Genetics

Song Li, Ph.D. Chancellor’s Professor, Bioengineering

April Pyle, Ph.D. Professor and Vice Chair, Microbiology, Immunology and Molecular Genetics

Lili Yang, Ph.D. Professor, Microbiology, Immunology and Molecular Genetics 5

Speaker Biographies Aparna Bhaduri, Ph.D. Assistant Professor, Biological Chemistry University of California, Los Angeles

Dr. Aparna Bhaduri earned a B.S. in Biochemistry and Cell Biology and a B.A. in Political Science from Rice University in 2010. She completed her doctoral studies in Cancer Biology at Stanford University in 2016, where she focused on epithelial tissue differentiation and neoplasms. She completed her postdoctoral training in Dr. Arnold Kriegstein's lab at the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at the University of California, San Francisco. She has used single-cell RNA sequencing to characterize cell types in the developing cortex across cortical areas in human and non-human primates, and in glioblastoma. Because experimental manipulations of the developing human cortex will require in vitro models, she has been using similar approaches to compare cells types in organoid models and primary tissues. Her long-term interest is in understanding how stem cells give rise to the human brain during cortical development, and how aspects of these developmental programs can be hijacked in cancers such as glioblastoma. In order to explore these questions, the Bhaduri Lab uses single-cell genomics, informatic analysis, and organoid models.

Shuibing Chen, Ph.D. Director, Center for Genomic Health Vice Chair, Innovation in Surgery Kilts Family Professor, Surgery and Biochemistry Cornell University Dr. Shuibing Chen is the Kilts Family Professor and Vice Chair of Innovation in the Department of Surgeryand the Director of the Center for Genomic Health (CGH) at Weill Cornell Medicine. Dr. Chen was trained as a biochemist in Dr. Peter Schultz's lab at Scripps. Following her postdoctoral training in Dr. Doug Melton's lab at Harvard, she transitioned to the stem cell field and began her independent career at WCM in 2011. Early in her career, Dr. Chen conducted chemical screens to identify compounds that control stem cell differentiation. In recent years, she has focused on using human stem cell-derived organoids for disease modeling and drug screening.

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Her laboratory has published in top peer-reviewed journals, including Nature, Nature Medicine, Nature Cell Biology, Cell Stem Cell, Cell Metabolism, etc. Dr. Chen serves on the Board of Directors for the International Society for Stem Cell Research and the International Chemical Biology Society. She is the President of the Chinese American Diabetes Association. She also serves on the editorial boards of leading stem cell journals, including Cell Stem Cell and Stem Cell Reports. Her exemplary contributions have been recognized with numerous prestigious awards, including the New York Stem Cell Foundation Robertson Investigator Award, the American Diabetes Association (ADA) Innovative Award, the NIH Director’s New Innovator Award, and the ISSCR Dr. Susan Lim Award for Outstanding Young Investigator, etc.

George Daley, M.D., Ph.D. Dean, Harvard Medical School Caroline Shields Walker Professor, Medicine Professor, Biological Chemistry and Molecular Pharmacology Harvard University Dr. George Daley is Dean, Caroline Shields Walker Professor of Medicine, and Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School. His research has focused on stem cell and cancer biology, with an emphasis on hematopoietic development and diseases of the bone marrow, blood and immune system. Daley earned his A.B. and M.D. degrees from Harvard and a Ph.D. in biology from MIT. Prior to becoming Dean at HMS, he was an investigator of the Howard Hughes Medical Institute and Director of the Pediatric Stem Cell Transplantation Program of the Dana-Farber Cancer Institute and Boston Children’s Hospital.

Mark Krasnow, M.D., Ph.D. Executive Director, Wall Center for Pulmonary Vascular Disease Paul and Mildred Berg Professor, Biochemistry Stanford University Dr. Mark Krasnow is the Paul and Mildred Berg Professor, Executive Director of the Wall Center for Pulmonary Vascular Disease, and past Chair of the Department of Biochemistry at Stanford University, and an Investigator of the Howard Hughes Medical Institute. He and his lab pioneered single-cell genetic and genomic approaches including single-cell RNA sequencing to elucidate organ development and stem cell programs, focusing on the respiratory system. They started with Drosophila where they defined the genes and molecular programs controlling each of the fundamental steps in organogenesis.

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They developed similar approaches for mouse lung and used them to define at single-cell resolution the progenitors, stem cells and their niches for many lung cell types. They created a molecular cell atlas of the human lung, identifying 58 molecular cell types, 14 of which are new, including specialized cells and stem cells on each side (air and blood) of the alveolar gas exchange surface. They are using this comprehensive atlas to map the targeted cell types and how they are transformed in major lung diseases including lung cancer, COPD/emphysema, pulmonary fibrosis, pulmonary hypertension, and respiratory infections such as COVID-19. They have also led establishment of a new genetic model organism, the mouse lemur, to study primate physiology, behavior, disease, and ecology, He is a member of the National Academy of Sciences and the National Academy of Medicine, and a fellow of the American Academy of Arts and Sciences.

Tippi MacKenzie, M.D. Director, The Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research Professor, Surgery University of California, San Francisco Dr. Tippi MacKenzie is a Professor of Surgery and Director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at the University of California, San Francisco. She is a pediatric and fetal surgeon who is focused on developing better ways to diagnose and treat genetic diseases before birth. She runs a translational research lab examining fetal immunology and maternalfetal tolerance, with the ultimate goal of inventing new fetal therapies for patients with genetic diseases or pregnancy complications. She has moved two fetal molecular therapies from the lab to the clinic as phase 1 clinical trials after obtaining FDA approval: in utero hematopoietic stem cell transplantation to treat fetuses with alpha thalassemia and in utero enzyme replacement therapy in fetuses with lysosomal storage disorders. Her research has been supported by the National Institutes of Health, the March of Dimes, the California Institute for Regenerative Medicine, and the Burroughs Wellcome Fund. Dr. MacKenzie has been elected to the American Society for Clinical Investigation and the National Academy of Medicine for her innovative work.

Dr. MacKenzie trained in classical piano at Juilliard before obtaining her undergraduate degree from Harvard College and her medical degree from Stanford University. She completed her surgical residency at Brigham and Women’s Hospital in Boston and obtained additional fellowships in Fetal Surgery and Pediatric Surgery at the Children’s Hospital of Philadelphia. She joined the faculty at the University of California, San Francisco in 2007 and is now a Professor of Surgery. She recently co-founded the Center for Maternal-Fetal Precision Medicine, with the aim of accelerating the processes that link basic research to clinical trials to improve maternal, fetal, and neonatal health. This Center is testing methods to improve prenatal diagnosis of birth defects and developing new cellular and molecular therapies for definitive fetal treatment.

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Charles Murry, M.D., Ph.D. Director, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research Professor and Chair, Stem Cell Biology and Regenerative Medicine University of Southern California Dr. Charles (Chuck) Murry received his bachelor’s degree in chemistry from the University of North Dakota, followed by M.D.-Ph.D. training at Duke University, where he studied myocardial ischemiareperfusion injury (heart attacks). He did residency training in Anatomic Pathology at the University of Washington, followed by fellowship training in vascular biology and diagnostic cardiovascular pathology.

At the UW, Murry was the founding director of the Center for Cardiovascular Biology, and he cofounded and for many years directed the Institute for Stem Cell and Regenerative Medicine. Murry recently was recruited to the University of Southern California Keck School of Medicine, where he chairs the department of Stem Cell Biology and Regenerative Medicine and directs the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research.

Dr. Murry’s research focuses on stem cell biology, with an emphasis on understanding differentiation of the human cardiovascular system and using these cells to study diseases and to regenerate damaged tissues. His group is a world leader in heart regeneration and is working toward a clinical trial using cardiomyocyte therapy. He has served on many local, national, and international committees, spoken widely about stem cells and cardiovascular medicine, and he has received numerous awards for teaching and scientific achievement. Dr. Murry is a past member of the International Society for Stem Cell Research Board of Directors and currently serves on its Manufacturing, Clinical Translation, and Industry Committee. In addition to his academic work, Murry has worked to promote commercialization of novel cardiovascular therapies. He cofounded BEAT Biotherapeutics, Sana Biotechnology, and most recently a Los Angeles-based startup called StemCardia Inc.

Samira Musah, Ph.D. Assistant Professor, Biomedical Engineering and Medicine, Nephrology Duke University Dr. Samira Musah is an Assistant Professor at Duke University, jointly appointed in the Department of Biomedical Engineering and the Department of Medicine, Division of Nephrology. Research in her laboratory aims to understand the roles of molecular and biophysical cues in organ development and how these processes can be harnessed to understand disease mechanisms and develop new therapeutic strategies.

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She is known for developing novel methods for stem cell differentiation and engineering functional models of human tissues and organs. Dr. Musah is a recipient of many prestigious research awards, including the NIH Director's New Innovator Award, the Whitehead Scholarship in Biomedical Research, the Burroughs Wellcome Fund Career Transition Award, the Baxter’s Young Investigator Award, the Genentech Research Award, the MEDx Biomechanics of Injury Repair Award, the Innovation & Entrepreneurship Initiative Award, and the Howard Hughes Medical Institute Gilliam Advisor Award. She was featured in Cell Stem Cell Early-Career Researchers and Nature Biotechnology Outstanding & Trailblazing Researchers. She was named among the inaugural "100 Inspiring Black Scientists in America" by Cell Press and named a Rising Star in Biomedical Engineering at MIT.

David V. Schaffer, Ph.D. Director, California Institute for Quantitative Biosciences Professor, Chemical and Biomolecular Engineering, Bioengineering, and Neuroscience University of California, Berkeley Dr. David Schaffer is the Hubbard Howe Professor, Chemical and Biomolecular Engineering, Bioengineering, and Molecular and Cell Biology at the University of California, Berkeley, and he also serves as the Executive Director of QB3 and the Director, Bakar BioEnginuity Hub, Bakar Labs, Bakar Fellows, and Bakar ClimatEnginuity Hub. He completed his B.S. in chemical engineering at Stanford University in 1993, his Ph.D. in chemical engineering at MIT in 1998, and a postdoctoral fellowship at the Salk Institute for Biological Studies in 1999 before joining Berkeley in 1999. There, he applies engineering principles to optimize gene and stem cell therapies, work that includes developing the concept of applying directed evolution to engineer targeted and efficient viral gene therapy vectors as well as new technologies to investigate and control stem cell function. He has published >250 papers, has advised >90 graduate students and postdoctoral fellows, is an inventor on >50 issued patents, and developed technologies that are being used in nine human clinical trials. In addition, he has co-founded eight companies, including 4D Molecular Therapeutics (NASDAQ: FDMT), Ignite Immunotherapies (acquired by Pfizer), and Rewrite (acquired by Intellia). He has received numerous recognitions, including election to the National Academy of Inventors and awards such as the Andreas Acrivos Award for Professional Progress, the American Institute of Chemical Engineers Pharmaceutical and Bioengineering Award, the American Chemical Society Marvin Johnson Award, and the Biomedical Engineering Society Rita Schaffer Young Investigator Award.

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Poster Abstracts Developing a rapid protocol to derive human lumbar/sacral spinal sensory interneurons for spinal cord regeneration Talin Demirjie UCLA – CSUN CIRM Bridges Trainee Lab PI: Samantha J. Butler, Ph.D. The spinal cord contains different populations of sensory interneurons, known as dorsal interneurons (dIs), crucial for processing touch, proprioception, pain, and itch. In spinal cord injuries (SCIs) or disease, dI dysfunction leads to sensory loss. To advance SCI therapies, we are developing human stem cell protocols to generate dIs through neuromesodermal progenitors (NMPs), the key progenitors that endogenously give rise to the spinal cord. However, no efficient protocols currently exist to rapidly induce the most posterior dIs, which relay gut and bladder sensations in vivo. We also want to ensure that the NMP protocols have wide patient applicability. Thus, we are using an African-American human-induced pluripotent stem cell (hiPSC) donor line (F3.5.2) to identify the conditions that induce human dIs with regional identities from the neck (brachial), trunk (thoracic), or tail (lumbar/sacral) spinal cord regions, thus facilitating region-specific transplantation. Specifically, we are testing whether NMPs can be posteriorized within 1-4 days by analyzing the effects of two growth factors: GDF11, which is highly expressed in the posterior spinal cord, or Notch, which also patterns this region. Spinal patterning was induced using retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) in 3D embryoid-body (EB) cultures. EBs were then analyzed with immunohistochemistry (IHC) to assess regional specificity through HOX expression and dI diversity for axial-specific identities. Preliminary results suggest high concentrations of GDF11 and Notch activation have limited effects on NMP posteriorization. However, we have unexpectedly found that treating the F3.5.2 line with BMP4, results in a wider range of cell fate decisions on day 20, than observed previously. These findings provide insight into critical time points in differentiation for generating specific dI subtypes with desired regional identities for regenerative therapies.

Modulating retinoic acid and WNT signaling to diversify human stem cell-derived sensory interneurons Yahir Verdin UCLA – CSUN CIRM Bridges Trainee Lab PI: Samantha J. Butler, Ph.D. Sensory modalities are relayed from the body to the brain by dorsal interneurons (dIs) in the spinal cord. Loss of these neurons after injury or disease can result in dysfunctional control over touch, pain, and motor output. Deciphering the mechanisms that direct embryonic stem cells (ESC) toward dI fates is essential to developing cellular regenerative therapies to replace impaired or injured sensory circuits. Previous studies in the Butler lab have shown that directing mouse ESC-derived neuromesodermal progenitors (NMP) towards dIs can generate in vitro-derived dIs that phenocopy their in vivo counterparts. These NMP protocols use retinoic acid (RA) ± bone morphogenic protein 4 (BMP4) to derive all dI populations, dI1-dI6. Moreover, these dIs largely resemble neck, and trunk regions, while the yield of most posterior dIs, such as tail regions is minimal. In developing embryos, RA and active Wnt signaling play critical roles in determining both the neuronal fates in the spinal cord and their lumbar/sacral identities by inducing posterior HOX gene expression. Thus, we tested if different concentrations of RA and WNTs can generate dIs with altered regional identities. To develop a human NMP protocol that can be applied to a wide patient base, we are deriving NMPs from ancestrally diverse human induced pluripotent stem cell (iPSC) lines. Spinal cord patterning was induced in NMP embryoid body (EB) cultures with low and high RA concentrations across a CHIR (WNT agonist) gradient. Immunohistochemical approaches were used to analyze if dIs with altered axial identities were generated. While we are still investigating the effects of these conditions on dI posteriorization, we unexpectedly discovered that low RA with high CHIR concentrations induced dI1, dI4-dI6. This is the first instance where we have identified the development of dorsal-most cell types without BMP4 treatment and suggests that WNT signaling has a critical role in human spinal cord patterning in vitro.

Cyto-Tag: A Tool to Rapidly Isolate Rare Cell Populations in Vivo Sara Frigui BSCRC – CIRM Predoctoral Trainee Lab PI: Heather Christofk, Ph.D. This project’s objective was to develop a technology to study the metabolism of stem cells in the in vivo niche to understand how metabolism drives cell fate decisions. Isolating rare cell types in vivo for downstream metabolomic analysis has posed an unsolved challenge. Current methods characterizing in vivo metabolism are lengthy and exert mechanical stress, resulting in alteration of the cellular metabolome and incompatibility with stable isotope-labeled nutrient tracing. We have developed the CYTO-Tag, a tool to isolate rare cell populations that is compatible with in vivo nutrient tracing. The CYTO-Tag is an affinity tag controlled by Cre-recombinase driven by a cell-type specific promoter.

Additionally, it can be used in conjunction with a new tissue digestion technique using proteases active at 4°C. Rapid cell isolation at cold temperatures should allow greater preservation of in vivo cellular metabolism.

We have validated CYTO-Tag mRNA expression in Lgr5+ intestinal stem cells (ISCs). We will further use CYTO-Tag and in vivo nutrient tracing to assess how fasting, diet, and pregnancy impact the metabolism of ISCs. Additionally, we have generated mice expressing the CYTO-Tag in Pax7+ Muscle stem cells (MuSCs) and will assess the impacts of injury and chronic exercise on MuSC metabolism.

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Investigating the Developmental Progression of Mouse Primordial Germ Cell-Like Cells in a Reconstituted Ovary Azra Cruz UCLA – CIRM COMPASS Scholar Lab PI: Amander T. Clark, Ph.D. Infertility affects around 15% of the global population, and existing clinical treatments fail to treat infertility at a germ cell level, leading to the importance of producing oocytes as a form of regenerative medicine. Though the reconstituted ovary (rOvary) is a model capable of creating oocytes, the process remains inefficient due to the high percentage of embryo loss. To begin improving the rOvary, it is essential to understand the development of germ cells in the in vitro system. Following the rOvary protocol, we generated rOvaries and collected them along their developmental trajectory. Utilizing immunostaining, we studied primordial germ cell-like cells (PGCLCs) as they developed into oocyte-like cells using green-fluorescent protein (GFP) to represent the transgenic reporter used, DAZL to mark PGCLCs competent of meiosis, and SCP3 to mark PGCLCs in meiosis. The expression of SCP3 led us to hypothesize that the inefficiency of the rOvary is due to an abnormal progression of meiosis in PGCLCs and a potential developmental misalignment. Expanding our knowledge of the rOvary is critical to gaining insight into the development of PGCLCs, improving the efficiency of IVO, creating higher-quality oocytes, and addressing infertility at a germ cell level.

A temporally resolved BMP4 response in the peri-implantation human embryonic disc is critical for the induction of multipotent TFAP2A + cells and EMT Auriana Arabpour BSCRC – Rose Hills Foundation Predoctoral Trainee Lab PI: Amander T. Clark, Ph.D. The unilaminar embryonic disc, a sheet of epithelized SOX2+ epiblast cells, undergoes complex morphogenesis to create a multi-dimensional human gastrula. This process endows early lineage specification and spatial identities that begin at the posterior end of the disc to create a coordinate system for embryogenesis. Here, we find that simply adding basement membrane extracts (BMEs) to the stem cell media is sufficient to model the early peri-gastrulating embryonic disc. We demonstrate that the emergence of amnion, primordial germ cells (PGCs), and mesoderm arise from BMP4-responsive disc cells that induce transient TFAP2A expression and SOX2 repression. We track the order of embryonic events that take place during disc morphogenesis and show that amnion-like cells (AMLCs) and PGC-like cells (PGCLCs) are specified first from epithelized TFAP2A+ progenitors. Shortly after, gastrulating mesoderm-like cells (MeLCs) arise from transiently expressed TFAP2A+ disc cells that then undergo EMT. We find that BMP4-responsiveness is critical to SOX2 repression, TFAP2A expression, lineage induction and EMT, while the process of EMT itself is not. Thus, we show that the extracellular matrix is necessary to promote disc morphogenesis and EMT.

Strategies for Analyzing Germ Cell Nest Breakdown, Follicle Formation, and Follicle Activation in the Reconstituted Ovary Autumn Jackson Lab PI: Amander T. Clark, Ph.D. Mouse reconstituted ovaries (rOvaries) are biomedical research tools used to model ovarian development in vitro. While many of the ovary’s cellular processes are reproducible in the rOvary, key differences between the in vitro and the in vivo system still exist. The process of follicle formation, which endows reproductive capacity in the female, is one such key event. Follicle formation happens when an egg cell (oocyte) is encapsulated by helper cells known as granulosa cells, which support the oocyte’s growth into maturity. Two classes of follicles exist: one that activates growth immediately after assembly, and another which remains dormant in the ovary until puberty. The rOvary is thought to only produce the first class of immediately growing follicles. However, this idea was only shown from the perspective of the oocyte. To gather a more holistic view of follicle assembly in the rOvary, we turned to in vivo ovaries. We performed immunofluorescence with a FOXL2 antibody to target granulosa cells, STELLA antibody for oocytes, SCP3 antibody for meiosis, and FOXO3 antibody to identify immediately growing or dormant oocytes in follicles. We combined our immunofluorescence with granulosa cell number quantification, which may be a reliable method to classify follicles as dormant or growing. We found that nuclear FOXO3 expression and an average granulosa cell count of 3-4 are reliable indicators for dormant follicles. However, FOXO3 was expressed in three classes of follicles over the course of growth. Therefore, follicle classification requires an integrated approach of protein markers and morphological indicators like granulosa cell count and shape. With the information gleaned from the in vivo ovary, we will be able to better understand in vitro follicle formation in the rOvary, and how to improve the process to make it more reflective of the in vivo system, increasing its relevance as a research and therapeutic tool.

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A 2D culture system for the efficient induction and study of early human germ cells Mark Larsen Lab PI: Amander T. Clark, Ph.D. During implantation, early human embryo cells diversify into several cell lineages including primordial germ cells, cells crucial for reproduction.

Our lab studies these cells to understand germ cell specification, which occurs 2-3 weeks post-conception, making them challenging to study in vivo. Valuable in vitro models of germ cell specification have been developed, but existing conditions generate cell line-dependent variability in germ cell induction. This variability makes it difficult to study molecular mechanisms of germ cell specification that are representative of their in vivo counterparts. To reduce variability, our lab has developed a highly defined embryo model that recapitulates differentiation of multiple human embryonic cells catalyzed by an overlay of basement membrane extract to cells plated in a xeno-free media. Here we have optimized this embryo model to induce a consistent percentage of germ cells for the study of germ cell specification in two separate cell lines.

Addition of a time-specific dose of BMP4 directs more cultured stem cells into specified germ cells, marked by SOX17 and TFAP2C expression.

To show the potential of the model for the mechanistic study of germ cell biology, we focused on the transcription factor TFAP2A, a known downstream effector of BMP4 involved in germ cell specification. Culturing stem cells harboring mutated TFAP2A led to significant reduction in germ cell percentages. This novel in vitro model provides the field with a reliable and reproducible means to study germ cell development.

Defining the Role of Autophagy-Deficient Bone Marrow-Derived Myeloid Cells on Tumor Growth Raymond Alvarado UCLA – CIRM COMPASS Scholar Lab PI: Hilary Coller, Ph.D. Autophagy, a cellular degradation process, is essential for maintaining cellular homeostasis and responding to stress. In cancer biology, autophagy plays a complex role as it can suppress tumor initiation and support tumor growth in established cancers. Furthermore, an essential element in cancer initiation and progression is the tumor microenvironment (TME), which includes immune cells, extracellular matrix proteins, and chemokines. Previous research found that a whole-body knockout (KO) of autophagy leads to smaller tumors. The role of autophagy in specific cell populations on tumor development remains poorly understood. My research addresses this gap by studying the effects of an autophagy KO in the immune cells within the TME. Macrophages and Dendritic Cells (DCs) play intricate roles within the TME, capable of either promoting or inhibiting tumor progression. As important cells in priming and activating T-cell responses, both cell types are targets for investigation. Our study investigates the tumor response of autophagy-deficient macrophage and DC. The macrophages and DCs were cultured in vitro from bone marrow that was isolated from either autophagy-proficient or deficient mice. The cultured macrophages and DCs were then treated with B16-F10, mouse melanoma, conditioned media to simulate a TIME. Then, RT qPCR was used to analyze their expression of proinflammatory cytokines. Our results demonstrate that autophagy-deficient macrophages and DCs express higher levels of proinflammatory cytokines than their autophagyproficient counterparts. These findings suggest that autophagy-deficient macrophages and DCs exhibit a heightened activity, which may play a role in slowing cancer progression. This study highlights the importance of studying immune cell responses in cancer biology. Our findings may pave the path for novel immunotherapy techniques that use macrophage and DC activity to battle tumor progression.

The Role of Senescent Macrophages as a Driver of Inflammaging and Aging-related Diseases Grasiela Torres Lab PI: Anthony J. Covarrubias, Ph.D. Aging is the number one risk factor for multiple diseases including cancer, diabetes, and neurodegeneration, creating urgency for solutions regarding health-span maintenance as lifespan increases. Cellular senescence is emerging as a main driver of age-related diseases due to the chronic low-grade inflammation that results from accumulation of senescent cells in various tissues throughout the body. Cells that undergo senescence are characterized by permanent cell cycle arrest, altered metabolism and secretion of inflammatory molecules, known as the senescent-associated secretory phenotype (SASP). Our lab has shown that senescent macrophages accumulate in aging tissues and can become senescent using DNA damaging stimuli such as irradiation and chemotherapeutic drugs. Our data suggest that macrophages represent a key source of senescent cells in aging tissues, however the molecular mechanisms driving senescence in macrophages and their link to age-related diseases remain unclear. Our study aims to understand role of senescent macrophages as a driver of inflammaging and aging-related diseases. To do so, we will identify genes and pathways that contribute to cellular senescence of macrophages. Our approach utilizes in vivo studies, use of a novel in vitro senescent macrophage system using primary bone marrow macrophages from WT and knockout mice, and CRISPR-Cas9 genomic editing to investigate how gene candidates regulate various features of senescence. Understanding the molecular mechanisms regulating macrophage senescence may provide insight into treating age-related diseases.

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Macrophages represent a source of p21+ senescent cells in aged and steatotic liver Ivan Salladay Lab PI: Anthony J. Covarrubias, Ph.D. Senescent cells play causal roles in sterile inflammation in aging tissues through the senescence-associated secretory phenotype (SASP).

Targeting senescent cells present promising strategies for treating age-related diseases linked to metabolic dysfunction; however, identifying specific senescent cell types, especially myeloid cells like macrophages, poses challenges in inflammatory conditions. To address this issue, an in vitro model of DNA damage-induced macrophage senescence was developed and facilitated the identification of unique biomarkers that distinguish senescence from other polarized states. Interestingly, excessive cholesterol storage is a distinctive feature and major driver of p21+ macrophage senescence, particularly in mice fed a high cholesterol diet. Treatment with senolytics reduced the levels of senescent macrophages and improved the fatty liver pathology. Furthermore, human senescent macrophages are also p21+ and are enriched in human liver cirrhosis samples. These findings provide evidence that senescent macrophages play pivotal roles in driving homeostatic dysfunction in aging tissues, potentially contributing to the development of liver failure. This underscores the potential of targeting macrophage senescence as a therapeutic approach to address the sources of age-related chronic inflammation.

Restoring Stemness of Surgically Procured Anti-Tumor T Cells Tyler McCaw, M.D., Ph.D. BSCRC – CIRM Clinical Fellow Lab PI: Joseph G. Crompton, M.D., Ph.D. Immunotherapy is a powerful cancer treatment strategy, occasionally resulting in cures; however, T cell exhaustion severely curtails its current clinical efficacy. Exhaustion is an epigenetically-mediated terminal differentiation wherein T cells lose stemness and tumor-killing ability. Thus, the problem is this: for immunotherapy to be effective, stem-like T cells are absolutely required but by the time of clinical presentation the vast majority of a patient’s available tumor-specific T cells are exhausted. We therefore hypothesized that transcription factor reprogramming can overcome the epigenetic mechanisms enforcing T cell exhaustion to regenerate stem-like tumor-specific T cells. Our approach is two-part.

First, exhausted tumor-specific CD8 T cells are dedifferentiated into induced pluripotent stem cells (iPSCs) via transduction with Oct3/4, Sox2, Klf4, and c-Myc transcription factors (OSKM; DE-differentiation). Second, CD8-derived iPSCs are re-differentiated to mature, stem-like CD8 T cells using an artificial thymic organoid (ATO; RE-differentiation). De-differentiating CD8 T cells procured from patients with soft tissue sarcomas (STS), we demonstrated that a T cell’s starting epigenome dictates its OSKM reprogramming potential. Notably, exhausted, polyclonal CD8 T cells isolated from surgically resected STS can generate stable iPSC colonies. Re-differentiation of these CD8-derived iPSC into mature CD8ab T cells was then accomplished using an ATO, reaching similar efficacies as iPSC derived from non-exhausted, monoclonal CD8 T cells. Our findings demonstrate that tumor-specific T cell exhaustion can be overcome and stemness restored using OSKM reprogramming. These stem-like CD8 T cells are then specific to the patient’s own tumor from which they were isolated and can then be reinfused into the patient as a potent immunotherapeutic platform.

Investigate the role of ancestry-associated germline variants in prostate cancer initiation Shile Zhang BSCRC – Rose Hills Foundation Predoctoral Trainee Lab PI: Andrew S. Goldstein, Ph.D. Prostate Cancer (PCa) is one of the most common and deadly cancers in American men. The PCa incidence and death rates are dispersed amongst minor ethnic groups, with African-American men having both higher incidence and mortality rates in PCa than Caucasian men. In addition to known factors associated with health disparities, including socioeconomic status, accessibility to healthcare, and racial bias, little is known about the genetic drivers of ancestry-associated disparities in PCa. Recent studies found that the heritable HOXB13 variants, or genetic mutations, increase PCa risks and progression in different ancestry groups. HOXB13 is a key gene for normal prostate development. Its variants of interest include G84E in Caucasians, G132E in Japanese, G135E in Chinese, and X285K in individuals of West African ancestry.

However, it remains unclear how the ancestry-specific HOXB13 variants influence the development of a prostate that eventually increases the risk of PCa. We employ a combination of 3D human prostate cell model and mouse embryonic prostate cell model to define the early role that these variants play in prostate tissue. Our data shows that some HOXB13 variants acquire stronger regenerative capacity and relevant functional changes in cell culture. Evaluating the underlying cause of the varying PCa risk in different ancestry groups is crucial to achieving a more equitable healthcare system in America. No studies have attempted to define the common mechanism behind multiple HOXB13 germline variants in pre-cancerous prostate tissues, largely due to the challenges in modeling early human prostate development. Our lab has developed both human and mouse cell-based model systems to strategically address the role of the heritable HOXB13 variants.

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Investigating Perilipin Function in Prostate Cancer Cell Lines to Explore Their Role in Lipid Metabolism and Androgen Receptor Signaling Javier Martinez UCLA – CIRM COMPASS Scholar Lab PI: Andrew S. Goldstein, Ph.D. Prostate cancer is the second most common cancer in men and a leading cause of cancer-related deaths. A key therapeutic approach targets the androgen receptor (AR) due to its role in tumor development. The AR, once activated by binding to androgens, translocates to the nucleus to act as a transcription factor for genes that drive cell survival and tumor growth. Androgen deprivation therapy (ADT) reduces androgen levels or blocks androgen binding to the AR. However, most tumors eventually become resistant to ADT, leading to castration-resistant prostate cancer (CRPC). Lipid metabolism is critical for tumorigenesis, as cells require enhanced lipid synthesis and uptake of lipids to fuel rapid cancer cell growth. Understanding lipidomic changes in CRPC can identify vulnerabilities to develop therapeutic targets. Using western blots, we aim to characterize endogenous perilipin (Plin2, Plin3, and Plin5P) expression in various prostate cancer (PCa) cell lines displaying a wide range of AR expression. Furthermore, to explore effects on lipid metabolism and AR signaling, we will overexpress Plin2, Plin3, and Plin5 in PCa cell lines and perform lipidomics and metabolomics. Currently, our preliminary results show that AR-low-expressing PCa cell lines display high levels of Plin2 expression. AR-low PCa cell lines are also marked by low levels of FASN and ACAC-A, two critical proteins important for lipogenesis. These experiments will help us understand how overexpression of each Plin affects lipid metabolism in AR high/low PCa cells.

This research will provide insight into lipid regulation and how it is interconnected with AR activity, which is crucial for developing strategies to target aggressive cancer cells.

Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids Mingxia Gu, M.D., Ph.D. Associate Professor, Anesthesiology and Perioperative Medicine The vasculature and mesenchyme exhibit distinct organ-specific characteristics adapted to meet local physiological demands. The microenvironment and cell-cell interactions are crucial in driving the adoption of organotypic features since the earliest developmental stages.

To recapitulate this entire process, we co-differentiated mesoderm and endoderm lineages within the same spheroid to vascularize lung and intestinal organoids from iPSCs. The ratio of endoderm and mesoderm lineages was fine-tuned by BMP signaling during the initial stage of differentiation, a critical step in generating the appropriate proportions of endothelial and epithelial progenitors with tissue specificity for subsequent organoid patterning and vascularization. Single-cell RNA-seq analysis further demonstrated the organ-specific gene signatures of endothelium and mesenchyme, and identified key ligands driving endothelial specification in both lung and intestine. The organotypic endothelium exhibited tissue-specific barrier function, enhanced organoid maturation, promoted cellular diversity, and supported alveolar structure formation on bioengineered scaffolds. Upon transplantation into mice, the vasculature retained its organ specificity and integrated with the host circulation, further enhancing the maturation and patterning of the organoids. Additionally, our model revealed abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease.

Distinct roles for NFκB signaling in the bone marrow niche and in hematopoietic stem cells upon hematopoietic aging Jennifer Chia, M.D., Ph.D. BSCRC – CIRM Clinical Fellow Lab PI: Alexander Hoffmann, Ph.D. Hematopoietic aging is characterized by chronic inflammation associated with myeloid bias, HSC accumulation, and functional HSC impairment. Yet it remains unclear how inflammation promotes aging phenotypes. NFκB both responds to and directs inflammation, and we present an experimental model of elevated NFκB activity (“IκB–”) to dissect its role in hematopoietic aging phenotypes. We found that while elevated NFκB activity is not sufficient for HSC accumulation, HSC-autonomous NFκB activity impairs their functionality, leading to reduced bone marrow reconstitution. In contrast, myeloid bias is driven by the IκB– proinflammatory bone marrow milieu as observed functionally, epigenomically, and transcriptomically. A new scRNA-seq HSPC labeling framework enabled further comparisons with aged murine and human HSC datasets, documenting an association between HSC-intrinsic NFκB activity and quiescence, but not myeloid bias. These findings delineate separate regulatory mechanisms that underlie the three hallmarks of hematopoietic aging, suggesting that they are specifically and independently therapeutically targetable.

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Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis Diego Sahagun UCLA – CIRM COMPASS Scholar Lab PI: Steve Jacobsen, Ph.D. Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. But most viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here, we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Elucidating the Regulation of a Critical Component of the SWI/SNF Chromatin Remodeling Complex Kalen Bunch UCLA – CIRM COMPASS Scholar Lab PI: Tracy L. Johnson, Ph.D. Stem cells require tight regulation of gene expression programs to maintain potency and differentiate at the right time. The SWI/SNF chromatin remodeling complex plays a critical role in this process by regulating genes required for growth and differentiation. This complex’s ability to integrate signals and control gene expression is conserved from mammals to yeast. While the mechanisms of SWI/SNF-dependent gene regulation are being studied, little is known about how the SWI/SNF complex is itself regulated. Here, we elucidate the mechanisms by which Snf2, the catalytic component of the SWI/SNF complex, is regulated in the biochemically tractable organism Saccharomyces cerevisiae.

Through random amplification of cDNA ends (RACE) we have found two distinct SNF2 RNA isoforms with the long isoform containing upstream open reading frames (uORFs). The transcription of these isoforms is influenced by glucose availability. To assess the effects of Snf2 RNA isoforms on protein expression we created a SNF2 5’UTR-Ub-GFP reporter system that allows for induction of long isoform-transcription.

Experiments with this system corroborate a model whereby SNF2’s two RNA isoforms have diverging effects on Snf2 protein expression.

A Proposed Murine Model to Study the Tumor Microenvironment Interaction of GBM and Microglia Amir Filabs UCLA – CSUN CIRM Bridges Trainee Lab PI: Harley Kornblum, M.D, Ph.D. Glioblastoma(GBM) is a highly aggressive brain cancer that is characterized by a complex tumor microenvironment (TME) that suppresses the immune system and reprograms immune cells into an immunosuppressive phenotype, allowing for unregulated cellular proliferation. Previous in-vitro studies such as conditioned media experiments have shown that microglia, the resident macrophages in the brain, are polarized to an anti-inflammatory, immunosuppressive phenotype in the GBM TME thereby permitting tumor growth. We hypothesize that this effect is partly due to cytokine secretion from nearby tumor endothelial cells’ (TECs) within the TME. Our study aims to investigate the mechanisms underlying this TEC-microglia interaction using a humanized microglia-mouse model. We will co-implant human microglia, tumor endothelial cells and patient-derived GBM cells into mouse brains, creating a physiologically relevant model of the GBM TME. Quantitative PCR data from in-vitro conditioned media experiments confirmed the expression of anti-inflammatory markers and identified key signaling pathways involved in this process, including JAK/STAT and TGF-β signaling. Therefore, we hypothesize that there will be similar results from our in vivo experiments.

Using single cell RNAseq, we will investigate the tumor-microglia and tumor-ECs interaction using UMAP plots, gene expression, and CellChat.

Our findings thus far highlight the potential for targeting TEC-derived signals to restore microglial anti-tumor activity, offering a novel therapeutic approach for improving GBM treatment outcomes. By understanding the mechanisms of TEC-microglia interactions, our work contributes to the broader understanding of the TME and its impact on tumor progression and therapy resistance.

Overcoming Glioblastoma Therapy Resistance by Interfering with intercellular vascular-to-cancer crosstalk Shane Yu Lab PI: Harley Kornblum, M.D, Ph.D. Glioblastoma (GBM), an aggressive form of brain cancer, is characterized by extensive neovascularization. In addition to supplying blood and nutrients, vascular endothelial (VE) cells provide trophic support to GBM cells via paracrine signaling, the precise mechanisms of which are being unraveled. Our lab has recently shown that one of the standard treatments for GBM, radiation, induced a phenotypic conversion of GSCs to vascular-like cells, which in turn, provide trophic support to the remaining tumor. In our current study, using patient-derived GBM and VE cells as well as orthotopic GBM mouse models, we demonstrated that Endocan (ESM1), an endothelial-secreted proteoglycan, promotes malignancy and radioresistance in GBM. Using Mass spectrometry analysis, we identified PDGFRA, a commonly dysregulated Receptor Tyrosine Kinase (RTK) in GBM, as a potential receptor for Endocan. Biacore experiment confirmed that Endocan binds to PDGFRA with nanomolar affinity.

Consistent with this result, we demonstrated that Endocan activates the PDGFRA pathway. Subsequent downstream signaling increases chromatin accessibility of the Myc promoter and upregulates Myc expression inducing highly stable phenotypic changes in GBM cells.

Inhibition of Endocan-PDGFRA signaling with ponatinib, a PDGFRA inhibitor increases survival in the Esm1 wild-type but not in the Esm1 knockout mouse GBM model. Identification of the Endocan/PDGFRA/Myc axis demonstrates an important role of VE cells in GBM malignancy while targeting this pathway might subdue the recurrence of GBM and further highlight the importance of vascular to tumor cell signaling for GBM biology.

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The Role of HDAC6 in Human Neurite Outgrowth Gabrielle Anne Go Lab PI: Harley Kornblum, M.D, Ph.D. Histone deacetylases (HDACs) are a large family of enzymes that diminish acetylation of histones and other proteins. HDAC inhibition has been investigated as a potential treatment to enhance neural repair, however, broad spectrum HDAC inhibitors have significant toxicities. In prior studies of rodent models, HDAC6 inhibition increased neurite outgrowth and survival. However, the potential role for targeting HDAC6 in human neural repair is still unknown. Treatment of NGN2-induced human neurons with either the broad spectrum HDAC inhibitor belinostat or the relatively selective HDAC6 inhibitor tubastatin A resulted in enhanced neurite outgrowth. However, while tubastatin A treatment resulted in elevated levels of acetylated α-tubulin, a direct target of HDAC6, belinostat treatment had no effect on tubulin acetylation, suggesting that the effect of belinostat on neurite outgrowth may be independent of HDAC6. To directly assess the role of HDAC6 in neurite growth, we knocked out HDAC6 in human ESCs using CRISPR/Cas9. NGN2-induced neurons were derived from these ESCs. Loss of HDAC6 enhanced neurite outgrowth and acetylated α-tubulin in the induced neurons, confirming the role of HDAC6 in neurite growth. These studies support a role for the study of selective HDAC6 inhibitors in neural repair.

Differences In Human Cell Phenotypes Between a Human PTEN Mutation Associated with Macrocephaly and Autism and a Complete PTEN Knockout Sofia Irvin Lab PI: Harley Kornblum, M.D, Ph.D. Heterozygous germline mutations in PTEN are associated with autism (ASD) and macrocephaly. While homozygous deletion of Pten in murine neural stem cells and their progeny is also associated with enlarged brains, there are few phenotypic changes in mice with heterozygous Pten deletions. This raises the possibility that human disease causing mutations play a dominant negative function, effectively eliminating PTEN functionality. To test this hypothesis, we utilized iPSCs derived from a patient carrying a heterozygous p.H93Y mutation in PTEN and who exhibited macrocephaly and ASD with isogenic CRISPR-corrected control lines and the same lines in which PTEN was fully deleted using CRISPR-Cas9. We measured activation of the AKT-mTOR pathway, the growth of cerebral organoids and neurite outgrowth in organoid-derived and NGN2-induced neurons. While full deletion resulted in enhanced organoid growth and neurite outgrowth in induced and organoid-derived neurons, there was very little effect of the heterozygous patient mutation on organoid growth compared to the CRISPR corrected line, and no clear effect on neurite outgrowth. Treatment with the selective mTOR inhibitor, rapamycin, inhibited neurite outgrowth in all conditions, suggesting that some level of tonic mTOR activation is present in all conditions and regulates human neurite outgrowth. Surprisingly, no condition resulted in elevated levels of pS6, while only the complete PTEN knockouts resulted in elevated pAKT, suggesting that chronic inhibition of PTEN function results in activation of mTORC2. These findings suggest that the mutation studied does not result in a full disruption of PTEN function, indicating that environmental or other factors may play a role in inactivating the WT allele in this patient. Future experiments will determine whether the same holds for other mutations associated with ASD and macrocephaly.

Characterization of a Microprotein in Saccharomyces cerevisiae Quinn Mortensen UCLA – CSUN CIRM Bridges Trainee Lab PI: Siavash K. Kurdistani, M.D. Small peptides, or microproteins, are being increasingly identified, but their roles in cellular biology remain mostly unknown. Our laboratory has discovered a novel 29-amino acid peptide, provisionally named CBP-29, that has not been previously characterized. This peptide is encoded in the ribosomal DNA (rDNA) locus of yeast within the non-transcribed spacer 2 (NTS2) region. While NTS2 was initially believed not to be transcribed, it is now known that NTS2 generates several mRNAs with unknown functions. Two proteins, the Sir2 lysine deacetylase, and the Set1 lysine methyltransferase, prevent high levels of transcription of NTS2 in rich media. Still, our preliminary data indicate that they do not affect the expression levels of CBP-29. My project focuses on characterizing the cellular impact of the loss of CBP-29 to delineate its function.

I have found that loss of CBP-29 may increase cellular resistance to starvation and may be involved in Zn homeostasis.

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Binary Colloidal Crystals Influence the Direct Conversion of Fibroblasts into Neurons Johnathan Horn UCLA – CIRM COMPASS Scholar Lab PI: Song Li, Ph.D. Direct reprogramming is the process of converting from one cell type into a very distantly related cell type without the use of an intermediate proliferative stem-cell-like stage. Studies have shown that somatic cells can be directly reprogrammed into neuronal subtypes using different combinations of transcription factors and microRNAs. Biochemical factors have also been shown to help reprogram cells into induced neuronal (iN) cells. Unlike biochemical factors, the role of biophysical factors during direct reprogramming is limited. Some biophysical factors have been shown to regulate varying cellular behaviors, and the use of binary colloidal crystals (BCCs) have been used as cell substrates for cell differentiation. Although the roles of transcription factors and soluble biochemical factors in reprogramming have been widely studied, how biophysical cues provided by BCCs regulate iN reprogramming is not well understood. Here we show that a specific BCC substrate, i.e.

BCC 6 (2 µm Si/ 70 nm PS), can enhance iN reprogramming after screening seven combinations of BCCs. We found that increasing the size of the polystyrene particles used in the BCC surfaces from 100 nm to 400 nm improved iN reprogramming efficiency and purity. On the other hand, decreasing the size of silica particles (5 µm to 2 µm) increased iN cell yield and purity. With regards to surface chemistry, we found that carboxylated polystyrene particles decreased both the reprogramming efficiency and iN purity by 1.4-fold. Findings from this study aim to broaden the understanding of cell-matrix interactions' influence on the fibroblast-to-neuron reprogramming process.

Viscoelastic Extracellular Matrix Enhances Epigenetic Remodeling and Cellular Plasticity Yifan Wu Lab PI: Song Li, Ph.D. Extracellular matrices of living tissues exhibit viscoelastic properties. Although several studies have investigated the cytoskeletal reorganization on viscoelastic substrates to connect extracellular mechanical changes with intracellular signaling, the roles of the cell nucleus as an important mechanosensory unit in response to matrix viscoelasticity is largely unknown. Deciphering whether and how matrix viscoelasticity affects the cell nucleus to modulate cell fate, especially chromatin and the epigenome, will shed lights on the development of smart materials to control cell phenotype conversion. To achieve this, we engineered the stiffness and viscoelasticity of cell-adhesive substrates, and investigated the genome-wide biophysical and biochemical changes globally and at specific sites. We found that viscoelastic substrates induce nuclear structural reorganization and epigenetic changes, with more pronounced effects on softer surfaces. Fibroblasts on viscoelastic substrates displayed larger nuclei, loosely organized chromatin, and differential expression of distinct sets of genes related to the cytoskeleton and nuclear function compared to those on purely elastic surfaces. Slow-relaxing viscoelastic substrates reduced lamin A/C expression and enhanced nuclear remodeling. These structural changes were accompanied by a global increase in euchromatin marks and local increase in chromatin accessibility at cis-regulatory elements associated with neuronal and pluripotent genes. Consequently, viscoelastic substrates improved the reprogramming efficiency from fibroblasts into neurons and induced pluripotent stem cells. Collectively, our findings unravel the roles of matrix viscoelasticity in epigenetic regulation and cell reprogramming, potentially leading to a new technology platform to engineer cell fate for tissue regeneration, disease modeling, and drug screening.

Viscoelastic Synthetic Cells Enhance TCR-T Cell Activation and Antitumor Efficacy Youcheng Yang Lab PI: Song Li, Ph.D. Recent advancements in TCR-T therapy have emphasized the importance of effective T cell activation for therapeutic applications. However, current methods for T cell activation and expansion, such as antibody-coated paramagnetic polystyrene beads (e.g., Dynabeads), often result in suboptimal cell activation. One reason is that Dynabeads are stiff and have a very different mechanical property in comparison to antigenpresenting cells (APCs) which are softer and have the viscoelastic property that is critical to mediate ligand-receptor engagement and activation. To address this, we explored the use of tunable viscoelastic synthetic cells as a novel platform for activating antigen-specific T cells. These synthetic cells, designed to mimic the mechanical properties of antigen-presenting cells, provide a customizable microenvironment that enhances T cell receptor (TCR) engagement and subsequent activation. We employed a click-chemistry based method to conjugate anti-CD28 and MHC (HLA-A2-NY-ESO1) on alginate microbeads to fabricate these synthetic cells. They can have the same stiffness but varying degrees of viscoelasticity, which are fast-relaxing beads (FRB), medium-relaxing beads (MRB), and slow-relaxing beads (SRB). Our experiments demonstrated that T cells cultured with these beads exhibit increased proliferation and cytokine production compared to cell-based activation methods. Moreover, the difference in the viscoelasticity of the beads allows for the change of T cell activation proximal signaling and metabolism, which may contribute to the better in vivo therapeutic effect. This innovative approach holds promise for improving the design of TCR-T based therapies, offering a versatile tool for immunological research and clinical applications.

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Exploring DNA Damage-Induced Senescence Pathways within CDKL5 Deficiency Disorder, Down Syndrome, Bohring-Opitz Syndrome, and Arboleda-Tham Syndrome neuronal cell models Leslie Rodriguez UCLA – CSUN CIRM Bridges Trainee Lab PI: William Lowry, Ph.D. Down Syndrome, Arboleda-Tham Syndrome, Bohring-Opitz Syndrome, and CDKL5 Deficiency Disorder are all neurological diseases caused by a single genetic mutation. This research aimed to determine whether the same senescence pathways contributed by elevated DNA damage observed in Rett Syndrome are also present in other intellectual diseases including Down Syndrome, Arboleda-Tham Syndrome, Bohring-Opitz Syndrome, and CDKL5 Deficiency Disorder. Using similar molecular profiling techniques as in the Rett Syndrome model, the research shown here takes advantage of the disease in a dish model to grow patient-derived induced pluripotent stem cells (iPSCs) into neurons to mimic these three neurodevelopmental disorders. Although the number of affected groups is high, molecular ramifications of these diseases have yet to be investigated. Throughout this process, we conducted a Beta-Galactosidase Assay, Comet Assay, and a series of Immunofluorescent staining to deduce whether the neural progenitor cells or neuronal cells were senescent and whether they had a higher incidence of DNA damage/breaks.

The preliminary results suggest elevated senescence patterns and DNA damage markers expression in some lines but not others. The Down Syndrome cells lines had an increase in cellular senescence and DNA damage. Arboleda-Than Syndrome and Bohring-Opitz Syndrome lines show competing results that do not fully support senescence and DNA damage as a component for the disease. CDKL5 deficiency disorder lines also showed crossing results between each DNA damage marker stains but preliminary senescence staining revealed higher incidence of senescence in mutant lines. This helped us characterize key cellular stress phenotypes for each disease. If a connection between these molecular mechanisms is made, we can identify possible treatments for the cells to further the research question and possibly provide an avenue for treatment options for patients.

Temporally distinct 3D multi-omic dynamics in the developing human brain Matthew Heffel Lab PI: Chongyuan Luo, Ph.D. The human hippocampus and prefrontal cortex play critical roles in learning and cognition, yet the dynamic molecular characteristics of their development remain enigmatic. Here we investigated the epigenomic and three-dimensional chromatin conformational reorganization during the development of the hippocampus and prefrontal cortex, using more than 53,000 joint single-nucleus profiles of chromatin conformation and DNA methylation generated by single-nucleus methyl-3C sequencing (snm3C-seq3). The remodelling of DNA methylation is temporally separated from chromatin conformation dynamics. Using single-cell profiling and multimodal single-molecule imaging approaches, we have found that short-range chromatin interactions are enriched in neurons, whereas long-range interactions are enriched in glial cells and non-brain tissues. We reconstructed the regulatory programs of cell-type development and differentiation from early radial glia stem-like progenitors to fully developed adult neurons and glial support cells, finding putatively causal common variants for schizophrenia strongly overlapping with chromatin loop-connected, cell-type-specific regulatory regions. Our data provide multimodal resources for studying gene regulatory dynamics in brain development and demonstrate that single-cell three-dimensional multi-omics is a powerful approach for dissecting neuropsychiatric risk loci.

Deciphering the functions of MLLT3 isoforms in hematopoietic and leukemia stem cell fate decisions Yuanyuan Wang, Ph.D. Lab PI: Hanna K.A. Mikkola, M.D., Ph.D. Leukemia stem cells (LSCs) and normal hematopoietic stem cells (HSCs) share the unique ability to self-renew, making it essential to distinguish their common mechanisms from LSC-specific pathways to develop targeted leukemia therapies. Our lab identified two MLLT3 isoforms—MLLT3-L (long) and MLLT3-S (short)—as key, yet opposing, regulators of human HSC stemness. MLLT3-L promotes stemness, while MLLT3-S serves as a potential inhibitor by sequestering key protein partners of MLLT3-L, such as DOT1L and the superelongation complex (SEC). MLLT3 is also involved in various leukemias and frequently serves as a fusion partner with MLL. However, little is known about the function of MLLT3 isoforms in initiating or maintaining LSCs. Our bulk, long-read, and single-cell RNA-Seq data revealed an imbalance of MLLT3 isoforms in aggressive leukemias, including MLL-MLLT3 and MECOM-rearranged leukemias, characterized by reduced or absent expression of MLLT3-S. In HSCs, MLLT3-L and MLLT3-S modulate MECOM expression via DOT1L-dependent H3K79me2 modification, whereas in MECOM-rearranged LSCs, full-length MLLT3 fuels MECOM overexpression, while MLLT3-S is absent. ChIP-seq data show that while MLLT3 binds the transcription start site (TSS) of both long and short MECOM isoforms in HSCs, it selectively binds the leukemogenic short MECOM (EVI1) isoform in LSCs. Furthermore, ChIP-seq identified a feedforward regulatory loop in which EVI1 binds the TSS of MLLT3-L, reinforcing the stemness program in MECOM LSCs. We are now defining the functional role of MLLT3 isoforms in LSC fate using lentiviral knockdown and overexpression, and will test whether MLLT3-S can disrupt MLLT3-L and MLL-MLLT3 complexes in LSCs. These findings will provide mechanistic insights into LSC regulation and suggest novel therapeutic strategies.

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Mimicking developmental niches to improve human PSC-HSC maturation and function Meng-Wei Ko, Ph.D. Lab PI: Hanna K.A. Mikkola, M.D., Ph.D. Hematopoietic stem cells (HSC) transplantation is a curative treatment for various blood disorders. However, many patients who could benefit from this treatment have a significant challenge of finding a matching donor, highlighting the need for alternatives that could make this lifesaving therapy available to all patients. To mitigate the hurdle, our long-term goal has been to generate HSCs in the laboratory from pluripotent stem cells (PSC). The most advanced protocols to date generate PSC-HSC that can be transplanted, but with low efficiency, which stems from their developmental immaturity. This poses a significant challenge to the clinical applicability of PSC-HSCs, as their capacity to engraft and produce blood cells over time is limited. To address this, our laboratory created a detailed map of human HSC development by employing single-cell RNA sequencing analysis. This map provides a comprehensive framework for understanding the various stages of human HSC development at the molecular level. When referencing to this developmental map, we found that PSC-HSC closely resemble the immature HSC generated early in development. Although these PSC-HSC represent an important step forward, they lack critical features of developmentally mature HSC, which likely contributes to their impaired function in transplantation settings. To promote the maturation of PSC-HSCs, we optimized a two-step culture system combining PSC-HSC generation with our established HSC expansion protocols. Interestingly, a subpopulation of PSC-HSCs gradually acquired hallmarks of more mature HSC in culture. We will now evaluate if these more mature PSC-HSCs show improved engraftment and blood cell production compared to early-stage PSC-HSCs. If we can enhance PSC HSC transplantation efficacy, this approach could provide a more accessible treatment for patients with blood cancers and hematological disorders.

Tracing the Origin and Evolution of Myeloid Leukemia in Down Syndrome Stephanie Frenz-Wiessner, Ph.D. Lab PI: Hanna K.A. Mikkola, M.D., Ph.D. Infants with Down syndrome (DS) have a 150-fold increased risk of developing myeloid leukemia (ML-DS) within the first years of life, attributed to aberrant hematopoietic stem/progenitor cell development. ML-DS has a favorable prognosis at diagnosis but is associated with significant treatment-related toxicities and poor outcomes after relapse, emphasizing the need for targeted therapies and molecular risk stratification. MLDS is preceded by transient abnormal myelopoiesis (TAM), a pre-leukemic condition arising in utero due to GATA1 mutations that result in the truncated protein GATA1s. This disrupts erythroid and megakaryocytic differentiation, leading to an accumulation of immature progenitors.

Although TAM resolves in most infants by 3 months, it is linked to high mortality, and 20% of cases progress to ML-DS without effective prevention strategies.

However, the developmental origin and leukemic evolution of TAM and ML-DS at the molecular level is not well understood.

We created a comprehensive single-cell map of disease origin, progression, treatment response, and relapse, by performing 5′ single-cell RNA sequencing of longitudinal samples from 5 TAM and 14 ML-DS patients, including 4 relapses, as well as 16 and 20-week fetal liver and bone marrow samples from Trisomy 21 (T21) and euploid tissues.

To trace ML-DS evolution and developmental origin we defined stage-specific molecular signatures and identified a TAM precursor cell population expanded in T21 fetal liver. We assessed GATA1 transcripts at the single-cell level and detected GATA1s with exon 2 skipping that enables clonal tracking and leukemia stem cell (LSC) characterization across disease stages. Flow cytometry validated novel blast surface markers associated with stemness programs. By tracing leukemia evolution from a fetal cell of origin through TAM, ML-DS, and relapse, our study bridges basic research and clinical application, offering insights into other infant leukemias originating in utero.

Utilizing small molecule inhibition to optimize cerebral organoid models for FOXP1 syndrome Isiah Estrada UCLA – CSUN CIRM Bridges Trainee Lab PI: Bennett Novitch, Ph.D. Cerebral organoids created from human induced pluripotent stem cells (hiPSC) have emerged as one of the best experimental platforms for recapitulating features of brain development in vivo and pathologies associated with neurological disease. In our study, cerebral organoids are used to model FOXP1 syndrome, a neurodevelopmental disorder associated with developmental delays in language milestones, epilepsy, and high incidence of autism spectrum disorder. A crucial aspect to cerebral brain organoid development is the manipulation of small molecule inhibitors, as they direct stem cell differentiation by manipulating developmental signaling pathways. Different concentrations of TGF- β, Wnt, and Ras/Raf/MAP Kinase pathway inhibitors were tested to establish which combinations could best yield the formation of well-structured organoids suitable for modeling the impact of FOXP1 gene loss on cortical development. Our preliminary findings show that the combined inhibition of TGF- β and Wnt pathways resulted in a large number of undifferentiated cells and excessive cell death. By contrast, Ras/Raf/MAP Kinase pathway inhibition alone increased the overall formation of neuroepithelial progenitors, though lower than expected numbers of neural rosette structures as visualized through immunohistochemistry. In our ongoing and future experiments, we aim to further optimize the organoid differentiation conditions by modifying the concentration and combinations of these and other signaling pathway modulators. In the future, we will seek to create WT and FOXP1-deficient hippocampal and ganglionic eminence organoids and use these to form assembloids in which functional neural circuits can be constructed to enable us to examine the impact of FOXP1 deficiency on neural network activities. These findings will provide a foundation to learn more about the underlying causes of FOXP1 syndrome and potentially therapeutic interventions to improve the well-being of patients with this disorder.

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Modeling Rett Syndrome with Patient Derived Brain Organoids Natella Baliaouri BSCRC – Rose Hills Foundation Predoctoral Trainee Lab PI: Bennett Novitch, Ph.D. Rett syndrome (RTT) is an X-linked neurodevelopmental disorder primarily caused by heterozygous mutations in the MECP2 gene. RTT is characterized by symptoms such as developmental delays, stereotyped movements, and seizures. Dysfunctional and functional MECP2 expression is mosaic due to the randomized pattern of X-chromosome inactivation in individual cells, meaning cells express either functional or dysfunctional MECP2. Both the proportion of dysfunctional cells and the specific mutation impact symptom severity. Prior organoid models used RTT-patient derived induced pluripotent stem cells to create organoids with homogenous cell populations. We have created and validated a mosaic organoid model for RTT to better model the disorder and examine the cell autonomous and non-cell autonomous effects of MECP2 dysfunction. Our mosaic models demonstrate distinct electrophysiological phenotypes relative to the fully mutant and control models. MECP2 dysfunction appears to impact neural network development by affecting the early neurodevelopmental timeline of mutant cells relative to control cells in mosaic organoids, which likely contributes to the electrophysiological differences in more mature organoids. We plan to further analyze the differences in cells with functional MECP2 in fully control organoids as compared to mosaic organoids to model the non-cell autonomous effects of cells with dysfunctional MECP2. Incorporating cellular mosaicism in the organoid model recapitulates the cellular composition of the brain of female RTT patients more accurately than any previous in vitro model. Additionally, mosaic organoids may have broader applicability to other disorders in which mosaicism is a notable feature. Mosaic organoid studies will help provide new insights into some of the key downstream effectors of MECP2 both intercellularly and within the cell.

Investigating the Effects of MECP2 Mutation on Neurogenesis in a Mosaic Brain Organoid Model for Rett Syndrome Maria Caballero UCLA – CIRM COMPASS Scholar Lab PI: Bennett Novitch, Ph.D. Human induced pluripotent stem cell (hiPSC)-derived brain organoids are increasingly becoming a reliable model to investigate neurodevelopmental disorders such as Rett syndrome, which is caused by heterozygous mutations in the MECP2 gene located in the X chromosome. In females, variability in phenotypes and symptom severity can result from random patterns of X-chromosome inactivation that lead to mosaic expression of wild-type and mutant MECP2 alleles in patient brains. To recapitulate this feature of Rett syndrome, we have successfully produced mosaic organoids by intermixing isogenic wild-type control and MECP2 mutant cells, which result in structures that exhibit abnormal neural network activities. To further refine our investigation, we assessed alterations in neurogenesis across wild-type, mutant, and a range of mosaic organoids by quantifying the expression of key markers reflecting distinct populations and developmental states of cells in the developing cortex. Our analysis reveals some significant shifts in the cellular composition across our mosaic models, providing insights into how MECP2 mosaicism might influence early cortical development in patients’ brains. This approach provides a relevant model for the study of the developmental basis of Rett syndrome, deepening our physiological understanding of the disorder.

Enhancing maturity of skeletal muscle progenitor cells using 3D hydrogels in vitro George Radionov UCLA – CSUN CIRM Bridges Trainee Lab PI: April Pyle, Ph.D. Volumetric muscle loss (VML) is characterized by a significant loss of skeletal muscle, resulting in permanent impairment due to the overwhelming demand for repair. With no standard treatment available, breakthroughs in stem cell therapies have sparked interest in using in vitro-generated muscle stem cells, called satellite cells, from human pluripotent stem cells (hPSCs) to combat muscle loss and enhance repair.

However, current in vitro protocols for generating skeletal muscle progenitor cells (SMPCs) are limited to modeling embryonic stages of development. These cells exhibit low niche occupancy and limited regenerative capacity when engrafted in vivo. To tackle these challenges, the Pyle Lab has created a protocol allowing for the co-differentiation of hiPSCs into vascularized muscle fibers innervated by motor neurons.

The integration of endothelial cells increases SMPC proliferation and fusion in addition to enhancing PAX7+ cell maturity. Although this new protocol produces fetal muscle MYH8 fibers and SMPCs, results have shown limitations in reaching full adult maturity. We hypothesize that integrating this model in a 3D environment will provide a more accurate setting for the capillarization of skeletal muscle and the formation of adult muscle MYH2 fibers and satellite cell niches. To achieve this, we created a 3D fibrinogen-thrombin-matrigel hydrogel to obtain long-term structural integrity and centralized suspension of the cells during differentiation, then compared to its 2D counterpart. Gene expression analysis was then optimized to extract RNA from the cells cultivated within the extracellular matrix (ECM) hydrogel at days 5 and 14. Our preliminary results showed high expression of myogenic and endothelial markers after 14 days. By integrating stage-specific ECM as discovered by the Pyle Lab in vivo, into the 3D model, we aim to generate more mature SMPCs with enhanced regenerative capabilities for cellular therapeutics to treat conditions like VML.

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Enhancing skeletal muscle progenitor cell maturity using endothelial cells to treat volumetric muscle loss Kilian Mazaleyrat, Ph.D. BSCRC – CIRM Postdoctoral Trainee Lab PI: April Pyle, Ph.D. Volumetric muscle loss (VML) injuries occur due to orthopedic trauma or the surgical removal of skeletal muscle (SkM) and results in irreversible damage in muscle structure and function due to the loss of a significant portion of SkM. Current clinical options to treat VML are inefficient, as advances in tissue engineered SkM offer a potential solution to address lost or damaged muscle tissue but are limited by donor availability and issues associated with immune incompatibility. The use of human induced pluripotent stem cells (hiPSCs) derived SkM is extremely promising for the treatment of VML due to their ability to generate personalized muscle cells in a dish. However, the Pyle lab has shown that currently available protocols for generating hiPSC muscle progenitors (SMPCs) are limited to modeling the embryonic stage of development. We hypothesized that introducing endothelial cells in our established protocol, allowing the co-differentiation of hPSCs into innervated muscle fibers, would enhance the proliferation and the maturity of the SMPCs. To optimize our model, we induced the codifferentiation of the lateral mesoderm along the paraxial mesoderm and the neuroectoderm and then induced the differentiation and proliferation of endothelial cells. After 60 days of differentiation, as shown by immunofluorescence, we obtained vascularized millimeter long fetal muscle bundles innervated by motor neurons capable of repeated contractions and surrounded by a high number of SMPCs. Our snRNAseq analysis, as well as the fusion index of the sorted SMPCs, suggests that the presence of endothelial cells increases the proliferation and enhance the maturity of our SMPCs aligning with our data of fetal in vivo SMPCs. The Pyle lab has shown that fetal SMPCs can better regenerate skeletal muscle compared to embryonic SMPCs and our ongoing studies are evaluating SMPCs and endothelial cells in mouse models of VML for future use in a cell-based therapy for treatment of VML injuries.

Phenotypic variations in the retinal pigment epithelial cells of patients with diverse ABCA4 mutations Arpita Dave, Ph.D. Lab PI: Roxana A. Radu, M.D. Recessive Stargardt disease (STGD1) is the most prevalent inherited retinal degenerative disorder characterized by progressive central and color vision loss and is currently untreatable. Mutations in the ABCA4 gene are the leading cause of STGD1. With over 2,000 known ABCA4 variants associated with STGD1, patients exhibit diverse disease severity and progression rates, creating challenges in understanding the precise pathophysiological mechanisms. While ABCA4 knockout mouse models have provided valuable insights into neural retina and retinal pigment epithelium (RPE) dysfunction, the translational relevance of these findings to human disease progression remains unclear. To bridge this gap, we developed induced pluripotent stem cell-derived RPE (iPSC-RPE) cells from skin biopsies of three STGD1 patients with distinct ABCA4 mutations. Our patient-derived iPSC-RPE cells demonstrated key pathological features previously observed in mouse models, including elevated lysosomal pH, reduced Cathepsin D activity, and impaired phagocytosis. Notably, we observed varying degrees of α-synuclein accumulation, lipid aggregation, and mitochondrial metabolic dysfunction across different patient samples, correlating with their unique ABCA4 mutations. These findings validate and extend our understanding of STGD1 pathophysiology in human cells, establishing a robust ""disease in a dish"" model that recapitulates patient-specific molecular signatures and provides a platform to evaluate new therapeutic approaches for ABCA4-mediated retinopathies.

Cortical Versus Hippocampal Network Dysfunction in a Human Brain Assembloid Model of Epilepsy and Intellectual Disability Colin McCrimmon, M.D., Ph.D. Lab PI: Ranmal A. Samarasinghe, M.D., Ph.D. Neurodevelopmental disorders often impair multiple cognitive domains. For instance, a genetic epilepsy syndrome might cause seizures due to cortical hyperexcitability and present with memory impairments arising from hippocampal dysfunction. This study examines how a single disorder differentially affects distinct brain regions by using human patient iPSC-derived cortical- and hippocampal-ganglionic eminence assembloids to model Developmental and Epileptic Encephalopathy 13 (DEE-13), a condition arising from gain-of-function mutations in the SCN8A gene. While cortical assembloids showed network hyperexcitability akin to epileptogenic tissue, hippocampal assembloids did not, and instead displayed network dysregulation patterns similar to in vivo hippocampal recordings from epilepsy patients. Predictive computational modeling, immunohistochemistry, and single-nucleus RNA sequencing revealed changes in excitatory and inhibitory neuron organization that were specific to hippocampal assembloids. These findings highlight the unique impacts of a single pathogenic variant across brain regions and establish hippocampal assembloids as a platform for studying neurodevelopmental disorders.

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Distinct nucleotide metabolism fluctuations define pluripotent stem cell fate transitions Mary Doan BSCRC – CIRM Predoctoral Trainee Lab PI: Michael Teitell, M.D., Ph.D. Changes in metabolite levels and fluxes guide human pluripotent stem cell (hPSC) self-renewal and differentiation. Differentiation begins with the loss of pluripotency and activation of lineage-specific transcription factors for ectoderm, endoderm, or mesoderm. Our recent findings reveal each lineage depends differently on glutamine (Gln) for fate transitions. Uniquely, in ectoderm, adequate acquired or generated Gln levels provide an essential fate-specifying signal that activates mTORC1 during the initial 14 hours of differentiation. Conversely, Gln levels are insufficient to activate mTORC1 in endoderm or mesoderm, and forced activation in these conditions reduces viability. Endoderm and mesoderm prioritize exogenous Gln for TCA cycle anaplerosis via GLS1, while ectoderm depends less on this process. Beyond energy, Gln supports anabolic pathways like protein and de novo nucleotide biosynthesis (DNS), with mTORC1-regulated DNS promoting cancer growth.

However, the role of DNS in hPSC fate decisions remains unclear, and whether DNS pathway activation downstream of Gln-mTORC1 signaling is necessary for ectoderm fate specification warrants investigation. Using nutrient-balanced media, we guided hPSCs into germ lineages and profiled nucleotide metabolism. We observed reduced nucleotide levels during the hPSC transition to endoderm, elevated levels in ectoderm, and no significant change in mesoderm. Supporting this, treatment with DNS inhibitors during trilineage differentiation selectively impacted hPSCs developing into ectoderm. Finally, we examined DNS inhibitor effects during the first 14 hours of ectoderm differentiation—a critical window for lineage specification—followed by washout until commitment completion. Early intervention in DNS pathways during this window impaired identity switching in ectoderm differentiation. Based on these results, we propose a refined model where Gln-driven nucleotide production in hPSCs is crucial for ectoderm lineage specification.

Investigating Nuclear Proteins and Metabolites During Pluripotent Stem Cell Differentiation Using a Novel Isolation System Vahan Martirosian, Ph.D. BSCRC – CIRM Postdoctoral Trainee Lab PI: Michael Teitell, M.D., Ph.D. Pluripotent stem cell differentiation is a complex process traditionally attributed to transcriptional changes specific to each germ lineage.

However, emerging evidence underscores the pivotal role of metabolites and metabolic enzymes in guiding differentiation trajectories. The subcellular localization of these mediators is critical to their function, with recent studies highlighting the involvement of tricarboxylic acid (TCA) cycle enzymes in nuclear processes. While nuclear proteins have been characterized to some extent, the specific composition of pluripotent stem cell nuclei, the dynamic changes in nuclear components during differentiation, and the quantification of nuclear metabolites remain largely unexplored. To address these challenges, we developed a novel model system based on previous methodologies to improve nuclear isolation for -omics analyses. Human pluripotent stem cells were transfected with a custom vector encoding chimeric proteins that label mitochondria and nuclei with distinct epitope tags. Immunoprecipitation using magnetically labeled beads specific to each tag demonstrated successful enrichment of mitochondrial and nuclear proteins in separate fractions, validating the reliability of our constructs for isolating these organelles. Our next steps involve leveraging this system to measure nuclear metabolites and quantify nuclear constituents throughout differentiation. This innovative approach promises to reveal new insights into the dynamic subcellular localization of proteins and metabolites, advancing our understanding of nuclear metabolic regulation in pluripotent stem cell differentiation.

PGC-1α drives small cell neuroendocrine cancer progression toward an ASCL1-expressing subtype with increased mitochondrial capacity Grigor Varuzhanyan, Ph.D. Lab PI: Owen N. Witte, M.D. Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers composed of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of proliferator-activatedreceptor gamma coactivator 1-alpha (PGC-1α), a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNCs. PGC-1α correlated tightly with increased expression of the lineage marker Achaete-scute homolog 1, (ASCL1) through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. Conversely, PGC-1α overexpression enhanced OXPHOS, validated by small-animal Positron Emission Tomography mitochondrial imaging, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These results establish PGC-1α as a driver of SCNC progression and subtype determination, highlighting metabolic vulnerabilities in SCNCs across different tissues.

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Functionally validating T cell receptors targeting prostatic acid phosphatase for prostate cancer Caitlin Gee Lab PI: Owen N. Witte, M.D. Prostate cancer remains one of the leading causes of cancer deaths in males, with recurrent and metastatic forms of the disease posing significant therapeutic challenges. Current therapeutics offer limited efficacy and are primarily palliative, highlighting the urgent need for novel approaches with curative intent and long-term immune surveillance. To address this gap, our project explores a T cell-based therapeutic approach. Specifically, we aim to use a defined T cell receptor (TCR) that activates after binding to cancer-associated antigen fragments presented by major histocompatibility complexes (MHCs) on the cancer cell surface. This interaction can induce tumor cell death and promote immune response against prostate cancer cells. Prior efforts of TCR-T cell-mediated therapeutics mainly focus on synovial sarcoma and metastatic melanoma, leading to the first FDA-approved TCR-T therapy, afamicel, in 2024. However, only limited success has been achieved against epithelial cancers such as prostate. Our lab has been dedicated to isolating, defining, and improving TCRs with the potential to effectively target prostatic acid phosphatase (PAP), a well-defined prostate cancer antigen. High serum PAP level often correlates with worse prognosis, making it an ideal target for early intervention. Our previous work has thoroughly defined the peptides of PAP on various HLA alleles, so called immunopeptidome. In this project, we use isolated and finely tuned TCRs specific to PAP to functionally validate and screen candidates via cytokine release measurements and cytotoxicity evaluation. Our findings indicate TCRs can effectively induce the killing of PAPpositive prostate cancer cells. These results highlight the potential of TCR-based immunotherapy in treating prostate cancer.

Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method Yan-Ruide Li, Ph.D. BSCRC – CIRM Postdoctoral Trainee Lab PI: Lili Yang, Ph.D. Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using ‘off-the-shelf’ products, such as allogeneic CAR natural killer T (AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced AlloCAR-NKT cells with high yield and purity. We generated AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of AlloCAR-NKT cells support their potential for clinical translation.

Induced pluripotent stem cell-derived CAR MAIT cells for cancer immunotherapy Kuangyi Zhou BSCRC – Rose Hills Foundation Predoctoral Trainee Lab PI: Lili Yang, Ph.D. Allogeneic chimeric antigen receptor (CAR)-T cells hold potential for more accessible and affordable cancer treatments, but challenges such as limited donor cells, manufacturing variability, and multiplex gene engineering persist. iPSCs provide a promising solution due to their unlimited self-renewal properties and the ability for single-instance genetic modifications. However, efficiently differentiating iPSCs into mature CAR-T cells remains a hurdle, particularly as CAR engineering may arrest T cell development from stem cells, and iPSC-derived CAR-T cells often lack in vivo persistence and clonal expansion needed for long-term tumor suppression.

Mucosal-associated invariant T (MAIT) cells, a subset of αβ T cells that recognize MR1 and do not cause graft-versus-host disease in allogeneic settings, have shown unique properties in tissue homing and tumor infiltration. Yet, their limited numbers in humans hinder their full therapeutic potential.

We have developed a feeder-free and serum-free ex vivo culture method capable of generating 1013 CAR MAIT cells from 106 iPSCs. Our culture method allows iPSCs to differentiate into mature CAR MAIT cells without the need of TRAC knockin, TCR knockout, or engineering iPSCs with CAR that contains compromised signaling domain. These iPSC-derived CAR MAIT cells, can be either single positive (SP)- CD4 or SP-CD8, exhibit potent antitumor capabilities and robust clonal expansion in vivo.

As a proof-of-concept for this innovative platform, we employed a BCMA-targeting CAR (BCAR) to treat BCMA+ multiple myeloma, and a mesothelin-targeting CAR (MCAR) to treat mesothelin+ mesothelioma.

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Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy Yichen Zhu Lab PI: Lili Yang, Ph.D. The clinical potential of current FDA-approved chimeric anti- gen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CARengineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor- associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies under- score the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Identifying Aberrant Cellular Phenotypes in Neural Cultures Derived from Arboleda-Tham Syndrome iPSCs through AI-Assisted Immunofluorescence Analysis Yaneth Perez UCLA – CIRM COMPASS Scholar Lab PI: Valerie A. Arboleda, M.D., Ph.D. Arboleda-Tham Syndrome (ARTHS) is a rare Mendelian neurodevelopmental disorder caused by dominant mutations in the KAT6A gene – with patients exhibiting variable degrees of developmental delay, intellectual disability, speech/language deficits, and craniofacial abnormalities.

Since its discovery in 2015, there have been over 200 identified cases. However, the mechanisms by which KAT6A mutations cause abnormal neurological phenotypes in ARTHS patients remain unknown. To better understand the developmental stages at which these phenotypic abnormalities arise, we first plated ARTHS/control induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and neurons on glass slides. We then performed immunofluorescence (IF) for cell fate and proliferation markers on these slides – followed by confocal imaging. The images from our IF experiments were then analyzed using an artificial intelligence (AI)-assisted quantification platform known as Biodock.

Statistical analyses, including t-tests, were performed using RStudio to create boxplots that compare the average fluorescent intensities between patient and control samples. Preliminary data shows significant differences in the expression of pluripotency between ARTHS and control samples. Understanding these early developmental variations may help develop future therapeutic strategies for Arboleda-Tham Syndrome.

A Meta-Atlas of the Developing Human Cortex Identifies Modules Driving Cell Subtype Specification Patricia Nano, Ph.D. BSCRC – CIRM Postdoctoral Trainee Lab PI: Aparna Bhaduri, Ph.D. Human brain development requires the generation of hundreds of diverse cell types, a process targeted by recent single-cell transcriptomic profiling efforts. Through parallel meta-analyses of the human cortex in development (7 datasets) and adulthood (16 datasets), we have generated over 500 gene co-expression networks that can describe mechanisms of cortical development. These “meta-modules” show dynamic cell subtype-specificities throughout cortical development, with several developmental meta-modules displaying spatiotemporal expression patterns that allude to potential roles in cell fate specification. We validated the expression of these modules in primary human cortical tissues. These include meta-module 20, a module elevated in FEZF2+ deep layer neurons that includes TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical chimeroid experiments validated that both factors are required to drive module 20 activity and deep layer neuron specification, but through distinct modalities. These studies demonstrate how our meta-atlases can engender further mechanistic analyses of cortical fate specification.

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Understanding the role of glucose metabolism in cell fate specification events during cortical development Jessenya Mil BSCRC – CIRM Predoctoral Trainee Lab PI: Aparna Bhaduri, Ph.D. Cortical organoids are revolutionary as a model for human cortical development. The 3D in vitro model provides a framework for the developmental architecture of the cerebral cortex. However, work from our lab and others have shown that cortical organoids display impaired cell subtype diversity and genetic maturation signatures in comparison to human primary cortical tissue. Moreover, these impairments have also been found to correlate with an upregulated glycolytic gene expression in cortical organoids. Considering that neural stem cells (NSCs) rely on aerobic glycolysis to retain their stemness and transition to utilizing mitochondrial oxidative phosphorylation (OXPHOS) during activation and differentiation into a neuronal lineage, I predict the upregulated glycolytic stress in cortical organoids may be a driving limitation of subtype specification and impaired correlation to gene maturation networks. Thus, the metabolic differences seen between cortical organoids and human primary cortical tissue provide us with a model to understand how the manipulation of glucose metabolism influences cell fate specification and neuronal maturation. I aim to establish the functional metabolic profile of both cortical organoids and human primary cortical tissue during key neurodifferentiation events, allowing for the comparison of active and inactive metabolic pathways throughout cortical development in both models. Additionally, I will introduce metabolic perturbations during key developmental time points to characterize its impact on cell subtype specification and genetic maturation signatures. By identifying the influential metabolic pathways during cortical development, we can provide new insights as to how dysregulation of cell fate and maturation may impact brain development and identify the etiology of neurodevelopmental disorders driven by metabolic mutations.

The interplay between Preinitiation and Cohesin complexes in Promoter-Enhancer Looping Xianglong Tan, Ph.D. Lab PI: Michael F. Carey, Ph.D. The organization and expression of genetic information within eukaryotic genomes rely on a highly compacted, three-dimensional chromatin architecture. This 3D genomic structure, hypothesized to orchestrate gene transcription predominantly through promoter-enhancer interactions, is carefully constructed and maintained by crucial molecular machineries, including the Preinitiation Complex (PIC), the cohesin complex, and others. Our lab has previously depleted the PIC, observing a drastic reduction in transcription but a surprisingly modest effect on the 3D genome structure. Despite a plethora of existing data, the exact roles these complexes play in mediating promoter-enhancer interactions controlling gene transcription remain somewhat elusive. To address this problem, we employed a robust experimental setup leveraging the 2nd generation auxin-induced degradation system to systematically deplete the Rad21 subunit of the cohesin complex and the Taf12 subunit of the TFIID complex in mouse embryonic stem cells individually and in combination. ChIP-Seq analysis of epigenomic hallmarks revealed a significant depletion of the PIC and Mediator components following Taf12 degradation, along with a depletion of cohesin components following Rad21 degradation. We further employed promoter-capture Hi-C, which enables us to examine chromatin conformational changes with 1.5-kb resolution. Our findings reveal that both the PIC and cohesin complexes contribute to promoter looping, though they impact different promoter loop groups. Additionally, by integrating with ChromHMM states, we uncovered a subtle yet important correlation between transcription and promoter looping, which disappears upon cohesin depletion, indicating only a certain group ofpromoter looping is necessary for gene transcription. Our research sheds new light on the dual roles of the PIC and cohesin complexes in mediating functional promoter-enhancer interactions.

The Role of Autophagy in Quiescence Prerana Kasanagottu Lab PI: Hilary Coller, Ph.D. Quiescence, a reversible exit from the cell cycle in G1, is required for proper formation of organs and tissues and the ability to heal after injury.

In response to nutrient deprivation, autophagy, a pathway in which cells sequester and degrade cellular material at lysosomes, can provide metabolites and energy. However, not all quiescent cells are nutrient deprived. We sought to understand how the autophagy pathway is regulated when cells become quiescent in response to signals besides nutrient deprivation. Imaging and immunoblotting revealed that serumstarved and contact-inhibited (CI) primary human dermal fibroblasts accumulate autophagosomes and autolysosomes. For CI fibroblasts, rather than degrading the engulfed material, it accumulates expanded lysosomes. When CI cells are restimulated, autophagosome levels are strongly reduced, lysosomes shrink, and the degradation rate of autophagy proteins becomes even more rapid than in proliferating fibroblasts.

The increase in LC3+ puncta and lipidated LC3 formed during CI likely reflects noncanonical pathway as LC3-II levels were lower in CI mouse embryonic fibroblasts unable to perform LC3-associated endocytosis. This pathway is important for the survival of CI fibroblasts as CI fibroblasts unable to perform it exhibit reduced viability. The findings suggest a model in which quiescent, CI fibroblasts activate noncanonical autophagy to engulf extracellular material that is stored in lysosomes until it is required for the cells’ bioenergetic needs.

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Clonal studies identify the effects of hematopoietic stem cell aging on T cell development Julia Gensheimer Lab PI: Gay M. Crooks, M.D. T cell production declines with age and contributes to decreased immune function in older adults. This decline in T cell output may be due to changes in aged progenitor cells and/or changes in the thymic microenvironment. During aging, the HSC pool is comprised of increasing proportions of myeloid-biased HSCs (My-HSCs) compared to lymphoid-biased HSCs (Ly-HSCs). Thus, whether reduced T cell potential is due to changes in the proportions of Ly- and My-HSCs or a reduced intrinsic capacity of each subtype is unclear. To better understand the effects of HSC aging on T cell output, we studied phenotypically-defined aged and young HSCs using the in vitro Artificial Thymic Organoid (ATO) system.

The ATO recapitulates all stages of T cell development from a single HSC, allowing for detailed dissection and quantification of thymopoiesis at a clonal level without the influence of an aged microenvironment. Our preliminary data suggest that the lineage bias of an HSC impacts its T cell potential regardless of age, with both young and aged Ly-HSCs generating more T committed clones than aged-matched My-HSCs. Further, ATOs derived from young and aged Ly-HSCs produce comparable cell numbers, which suggests that removing aged HSCs from their microenvironment restores their T potential.

Development and characterization of non-alloreactive T and CAR T cells from iPSCs Lindsay Lathrop Lab PI: Gay M. Crooks, M.D. A primary barrier to Chimeric Antigen Receptor (CAR) T cell therapies is the current reliance on autologous T cell sources and the resulting unpredictable response rates due to production artifacts. A potential solution is to use induced-pluripotent stem cells (iPSCs) as the initial source for developing these therapies. Compared to other allogeneic sources, iPSCs allow for complex multi-gene editing that can be used to change the nature of the T cells to create a universal renewable CAR T cell product that can be readily and widely available to all patients. As iPSCs are an allogenic source the endogenous TCR and MHC must be removed to prevent GvHD and/or rejection respectively. However, since these genetic alterations are introduced at the stem cell stage they will affect development of the T cells. Removing these elements creates a block in development as TCR stimulation by MHC-peptide antigen presentation mediates positive selection of double positive (DP) T cells into single positive CD4+ (SP4s) and CD8+ (SP8s). Here our group demonstrates how stage-specific transgenic TCR and CAR expression overcomes this block in positive selection and allows for development of TCR-less CAR T cells. In future studies, we aim to elucidate how single CAR-mediated positive selection affects T cell intracellular signaling, phenotype, transcription and function.

Investigating the Development of Tregs from iPSCs through FOXP3 Expression Anthony Azzun Lab PI: Gay M. Crooks, M.D. Regulatory T cells (Tregs) are essential for maintaining immune balance and preventing autoimmune diseases like lupus and type 1 diabetes.

Despite their therapeutic potential, current strategies for generating Tregs rely on converting mature T cells into induced Tregs, which often exhibit instability and loss of function over time. Our research seeks to address this limitation by developing a novel method to generate stable Tregs from induced pluripotent stem cells (iPSCs), providing an unlimited, renewable source of these crucial immune cells.

We focus on FOXP3, the master regulator of Treg development, to understand how its expression influences T cell fate. Using an artificial thymic organoid (ATO) system, the only in vitro model capable of producing mature T cells from stem cells, we investigate how introducing FOXP3 into iPSCs affects their differentiation. We employ multiple strategies, including constitutive FOXP3 overexpression via lentiviral transduction and targeted insertion using CRISPR-Cas9 to mimic physiological expression. Additionally, we explore small-molecule modulation to enhance endogenous FOXP3 expression.

Our preliminary findings reveal that thymic levels of FOXP3 expression block normal T cell development at early differentiation stages in iPSCs while restricting FOXP3 to later stages preserves mature T cell formation. These insights pave the way for optimized Treg generation and functional testing in suppressor assays. By uncovering mechanisms that control FOXP3 expression in iPSC-derived T cells, our work advances the field of stem cell-based immunotherapy. This research holds promise for developing more effective Treg-based treatments for autoimmune diseases, potentially revolutionizing personalized cell therapies and improving outcomes for millions of patients.

Effects on Neurogenesis and Cell Survival Following the Knockdown of CARHSP1 in Primary Human Neural Progenitor Cells Nolan Fernandez UCLA – CIRM COMPASS Scholar Lab PI: Luis de la Torre-Ubieta, Ph.D. The development of the human neocortex, responsible for our most distinguishing cognitive and social capabilities, is precisely regulated by genetic programs driving cell type-specific molecular mechanisms. However, many gene-regulatory mechanisms governing cortical neurogenesis remain to be characterized. Previously, we identified the RNA-binding protein and putative transcriptional regulator CARHSP1 as a novel cell-specific marker and potential regulator of radial glial progenitor identity. Leveraging CRISPR interference (CRISPRi) we knocked down CARHSP1 in differentiating primary human neural progenitor cells (phNPCs) and assessed neurogenesis using immunocytochemistry for known markers of neurons and progenitors. We did not observe significant changes in neurogenesis with loss of CARHSP1, but observed a trend towards reduced infection rate and number of knockdown cells suggesting changes in progenitor proliferation and/or survival. In ongoing work, we aim to define whether CARHSP1 affects neuronal progenitor proliferation or survival. Altogether, our findings suggest CARHSP1 is essential for progenitor survival or proliferation dynamics.

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Defining molecular and gene-regulatory dysregulation in Down Syndrome neocortex Alexis Weber Lab PI: Luis de la Torre-Ubieta, Ph.D. Down syndrome (DS) is the most common form of genetic intellectual disability, occurring in 1/700 newborns and presenting with cognitive, learning, memory, and language deficits. DS results from trisomy of chromosome 21 (hsa21), causing impaired cortical development and brain architecture. However, the cellular and molecular mechanisms driving these pathologies remain unclear. We hypothesize that increased dosage of hsa21-encoded transcription factors (TFs) and epigenetic regulators alters global gene expression leading to changes in neurogenesis and neural specification. To address this gap in knowledge, we profiled ~110K cells from 26 age/sex-matched neurotypical (NT) and DS mid-gestation neocortices via snMultiome technology. In DS tissue, we observed a significant reduction in neural progenitors and deep layer corticothalamic (CT) neurons and concomitant increase of inter-telencephalic (IT) neurons, accompanied by accelerated time to neural specification as demonstrated by lineage trajectory analysis. In addition to the 25% of hsa21-encoded genes detected as exclusively upregulated, we found widespread changes in non-hsa21 gene expression and chromatin accessibility. Using gene co-expression (hdWGCNA), we identified cell type-specific changes in modules involved in neurogenesis and axon development. To understand the molecular mechanisms driving these changes, we performed gene regulatory network analysis (SCENIC+), finding significant changes in the activity of regulons controlling CT vs IT neuron specification that are downstream of hsa21-encoded TFs, including BACH1. Together, our data reveals a cascade of molecular dysregulation originating from hsa21-encoded TFs leading to alterations in neurogenesis and maturation, providing a foundation for developing future therapeutic targets.

Machine learning-assisted quantification of cell composition in developing Down Syndrome neocortex Moonkyu (Patrick) Seong Lab PI: Luis de la Torre-Ubieta, Ph.D. Down syndrome (DS),resulting from trisomy of chromosome 21, is the most common genetic cause of intellectual disability, affecting approximately 1 in 700 live births. DS is associated with disruptions in the development of the cerebral cortex, leading to cognitive, learning, and memory deficits. Histological and bioinformatic studies in postmortem DS brain tissue have found conflicting results with regards to changes in cellular composition and identity. While pathological imaging studies are often thought as the gold standard, they are often limited by sample size and laborious manual quantification. Work in our laboratory using single-nuclear multiomics of mid-gestation DS neocortex identified changes in neuronal composition, in particular deep-layer corticothalamic (CT) and inter-telencephalic (IT) neuron specification. To validate these findings, we performed immunohistochemistry in developing DS and neurotypical (NT) neocortical tissue using markers labelling CT (BCL11B/CTIP2) and IT (SATB2) neurons. We implemented a machine learning-assisted cell segmentation (CellPose) and automated thresholding pipeline to quantify thousands of cells across 6 age-matched NT and DS samples.We validated our pipeline by comparing their results to those of manual quantifications on smaller regions of neocortical tissue stains. Confirming the accuracy of these methods, we then scaled up the automatic approach to larger regions of cortical tissue, efficiently classifying many nuclei to determine the neuronal composition of our samples. Our work provides a pipeline to quantify cell composition with nuclear markers in brain tissue sections at scale. Together, these efforts contribute to a deeper understanding of the cellular alterations underlying DS and ultimately the search for targeted therapeutic interventions.

Developing a Novel Therapeutic for Smokers Infected with SARS-CoV-2 Summer Parr UCLA – CIRM COMPASS Scholar Lab PI: Brigitte Gomperts, M.D. Viruses are dependent on host cellular proteins for each step of their life cycle, and they hijack many of the host cell proteins for their function.

Many RNA viruses can induce a significant DNA damage response (DDR) which can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis and stimulation of inflammatory immune responses. Cigarette smoke (CS) can also induce DDR and it is known that patients who smoke are at increased risk for severe COVID-19 lung disease and death. We identified berzosertib as an inhibitor of SARS-CoV-2 infection. Berzosertib exhibited anti-SARS-CoV-2 activity and was effective at reducing SARS-CoV-2 infection in the human air-liquid interface (ALI) culture model. We exposed human alveolar type II (AT II) cells grown in organoids and ALI cultures to CS followed by live SARS-Cov-2 viral infection and found that CS increased the number of infected cells 2-3-fold. ATII organoids exposed to cigarette smoke extract (CSE) also showed increased proliferation and a five-fold increase in SARS-CoV-2 infection. As both cigarette smoking and viral infection induce the DDR, we hypothesized that berzosertib, a DDR inhibitor, would be an especially effective therapy to block SARS-CoV-2 replication. Treatment with Berzosertib during CS exposure and SARS-CoV-2 infection reduced viral replication in human ALI and ATII cell cultures. In addition, K18-ACE2 mice exposed to CS and infected with SARS-CoV-2 showed less lung consolidation when treated with Berzosertib. Berzosertib is effective at reducing SARS-CoV-2 infection in primary human airway and alveolar lung cultures and the K18-ACE2 mouse model. We are currently elucidating the toxicity and efficacy of berzosertib during smoking in these three models. This data will help to establish the efficacy of berzosertib in the context of SARS-CoV2, especially in vulnerable patient populations such as smokers.

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Profiling Regenerative Potential And Lineage Specification Of Human Airway Submucosal Gland Stem Populations In Organoid Cultures Andrew Lund Lab PI: Brigitte Gomperts, M.D. Airway submucosal glands (SMGs) house at least two distinct stem populations, namely the duct basal and myoepithelial cells (MECs). Based on studies utilizing severe airway injury modalities in murine models, both populations are seemingly competent to regenerate SMG and surface airway epithelium (SAE). However, equivalent regenerative capacity has not been established for the human counterparts to these stem populations.

Sorted SMG duct basal, surface basal, MECs, and unsorted SMG isolates were seeded to initiate organoid cultures. Organoid-forming efficiencies and organoid area were measured. Single-cell gene expression of organoids from each population was interrogated.

Sorted MECs exhibited diminished organoid-forming efficiencies compared to unsorted SMG isolates, sorted duct basal, and sorted surface basal cells. Single-cell fixed RNA profiling revealed that MECs preferentially differentiate into secretory lineages at the expense of stem maintenance, instead generating cycling secretory intermediates that are fate-committed. Conversely, surface basal cells rapidly mobilize under expansion conditions in culture, accounting for the greatest organoid-forming efficiency and total biomass of the sorted populations, whereas duct basal cells demonstrate growth dynamics intermediate between MECs and surface basal cells. Moreover, surface and duct basal cells are more proficient in maintenance of “stemness” compared to MECs and have a more balanced distribution across fate-committed clusters, including progeny that are directed towards surface/duct-like luminal cell types.

Identification of a long-lived airway stem population with broad multipotency would produce an ideal candidate for therapeutic concerns. SMG duct basal cells isolated from human airways demonstrated excellent self-renewal capacity and multipotency in organoid cultures, marking this population as an attractive prospect for advancing regenerative medicine in the respiratory system.

Woodfire Smoke Induced Mucociliary Airway Injury is Preventable with MEK Inhibition Caroline Cherry Lab PI: Brigitte Gomperts, M.D. Woodfire smoke (WFS) permeates our environment, but the health risks from these exposures are not known. Although the relationship between lung disease and ciliary dysfunction has been established, little work has been done to identify the effects of environmental exposures on airway cilia. Of note, the MAP Kinase (MAPK) signaling pathway has been shown to be associated with ciliary motor behavior and cytoskeletal rearrangement. Therefore, this proposal aimed to understand the mechanisms of ciliary injury following WFS exposure and identify potential therapeutic agents to mitigate this injury. Utilizing healthy human bronchial samples, we exposed cell cultures to WFS. Airway basal cell injury following WFS exposure was assessed through bulk-omics analysis, ciliary beat analysis, and transmission electron microscopy (TEM). We identified global downregulation of transcription of cilia related pathways and an upregulation of MAPK. Proteomic analysis revealed little change in protein abundance, coupled with a global downregulation of phosphorylation of cilia related proteins. WFS exposure for 5 days abolished ciliary movement in a dose dependent manner. Interestingly, treatment with various MEK1/2 inhibitors increased ciliary movement by approximately 70%. Furthermore, we investigated the ultrastructure of ciliated cells following WFS exposures and MEK inhibitors.

TEM analysis revealed normal ciliary axonemes across control samples, and ~90% loss of the central pair following WFS, which was rescued

by MEK inhibition. Together, we have created an exposure chamber to mimic the human airway exposure to WFS. We show that WFS is responsible for reducing ciliary movement in the airway by damaging the central pair of proteins in ciliary axonemes and that MAPK signaling regulates these ciliary proteins. Inhibition of MEK preserves ciliary ultrastructure and ciliary movement, therefore, posing potential to be used as a mitigator of airway disease in WFS exposed individuals.

Designing Targeting Lipid Nanoparticles for in vivo CAR T Cell Generation Itzel Huerta UCLA – CSUN CIRM Bridges Trainee Lab PI: Steven J. Jonas, M.D., Ph.D. Cell based immunotherapies in the form of T cells genetically modified to express chimeric antigen receptors (CAR) are providing additional treatment options for relapsed and/or refractory pediatric cancers; however, current CAR T cell therapies require costly and timely viral ex vivo modification. Lipid nanoparticle (LNP) technologies have shown significant promise in the non-viral engineering of CAR T cells, yet their clinical, in vivo translation is limited by their non-specific delivery.

Here we report the design of a modular, antibody conjugated LNP platform and its optimization for targeted engineering of primary, human T Cells. Optimization of our LNP platform was achieved through microfluidic synthesis of several formulations integrating different ionizable lipids for mRNA delivery, with the most efficient formulas displaying nanoparticle diameters 80% by Ribogreen fluorescence-based assay. The surface of the LNPs were then decorated with αCD3 monoclonal antibodies utilizing a bioorthogonal click chemistry conjugation strategy, and size exclusion chromatography (SEC) enabled removal of unbound antibodies confirmed via fluorescent protein labelling. αCD3-modified LNPs showed comparable transfection of activated, primary human T cells to that of optimized, non-targeting LNPs with at least 70% of treated cells expressing green fluorescence protein (GFP) 24 hours post LNP treatment. Our optimized αCD3-LNPs enabled the stimulation and transfection of resting T cells, with 10% of treated cells expressing GFP after 24 hours, increasing to 20% after 48 hours. These nanotechnologies serve as a modular, foundational platform for T cell engineering, enabling target specificity to empower next generation cellular immunotherapeutic approaches.

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Tools for maintaining hematopoietic stem cells in 3D matrices for stem cell gene therapies: collagen-based hydrogels for HSC culturing and recovery Luis Reyes UCLA – CSUN CIRM Bridges Trainee Lab PI: Steven J. Jonas, M.D., Ph.D. Hematopoietic stem cells (HSCs) can be used to treat malignant and non-malignant diseases. While the differentiation and self-renewal of hematopoietic stem cells (HSCs) in bone marrow has been extensively studied, the impact of ex-vivo culturing and the ability to maintain the stem-like nature of these cells must be improved to advance HSC gene and cell therapies. A challenge with HSCs is the absence of in vitro models that can effectively mimic the human bone marrow microenvironment. Here we present the development of tools to enable the culturing of HSCs in a 3D hydrogel matrix, and their impact on HSC self-renewal. Our preliminary studies indicate that the successful encapsulation, maintenance and recovery of HSCs from these 3D cultures enables the holding of HSC markers when compared to standard 2D culture practices. This research project primarily aims to investigate whether HSCs can be maintained in a 3D culture environment that prevents differentiation and preserves their stem-like properties. For our experiments, we utilized the KG-1a cell line as a model to study HSC behavior before experimenting with the HSCs in hydrogels. The hydrogels were fine-tuned by evaluating different collagen and fibrinogen formulations that most effectively kept viability above 90%, regulated proliferation of KG-1a cells, and allowed for the most amount of cells to be recovered after melting. The viability of HSCs was assessed through flow cytometry both at the time of encapsulation and 24 hours after.

Our findings indicated that a combination of collagen and fibrinogen in the hydrogels led to a decrease in the proliferation of KG-1a cells and HSC populations while keeping viability levels above 90%. This work will enable further investigation into the mechanisms of HSC self-renewal regulation and fate decision of committed progenitors to improve current in vitro maintenance of HSCs for gene and cellular therapy manufacturing.

Lipid Nanoparticle Delivery of CRISPR/Cas9 Reagents for Disruption of Oncogenic Translocations in Alveolar Rhabdomyosarcoma Amadu Tadesse Lab PI: Steven J. Jonas, M.D., Ph.D. Alveolar rhabdomyosarcoma (aRMS) is a rare and aggressive soft tissue cancer arising from skeletal muscle stem cells in children and young adults. This project leverages nanotechnology-based strategies to develop a lipid nanoparticle (LNP) platform designed to deliver CRISPR/Cas9 reagents configured to disrupt the PAX3-FOXO1 translocation that drives the most aggressive phenotypes of aRMS and mitigate its oncogenic effects. Here we present a two-pronged approach: first, designing and validating single guide RNA (sgRNA) sequences configured for the targeted knock out of PAX3-FOXO1 translocations; second, engineering LNPs for the efficient delivery of these gene-editing reagents for direct therapeutic use on aRMS tumors. PAX3-FOXO1 sgRNAs were designed using conserved fusion regions identified from ChimerDB 4.0 and the UCSC Genome database. Guides were optimized using Benchling and Integrated DNA Technology tools to maximize ontarget efficiency while minimizing off-target risks. Guide candidates were then tested in a representative PAX3-FOXO1 positive aRMS cell line (RH30) by electroporating Cas9 ribonucleoprotein complexes assembled with PAX3-FOXO1 sgRNA constructs and validated via qPCR analysis.

Green fluorescent protein (GFP)-encoding mRNA was initially encapsulated in LNPs to optimize formulation delivery efficiency. Nanoparticles were prepared via microfluidic mixing with mean diameters 80% determined by Ribogreen fluorescence-based assay. Our best performing formulations achieved >90% GFP expression 24 h post treatment. Preliminary studies applying LNPs carrying PAX3-FOXO1 CRISPR/Cas9 cargoes, observed a >50% reduction in PAX3-FOXO1 expression in targeted aRMS cells, as confirmed by qPCR. These findings demonstrate the potential of LNP-based CRISPR/Cas9 strategies to target oncogenic mutations therapeutically in high-risk childhood malignancies like aRMS.

Augmenting Natural Killer Cell Function Through Chimeric Antigen Receptor and Endogenous Signaling Pathways Jedric Gonzales UCLA – CIRM COMPASS Scholar Lab PI: Scott G. Kitchen, Ph.D. Current research has established the effect which the transduction of chimeric antigen receptor (CAR) machinery into natural killer (NK) cells and T-cells (TC) has on the cytotoxic capabilities of such cells. Simultaneously, research shows that the endogenous pathways combined with CAR machinery helps to increase the cytotoxic efficiency of both T and NK cells. While these studies have shown the potential for adjoined engineering of CAR therapy with endogenous elements, research on adjacent, NK-specific forms of endogenous signaling in the context of CAR therapy is not as thorough, despite such activation playing a major role in the activation of NK cells. Thus, this study focuses on how the functionality of NK cells can be improved by combining the elements of CAR machinery alongside important NK-specific signaling pathways.

To conduct this study, we will use four different genetic constructs that will be incorporated into four NK92 cell lines via lentiviral transduction.

Each construct contains CD4, CAR, CD3(𝛇) machinery, and the fluorocomb MScarlet3, which aids in cell staining procedures and verification of transduction. Three constructs contain combinations of NK signaling: one containing DAP12, another containing DAP12, CD40, and CD3(ε) instead of CD3(𝛇), and the last containing DAP12 and NKG2C. Current analysis of the control cell line confirms the strong efficacy of current transduction methods and establishes the baseline of unmodified CAR-NK function. Future procedures will focus on the transduction of the three experimental constructs into respective NK92 cell lines before conducting cellular staining, cytokine assay, and cytotoxicity procedures.

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Optimization of BTK Donor for CRISPR/Cas9 Gene Therapy of X-Linked Agammaglobulinemia (XLA) Elyce Velazquez UCLA – CSUN CIRM Bridges Trainee Lab PI: Donald B. Kohn, M.D.

The Bruton’s Tyrosine Kinase (BTK) gene encodes for a protein that plays a crucial role in B cell signaling and is essential for B cell development, proliferation, and differentiation. Mutations in the BTK gene causes X-Linked Agammaglobulinemia (XLA), a primary immunodeficiency disorder leading to the inability to produce B cells or immunoglobulins, conferring the susceptibility to infections. XLA is treated through regular and frequent intravenous immunoglobulin replacement therapy, however this treatment is costly and does not provide a permanent cure for patients.

Previous research has shown it is possible to edit the BTK locus to restore function to the BTK gene, however further research is necessary to find the optimal expression and integration of the BTK donor DNA. It is especially important to have a precise level of BTK expression as overexpression is correlated with B lymphoid leukemias. Using intronic regulators in the gene therapy of XLA may restore the proper expression patterns, thus we will optimize the repair template to reach this correct level of expression of the BTK gene. Several candidate sequences have been cloned into the BTK donor template in previous studies and intron 4 has shown an improvement of the expression level. Intron 4 and 5 of the endogenous BTK gene have been cloned and tested for expression level induced. At the same time removing of the WPRE sequence was tested to see if the intronic sequence would maintain the expression in the absence of this viral regulator. Intron 4’s sequence is too large to be packaged into an AAV vector, so we will design and test 3 different constructs generated with subsequent deletions and containing the necessary portions of Intron 4 to fit into an AAV vector. These 3 DNA constructs will then be tested for the induced expression of the BTK transgene.

IDLV vs AAV: Optimizing Vector Delivery for BTK Gene Editing Patience Akok UCLA – CIRM COMPASS Scholar Lab PI: Donald B. Kohn, M.D.

X-linked agammaglobulinemia (XLA) is a disease affecting 1 in 200,000 males caused by a mutation of the Bruton tyrosine kinase (BTK) gene, responsible for the development of B cells. Since B cells produce antibodies, patients affected by XLA have an increased susceptibility to infections. Allogeneic transplant of bone marrow from a healthy donor to the patient would result in a cure. However, it poses the threat of graft vs host disease. Alternatively, gene editing the patient’s hematopoietic stem cells (HSCs) would allow for correction of the mutation and restore the HSCs ability to differentiate into functional B cells. Before clinical applications, this must be tested and optimized in vitro. We focused on the use of viral vectors when delivering a healthy BTK donor sequence. Adeno-associated viral vectors (AAV) have previously led to high integration of the BTK donor, however it was toxic to cells in-vitro. On the other hand, integration-defective lentiviral vectors (IDLV) had less integration potential, but preserved cell viability. In order to test optimal conditions for gene editing, we electroporated HSCs with a CRISPR/Cas9 complex and transduced them with a DNA donor using either AAV or IDLV. Then, we compared BTK integration by ddPCR and BTK expression using flow cytometry.

Preliminary findings concluded that AAV integrated BTK more efficiently and had higher BTK expression, but more optimizations of both vectors must be done to confirm. Once done successfully, it will be possible to select the best vector for delivery in patient cells. iPSC-derived blood-brain barrier cellular model reveals distinct mechanisms of Old World alphavirus neuroinvasion Pablo Alvarez Lab PI: Melody Man Hing Li, Ph.D. Several alphaviruses bypass the blood-brain barrier (BBB), causing debilitating or fatal encephalitis. Sindbis virus (SINV) has been extensively studied in vivo to understand alphavirus neuropathogenesis; yet the molecular details of neuroinvasion at the BBB remain poorly understood. We investigated alphavirus-BBB interactions by pairing a physiologically relevant, human pluripotent stem cell derived model of brain microvascular endothelial cells (BMECs) with SINV strains of opposite neuroinvasiveness. Our system demonstrates that SINV neuroinvasion correlates with robust infection of the BBB. Specifically, SINV genetic determinants of neuroinvasion enhance viral entry into BMECs. We also identify solute carrier family 2 member 3 (SLC2A3, also named GLUT3) as a potential BMEC-specific entry factor exploited for neuroinvasion. Strikingly, efficient BBB infection is a conserved phenotype that correlates with the neuroinvasive capacity of several Old World alphaviruses, including chikungunya virus. Here, we reveal BBB infection as a shared pathway for alphavirus neuroinvasion that can be targeted for preventing alphavirus-induced encephalitis.

Unraveling the SLC6A8 Mutation: Insights into Neurodevelopment through Organoid Research Maya Moreno UCLA – CIRM COMPASS Scholar Lab PI: Gerald S. Lipshutz, M.D. Creatine is a fundamental nitrogenous metabolite essential for ATP production, osmoregulation, and neurotransmission. Disruptions in creatine metabolism, arising from mutations affecting its synthesis (AGAT, GAMT) or transport (SLC6A8), lead to severe intellectual and physical disabilities, collectively known as Cerebral Creatine Deficiency Syndromes (CCDS). Despite the profound neurological impact of SLC6A8 deficiencies, the underlying disease mechanisms remain poorly understood. This study aims to elucidate the cellular and molecular pathways disrupted by SLC6A8 mutations and explore potential therapeutic strategies. We will generate and analyze patient-derived organoids, isogenic corrected-cell lines, and control cell lines to model CCDS in a human neurodevelopmental context. Additionally, we will assess the efficacy of current but limited therapeutic interventions on cellular function to determine their corrective potential. By bridging critical knowledge gaps in SLC6A8-related neurodevelopmental deficits, this research will advance our understanding of CCDS pathology and contribute to the development of targeted therapies. Ultimately, these insights have the potential to drive innovative treatments that improve the quality of life for affected individuals and their families.

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Mechanotransduction in the Gut: Investigating Piezo1/2 Function in PDGFRα⁺ Cells Allison Flores Lab PI: Martín G. Martín, M.D., M.P.P. Efficient gastrointestinal (GI) motility relies on coordinated interactions between smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and PDGFRα+ cells within the subintestinal plexus (SIP) syncytium. PDGFRα+ cells function as fibroblast-like interstitial cells, modulating SMC excitability, mechanosensation, and contractions. However, the molecular mechanisms through which Piezo1/2 mechanosensitive calcium channels contribute to these functions remain unclear. To investigate Piezo1/2 function, we utilized a Piezo1/2;mTmG;PDGFRα-ERT2-Cre/LoxP model to generate Piezo1/2 control (Piezo1/2WT) and knockout (Piezo1/2ΔPDGFRα) mice. We confirmed membrane GFP (mG+) expression and mapped PDGFRα+ cell localization within the intestinal muscularis. Our findings revealed Piezo1/2WT mice showed dense clustering of mG+ PDGFRα+ cells within the horizontal and circular muscularis, forming a web-like structure in the inner longitudinal muscle. In contrast, Piezo1/2ΔPDGFRα mice exhibited a continuous PDGFRα+ cell distribution from the serosa to the submucosa, with unique serosal patterns.

Both groups displayed elongated PDGFRα+ cells near the circular muscle, likely shaped by tissue orientation, with no observed sex differences.

Immunostaining confirmed Piezo1/2 expression in PDGFRα+ GFP+ cells, suggesting a regulatory role in mechanosensitive ion channel signaling. Future studies will use a SALSA reporter line to assess Piezo1/2 depletion effects on cell morphology, Ca²⁺ flux, and contractility. By elucidating Piezo1/2 function in PDGFRα+ cells, this study advances our understanding of SIPgenic modulation in GI motility and may reveal new mechanistic pathways underlying intestinal motility regulation.

Investigating the Impact of Piezo1 Depletion in Smooth Muscle Cells on Intestinal Function Mireya Cruz UCLA – CSUN CIRM Bridges Trainee Lab PI: Martín G. Martín, M.D., M.P.P. Piezo1/2 are mechanosensitive channels that are responsive to stretch and lead to the release of di-cationic elements such as Ca++. To investigate the specific functions of these channels in GI motility and homeostasis, we generated four general mouse strains that allow for targeted deletion of Piezo1/2 in particular cell types in the small intestines. This includes epithelial cells, smooth muscle cells (SMCs), Interstitial cells of Cajal and PDGFRα cells. Enteric SMC contraction results from the influx of Ca++ in the cytoplasm. In the SMCs, Piezo1, not Piezo2, is expressed at high levels. Here, we determined the role of Piezo1 in SMCs in mediating intestinal muscularis function. We hypothesized that loss of Piezo1 in the SMCs will lead to intestinal dysmotility. Using the Piezo1/mTmG/Myh11-ERT2/Cre-LoxP system, we generated a model with tamoxifen (Tam)-Piezo1 control (Piezo1WT ) and KO (Piezo1ΔSMC) in SMCs. Tam was given to 6-wk-old mice and assessed 21 days later. We have performed a comprehensive assessment of the role of Piezo1 in enteric SMCs, and have found that selective depletion of Piezo1 in SMCs of the small bowel leads to significant changes in intestinal motility and function, as evidenced by 1) delayed gut transit and weight loss without an increase in mortality; 2) thinning of the muscularis and a loss of SMCs; 3) diminished contractile pattern and strength; 4) alterations in tissue properties including tonicity and viscoelastic properties; 5) dysregulated expression of ion channels in SMCs; and 6) predominant localization of Piezo1 in the SR of SMCs with reduced IP3R expression. This study was limited to 21 days post-tamoxifen.

Despite muscularis thinning, no changes in SMC apoptosis or proliferation were found. To investigate early cellular changes, we are performing a time-course analysis using immunofluorescence on whole-mount distal bowel to assess SMC abundance, apoptosis (CC3), and proliferation (Ki67) within the first 21 days post-tamoxifen.

Investigating the Role of Piezo1/2 in c-Kit+ Interstitial Cells of Cajal and Their Contribution to Gastrointestinal Motility Nicole Kushnir Lab PI: Martín G. Martín, M.D., M.P.P. The gastrointestinal (GI) tract relies on coordinated mechanical properties to facilitate nutrient assimilation, regulated by smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and Pdgfrα+ cells within the SIP syncytium. As pacemaker cells, ICCs generate slow waves, rhythmic contractions, and mediate neurotransmission to regulate motility. All cellular components of the SIP syncytium richly express the mechanosensitive calcium channels, Piezo1 & 2, yet their role in these cell types remains unclear. Piezo1 & 2 are strong candidates for modulating intracellular Ca2+ dynamics. We hypothesized that the loss of Piezo1/2 in ICCs would impair intestinal motility. To further assess Piezo1/2 function in ICC cells, we have established a c-Kit-CreERT2; Piezo1/2 LoxP; mTmG or Salsa system to develop Piezo1WT and Piezo1/2ΔICC knockout model in ICCs. The mTmG reporter marks Cre by changing membrane tdTomato (mT) to GFP (mG), while the Salsa reporter is tdTomato linked to GCaMP6f. Tamoxifen was administered to 6-week-old mice and analyzed after 21 days. Membrane GFP expression confirmed c-Kit+ cells were localized within the upper longitudinal, inner longitudinal, circular muscularis layers, and epithelial cells along the crypts and villi. Co-localization with a c-Kit antibody validated their identity. Male and female assessments showed no significant differences in ICC distribution. Immunostaining confirmed Piezo1/2 in ICCs, supporting their potential role in calcium modulation within ICCs. Analysis of Piezo1/2ΔICC knockout mice has just begun. Ongoing studies will examine how Piezo1/2 deletion affects ICC Ca2+ flux and contractility using a SALSA reporter line that will allow us to measure that pattern of Ca+ flux in live cells isolated from pups and adult mice.

This study advances our understanding of ICC function in SIPgenic GI motility and provides insights into mechanistic pathways that could drive research on therapeutic targets for motility disorders.

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Characterization of Platinum-Resistant High-Grade Serous Ovarian Cancer (HGSOC) Patient-Derived Xenograft Models (PDX): Investigating Cancer Stem Cell Populations and Their Role in Tumor Recurrence and Chemoresistance Jessica Gebreel UCLA – CIRM COMPASS Scholar Lab PI: Sanaz Memarzadeh, M.D., Ph.D. High-grade serous ovarian cancer (HGSOC) is the most common type of ovarian cancer and a leading cause of death from gynecological tumors. After initial treatment, up to 85% of patients experience a recurrent, and typically more aggressive form of the disease. Recurrent tumors are often resistant to standard platinum chemotherapy, resulting in and aggressive disease progression and poor patient outcomes.

Consequently, improved treatments and a better understanding of HGSOC are needed. Patient-derived xenograft (PDX) models are a viable model for cancer research, due to their ability to retain similar characteristics to their parent tumor and can be used as pretherapy models for novel therapeutic testing. We aimed to establish and characterize platinum resistant HGSOC PDX models and further our understanding of cancer stem cell (CSC) populations and their role in driving tumor recurrence and resistance. HGSOC cells were isolated from platinumresistant patient samples and injected into NSG mice intraperitoneally (n=9). Tumor cells and major organs were collected from mice and used for downstream analyses. Immunohistochemistry (IHC) demonstrated the expression of HGSOC markers including PAX8 and p53, in parent HGSOC cells, tumor cells, and organs collected from mice. Invasive metastatic spread was observed in all PDX mice. To advance this project, an emphasis on CSC markers like ALDH (aldehyde dehydrogenase), CD44, CD133, and CD117 will be studied via flow cytometry. We plan to sort ALDH+ and ALDH- populations and subcutaneously inject these into mice to assess tumorigenesis. Tumor growth will be monitored, followed by analysis using IHC and flow cytometry to assess phenotypic and functional differences between these populations. Utilizing these CSC markers will enable identification of cancer stem cell populations with the hope to target these cells with novel immunotherapies and consequently improve patient outcomes in ovarian cancer.

Profiling of gynecologic carcinosarcomas reveals unique vulnerabilities to NK cell-based immunotherapies Christopher Ochoa BSCRC – Rose Hills Foundation Predoctoral Trainee Lab PI: Sanaz Memarzadeh, M.D., Ph.D. Gynecologic carcinosarcomas (GyCS) are rare, aggressive tumors originating in the uterus or ovaries, characterized by both carcinoma and sarcoma elements. These tumors exhibit high recurrence rates, resistance to conventional treatments, and limited therapeutic options. We investigated whether a stem-like cell population and an immunoevasive phenotype drive GyCS progression.

Single-cell RNA sequencing of eight primary GyCS patient samples identified a potential progenitor population with a high stemness signature, supported by CytoTRACE and pseudotime analyses. This stem-like phenotype was further validated by flow cytometry, which revealed heterogeneous expression of cancer stem cell (CSC) markers. Functional studies in a uterine carcinosarcoma patient-derived xenograft model showed that ALDH, an enzyme linked to maintaining stemness, marks a highly tumorigenic population. ALDH+ cells outgrew ALDH- cells in vitro and in vivo, confirming a stem-like role in GyCS progression.

We also found evidence of an immunoevasive phenotype in GyCS tumors, marked by reduced natural killer (NK) cell infiltration. Despite this, tumor cells expressed NK-activating ligands (CD112, CD155) and the tumor-associated antigen HER2, suggesting their potential susceptibility to NK-based therapies. In vitro and in vivo studies demonstrated that combining NK cell therapies with HER2 monoclonal antibody treatment significantly outperformed HER2 antibody-drug conjugates, resulting in improved tumor clearance and survival.

Our findings confirm that GyCS tumors possess a stem-like, immunoevasive phenotype and highlight NK cell-based immunotherapies as a potential strategy to improve patient outcomes.

Impaired Cardiomyocyte Maturation and Mitochondrial Dysfunction in the embryonic heart of diabetic pregnancy Haruko Nakano Lab PI: Atsushi (Austin) Nakano, M.D., Ph.D. Glucose is a crucial energy source for a developing baby's heart. However, when a mother has high blood sugar (a condition called maternal hyperglycemia), it can lead to heart defects in the baby. Our previous research found that too much glucose interferes with the heart cells' ability to mature properly, partly because it increases the production of certain cellular building blocks called nucleotides. To better understand how high maternal blood sugar affects organ development, we analyzed fetal tissue samples from normal and diabetic pregnancies. Using advanced techniques to track how glucose is used in the body, we found that fetal organs from diabetic pregnancies relied more on a process called glycolysis (which breaks down glucose for energy) but did not efficiently connect to another important energy pathway, the TCA cycle.

We also saw consistent changes in another metabolic pathway called the pentose phosphate pathway.

Further investigation of individual heart cells confirmed that fetal heart cells from diabetic pregnancies showed reduced signs of maturity and an increased tendency toward mitochondrial fragmentation (which is associated with stress and dysfunction). However, when we activated AMPK using a chemical called AICAR, we observed improved mitochondrial content and increased expression of a heart maturity marker, even in high glucose conditions.

In summary, our study suggests that an excess of nucleotides and the suppression of AMPK signaling are key reasons why fetal heart cells fail to mature properly in diabetic pregnancies. This insight could lead to new strategies for preventing heart defects in babies of mothers with high blood sugar.

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The chiral nematic pattern underlies cardiac tissue organization and function Kawahira Naofumi, Ph.D. Lab PI: Atsushi (Austin) Nakano, M.D., Ph.D. The helical motion during the macroscopic contraction of the heart is driven by the twisted arrangement of cardiomyocytes. The cardiac fibers exhibit a distinct chirality characterized by a counterclockwise rotation from the epicardial to the endocardial surface. This unique structure of the cardiac fibers forms the nematic pattern akin to that observed in liquid crystal. Although the nematic pattern of cardiac fibers has been studied in 2D, the absence of 3D characterization has limited our understanding of how the microscopic cell chirality translates into macroscopic contractile motion of the heart.

Analyses of the left ventricular free wall of light-sheet and confocal imaging of cleared mouse hearts and diffusion tensor MRI data from human hearts revealed a counterclockwise rotation of cardiomyocytes. This chiral pattern is established during stages E12.5-14.5 concomitant with the development of compact layer in mouse embryo. The formation of the local tissue chirality is independent of systemic Left-Right signal as heterotaxy hearts display the same counterclockwise rotation in mice. A prominent feature of liquid crystals is the formation of topological defects, discontinuities of orientation field with nematic order. Our 3D analyses revealed distinct topological defects in the left apex and in the anterior interventricular septum. Mechanical simulation based on these data showed that the 135-degree counterclockwise fiber rotation is advantageous for the macroscopic contractile function and that the work production is reduced in the topological defect regions.

Together, this first 3D analysis of the muscle fiber orientation in mammalian heart provides a solid basis for how chiral nematic behavior of the cardiomyocytes shapes the architecture and function of the heart. The demonstration of the theoretical foundation for the organ function by cellular topology is of great significance to our understanding of the physiology and pathology of the heart.

Preventing erosion of the inactive X chromosome with F repeat in female human pluripotent stem cells Liang-Wei Huang BSCRC – CIRM Predoctoral Trainee Lab PI: Kathrin Plath, Ph.D. X-chromosome inactivation (XCI) is essential in female mammals to balance the dosage of genes on the X chromosome with males. XCI is achieved through the expression of XIST, a long non-coding RNA that coats and silences one X chromosome. However, in human female induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), prolonged culture leads to the silencing of XIST and erosion of gene silencing of the inactive X chromosome (Xi), resulting in reactivation of a subset of genes and further affecting differentiation efficiency.

Hence, it is critical to understand the mechanism of the silence of XIST in human as it is the leading cause of Xi erosion. However, it is unclear how the XIST is silenced in female human iPSCs. We found that similar epigenetic patterns between a subregion: F repeat of XIST and FIRRE locus during Xi erosion from the published data. In addition, we found similar transcription factors bind to XIST and FIRRE locus only in Xi. Consequently, we are interested in how these region regulate the process of XIST silencing. Importantly, these two regions form long-range interaction, termed superloop, exclusively in the Xi. Therefore, we are interested in whether the superloop is disrupted during Xi erosion, and whether the structure of the superloop facilitates the maintenance of XIST expression. From the Hi-C result generated from the early and late passages human iPSCs, we found that FIRRE locus forms superloop with DXZ4, another locus known to form super-loops on the Xi, only in the early passage iPSCs. Hence, the super-loop was disrupted during Xi erosion, indicating the contribution of Xi maintenance from higher-order structure of the Xi chromosome. We will further decipher the role of the "common" transcription factors between XIST and FIRRE regarding to the XIST expression and superloop formation on Xi. Our findings will clarify the mechanisms of XIST silencing and may help prevent Xi erosion in human iPSCs.

Metabolic regulation of stem cell fate through the modulation of the extracellular matrix Zeenat Rashida, Ph.D. BSCRC – CIRM Postdoctoral Trainee Lab PI: Kathrin Plath, Ph.D. The various functions of stem cells during development, regeneration and in homeostasis are determined by their trajectory towards differentiation, self-renewal, or quiescence, and disrupting these paths can lead to a variety of diseases. While genetic factors regulate pluripotency and cell fate, increasing evidence shows that extrinsic factors like the nutrient environment and the extracellular matrix (ECM) can regulate stem cell functions. The ECM is a complex network of proteins that surrounds the cells, provides structural support and transduces mechanical and biochemical signals to influence cell behavior, including proliferation, migration, and differentiation. Despite its importance, the role of ECM in regulating stem cell function, particularly during induced pluripotent stem cell (iPSC) reprogramming, remains inadequately explored. Our work aims to address this gap by investigating how nutrient-driven changes in metabolism influence ECM composition and stem cell reprogramming. Specifically, we focus on Vitamin C (VitC), an essential nutrient that enhances pluripotency induction and is commonly used in iPSC culture media. We propose that VitC modulates ECM deposition through its effect on cellular metabolism, particularly by enhancing glycosylation pathways necessary for glycosaminoglycan and collagen production. This, in turn, alters ECM composition and promotes iPSC generation. These findings provide novel insights into how metabolism and ECM interact to regulate stem cell fate transitions.

Our work also advances the understanding of ECM’s role in stem cell function, with broad implications for improving stem cell therapies, tissue engineering, and regenerative medicine, as well as addressing diseases linked to ECM dysregulation.

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Investigating Erosion of the Inactive X Chromosome in Human Pluripotent Stem Cells Sophia Peavy UCLA – CIRM COMPASS Scholar Lab PI: Kathrin Plath, Ph.D. Reprogramming female human fibroblasts to induced pluripotent stem cells (hiPSCs) has enabled new disease studies and possibilities in regenerative medicine. However, all female hiPSCs display a culture-induced erosion of X-chromosome inactivation (XCI) which impairs differentiation mechanisms therefore limiting the utilization of female hiPSCs. In this process, XIST RNA, which is responsible for silencing the genes on the inactive X chromosome (Xi), is lost, which leads to the Xi re-exhibiting gene expression. The cause of Xi erosion is ultimately XIST loss, but the mechanisms leading to its downregulation are still unknown. This project aims to find a practical solution to prevent Xi erosion and to establish a better understanding of the mechanisms contributing to the process.The Polycomb Repressive Complex 2 (PRC2) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3) and is highly enriched on the Xi in hiPSCs but not in fibroblasts, albeit H3K27me3 accumulates on the Xi in both. A time course of reprogramming intermediates reveals strong Xi localized Ezh2 enrichment occurs late in the reprogramming process after mesenchymal to epithelial transition. Since PRC2 and H3K27me3 facilitate gene silencing on the Xi downstream to XIST on the normal Xi, we hypothesize that the high PRC2 enrichment on the Xi, which is specific to hiPSCs, contributes to theXCI-erosion process. We cultured hiPSCs with PRC2 inhibitors to address this question and followed Xi erosion over time. There was no evidence of a significant effect of PRC2 inhibition on erosion, however, we demonstrated the ability to dramatically deplete H3K27me3 globally and locally on the Xi in female hiPSCs. Additionally, we have begun investigating both cis and trans elements involved in XCI to better understand their relationship to Xi erosion. Xi erosion is a deeply complex process that appears to be established by many different factors and conditions.

Small Molecule Mediated Somatic Cell Reprogramming Alexander Lee, Ph.D. Lab PI: Kathrin Plath, Ph.D. Adipocyte-derived stem cells (ADSCs) are a promising source of stem cells for regenerative medicine due to their relative ease of isolation and multipotent differentiation capacity. However, their limited pluripotency restricts their broader clinical applications. Induced pluripotent stem cells (iPSCs) offer a more versatile alternative but typically require overexpression of oncogenes like c-Myc via viral vectors to achieve pluripotency, reducing their clinical utility. In this study, we explore a small-molecule-based approach to directly reprogram ADSCs into iPSCs, following the methodology outlined by Liuyang et al. (2023). Our goal was to overcome the challenges of current reprogramming techniques by utilizing small molecules alone, thus enhancing clinical safety. By applying a cocktail of epigenetic modulators and signaling molecules, we successfully reprogrammed ADSCs into iPSCs that exhibit characteristic pluripotent markers and self-renewal. Our results demonstrate that small-molecule-based reprogramming can potentially serve as an alternative to conventional viral reprogramming methods, which could be more suitable for clinical applications. These findings contribute to advancing cell-based therapies by enabling the generation of pluripotent stem cells from a readily accessible and clinically relevant cell source. The broader implications of this work may include improved strategies for personalized medicine and regenerative therapies, facilitating the use of patient-specific iPSCs for disease modeling and therapeutic interventions.

Regulation of Cardiac Fibroblast Activation and Angiogenic Potential with Recombinant SLIT2 Ana Hernandez UCLA – CIRM COMPASS Scholar Pearl Quijada, Ph.D. Slit2 is a glycoprotein known for its role in axon guidance and angiogenesis in the central nervous system. However, its function in adult cardiac fibroblasts has not been well defined. We hypothesize that Slit2 regulates the expression of critical genes involved in extracellular matrix (ECM) production and angiogenesis, regulating fibrotic remodeling in the heart. We isolated adult cardiac fibroblasts to investigate the role of Slit2 in the cardiac fibroblasts. We treated them with either vehicle or transforming growth factor b-1 (Tgfb1) and Angiotensin II (AngII) to induce fibroblast activation and with or without recombinant SLIT2 protein for 48 hours. To test our hypothesis, RNA was isolated from adult cardiac fibroblast treatment groups and subjected to quantitative qPCR to measure the expression levels of angiogenic and fibrosis-related genes. Our results show that Slit2 enhances the upregulation of Postn, Acta2, and Angpt1, indicating its significant regulatory role in ECM production and angiogenesis during fibroblast activation. Conversely, expression levels of genes like Vegfc and Pdfga were downregulated or unchanged. Our data suggests that Slit2 may significantly regulate ECM production and angiogenesis during ischemia-induced cardiac remodeling in vivo.

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In vivo partial cellular reprogramming of myofibers leads to systemic rejuvenation across other tissues Minori Ohashi, M.D., Ph.D. BSCRC – CIRM Clinical Fellow Lab PI: Thomas A. Rando, M.D., Ph.D. Skeletal muscle health is a critical predictor of longevity and has a profound impact on organismal aging. The progressive decline in skeletal muscle function with aging contributes to frailty and age-related disorders. Importantly, muscle specific interventions have demonstrated significant potential in slowing down or even reversing the aging process. Partial cellular reprogramming is an innovative anti-aging technique that resets the biological clock and restores youthful cellular features by overexpressing key reprogramming factors. Skeletal muscle specific reprogramming has shown promise in promoting skeletal muscle rejuvenation and restoring regenerative function. In this study, we tested the hypothesis that skeletal muscle plays a central role in regulating aging. We proposed that rejuvenating skeletal muscle using partial cellular reprogramming could lead to systemic anti-aging effects across the body. Using skeletal muscle-specific partial cellular reprogramming techniques in aged mice, we observed rejuvenation not only in skeletal muscle but also in other organs, including the spleen, kidney, and skin.

These systemic effects are likely mediated by muscle-derived secretory factors that enter the bloodstream and influence other tissues. Our findings underscore the potential of targeting skeletal muscle to achieve systemic rejuvenation. Furthermore, identifying and characterizing the muscle-secreted factors induced by partial cellular reprogramming could pave the way for the development of novel therapeutic candidates for systemic anti-aging treatments.

The Role of Lipid Droplets in Satellite Cells Differentiation and Fusion Yutzil Herrera UCLA – CIRM COMPASS Scholar Lab PI: Thomas A. Rando, M.D., Ph.D. Lipid Droplets (LDs) are organelles present in the cytoplasm across various cell types. Their primary role involves storing free fatty acids (FFAs) for subsequent use in many metabolic pathways, as a source of energy, and for membrane synthesis. The goal of the study is to investigate whether changes in LD dynamics impact SC differentiation and the ability to fuse. We targeted enzymes involved in LD formation and breakdown by using specific inhibitors: Atglistatin (ATGLin) to increase the number of LDs, and A922500 and PF-06424439 (DGATin) to inhibit LD formation. We found that ATGLin significantly decreased myoblast fusion by 79% (p= 0.0003) at 3 hours and 38% (p=0.0061) at 5 hours, and their ability to grow when compared to control (36% MHC+ area vs 27%, p < 0.0001). Incubation of SCs with DGATin resulted in an increase of myosin heavy chain (MHC) positive area by 18% when compared to the control (p = 0.410). DGATin, Linoleic Acid (LA), and Ferrostatin-1 partially reversed the negative effects of ATGLin. Those data indicate that LD dynamics may play an important role in myogenic progression. The observed results likely arise from an augmented cellular demand for FFAs during periods of heightened growth and the capability of Ferrostatin-1 to prevent ferroptosis, cell death due to lipid peroxidation. The exact mechanism underlying this relationship and its potential implications for the myogenic progression. This data might help develop new therapies targeting conditions resulting in reduced muscle regeneration capacity.

Developing a human iPSC-derived model of white adipocytes to study the contribution of genetic risk factors to metabolic diseases Hanna Liliom, Ph.D. Lab PI: Débora Sobreira, Ph.D Metabolic disorders such as obesity and type 2 diabetes present major health problems worldwide and the risk of their development is to a certain extent heritable. Genome-wide association studies (GWAS) have identified numerous genetic variants linked to these conditions, but comprehending their molecular mechanisms remains challenging, largely due to many disease-associated variants being non-coding. Tissue type, cell type and developmental stage are important determinants of how risk loci exert their effects, emphasizing the importance of developing accurate human cellular models for in vitro experiments to identify causal risk variants, as well as to elucidate their mechanism of action. Adipocytes of white adipose tissue are crucial players in maintaining whole-body energy homeostasis and regulating the metabolic response through various pathways, and their function is impaired in various metabolic disorders. Direct study of human white adipocytes is challenging since the mature cells are post-mitotic, as well as fragile and buoyant due to their large intracellular lipid droplet.

To overcome these challenges, we are developing an in vitro model of white adipocytes derived from induced pluripotent stem cells (iPSCs).

Utilizing the known functional-phenotypic plasticity of beige adipocytes, we adapted a published directed differentiation protocol to generate mature beige adipocytes from human iPSCs through a mesenchymal stem cell phase, and tested whether we could shift the mature beige adipocytes towards a white adipocyte-like identity through different media and reagent compositions. We compared cell morphology and the molecular phenotype of the iPSC-derived beige and white-like adipocytes. Our results demonstrate that ‘whitening’ induced drastic increases of the intracellular lipid droplet sizes and downregulation of key markers associated with beige adipocyte identity in the ‘whitened’ adipocytes.

Our goal is to refine our cellular model and establish the framework of its use as a tool for studying GWAS candidates implicated in metabolic disorders, enabling us to understand how risk variants exert their effect within the wider genetic context of a diverse set of individuals.

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Identifying Muscle Stem Cell Tropic AAV Variants Through Directed Evolution Harry Chen Lab PI: Melissa J. Spencer, Ph.D. Duchenne Muscular Dystrophy (DMD) is a rare genetic disease caused by frameshifting mutations in DMD, resulting in a lack of dystrophin protein production. While AAV-microdystrophin gene replacement therapy was recently given accelerated FDA approval for DMD, the therapeutic efficacy appears to reduce with time, likely due to turnover of skeletal muscle and replacement by muscle stem cells. Redosing AAV is not an option; however, a strategy to retain durable dystrophin is to target Muscle Stem Cells (MuSCs) with CRISPR/Cas9 to correct genomic DNA in the proliferating cell population. We have previously identified 5 MuSC targeting and 5 Skeletal Muscle (SkMu) targeting AAVs through directed evolution of a 7 amino acid sequence in vivo, inserted between amino acids 589A and 590Q in the surface region VIII (VR-VIII) of AAV9. Individual validation of these variants in human muscle cells has revealed that they perform nearly 40x better than wtAAV9 and 15x better than MyoAAV1A in MuSC targeting while retaining their myotube tropism. In addition, in vivo studies in mice have demonstrated a similar enhanced tropism compared to wtAAV9. These data have been confirmed through flow cytometry, luciferase, and qPCR of viral genomes. While further characterization must be done, these new variants provide a potential new platform for achieving long-term dystrophin restoration and durability when AAV is used to deliver CRISPR components in DMD and other myogenic diseases that involve muscle degeneration and regeneration.

Stimulus-dependent neurodevelopmental mechanisms in chromosome 16p11.2 microdeletion syndrome Rachel Fox BSCRC – CIRM Predoctoral Trainee Lab PI: Michael F. Wells, Ph.D. Copy number variation (CNV) at the chromosome 16p11.2 locus is associated with neuropsychiatric and neurodevelopmental disorders (NDDs). The heterozygous proximal deletion of chromosome 16p11.2 (16pdel) accounts for ~0.5% of autism spectrum disorder (ASD) cases and often co-occurs with macrocephaly, developmental delay, language disorders, and obesity. The mechanisms that link 16pdel to ASD and drive associated phenotypes remain unknown, and no clear therapeutic targets have been identified. Neural progenitor cells (NPCs) respond to spatial and temporal cues as they proliferate and differentiate, establishing regional identity and determining the proportion of neurons and glia in the developing brain. Interconnected and highly-coordinated signaling pathways, such as sonic hedgehog (SHH), regulate this process in NPCs, and genetic mutations in these and related signaling pathways are also associated with NDDs. In 16pdel iPSCs, researchers have observed differential expression of developmental signal transduction related genes, but the extent to which aberrant response to pathways themselves drives downstream 16pdel dysfunction has not been interrogated. In a cell village of 16pdel and control iPSC-derived NPCs treated with SHH, we have observed that 16pdel NPCs appear hyperresponsive to SHH stimulation. Exposure to SHH during neurodevelopment therefore provides a context in which 16pdel cellular dysfunction may be most dysregulated and may underlie phenotypes like macrocephaly.

VAMP2/3-dependent exocytosis in oligodendrocytes is required for maintaining oligodendrocyte plasma membrane integrity and the homeostatic state of microglia and astrocytes Caitlin Hooper UCLA – CIRM COMPASS Scholar Lab PI: Ye Zhang, Ph.D. Oligodendroccytes produce extensive myelin membranes to insulate axons. As axons and myelin expand during development, the oligodendrocytes’ plasma membranes undergo physical stretching. In multiple sclerosis, the complement membrane attack complex forms pores in the myelin membrane. Given the challenges to plasma membrane integrity, maintaining this integrity is vital for oligodendrocyte functionality and survival, although the underlying mechanisms are unknown. We hypothesized that VAMP2/3-dependent exocytosis actively repairs plasma membrane damage in mature oligodendrocytes. To test this, we employed genetic tools to inhibit VAMP2/3-dependent exocytosis in these cells. Our findings indicate that inhibiting VAMP2/3-dependent exocytosis compromises oligodendrocyte membrane repair following laser-induced membrane injury in vitro. Intriguingly, blocking oligodendrocyte exocytosis in vivo results in the prolonged reactivity of microglia and astrocytes. Ongoing studies aim to determine whether oligodendrocyte membrane integrity is compromised in vivo and how oligodendrocyte exocytosis contributes to maintaining the homeostatic state of microglia and astrocytes. This research sheds light on the underexplored domain of myelin plasma membrane repair, revealing potential therapeutic targets for demyelinating disorders. Additionally, it unveils new insights into the interaction between oligodendrocytes, microglia, and astrocytes.

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22ND ANNUAL

STEM CELL SYMPOSIUM FRIDAY, FEBRUARY 6, 2026

Human stem cell-derived sensory nerve cells of the spinal cord.

Credit: Piunik Shahgholian/ Samantha Butler Lab