20th Annual Stem Cell Symposium Program

20TH ANNUAL STEM CELL SYMPOSIUM

Celebrating 20 Years of Excellence in Stem Cell Research Friday, February 2nd, 2024 8:00AM - 4:35PM UCLA Covel Commons ● 3rd Floor

Photo: Motor neurons and glial cells from mESCs Source: BSCRC Microscopy Core

Sponsored 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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JAMES BRISCOE, PH.D.

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MARIANNE BRONNER, PH.D.

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DINO DI CARLO, PH.D.

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CARLA F. KIM, PH.D.

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KARA MCKINLEY, PH.D.

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APRIL D. PYLE, PH.D.

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OWEN WITTE, M.D.

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YUKIKO YAMASHITA, PH.D.

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POSTER ABSTRACTS

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Welcome to the 20th Annual Stem Cell Symposium!

Celebrating 20 Years of Excellence in Stem Cell Research Dear Colleagues and Friends: On behalf of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, we are delighted to welcome you to our 20th Annual Stem Cell Symposium. It is a pleasure to celebrate the center’s milestone year, together, with this vibrant stem cell community that has been cultivated here at UCLA. Since our first symposium, we have convened nearly 200 featured speakers from all over the world, created a platform for UCLA students to present over 1,000 posters, and hosted tens of thousands of audience members. Year after year, we are proud to showcase the incredible breadth of stem cell research and the many ways in which the field is translating promise to progress by bringing new therapies to the clinic.

Today, we have assembled an incredible lineup of speakers who will highlight regenerative medicine’s potential to transform human health. The program is designed to encourage interaction among attendees, with particular attention given to the poster session, in which nearly 60 stem cell researchers in training will present their projects. These scholars represent a range of departments and research areas and reflect our commitment to support the next generation of stem cell scientists through a pathway for education and career development. We are excited to strengthen this pipeline by expanding our high school outreach program and welcoming more students into our labs and classrooms.

With gratitude for your contributions to the UCLA Broad Stem Cell Research Center, we celebrate 20 years of excellence and look forward to many more. Thank you for your invaluable support of our programs and initiatives—we could not do it without you.

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

20TH ANNUAL STEM CELL SYMPOSIUM

Celebrating 20 Years of Excellence in Stem Cell Research Friday, February 2nd, 2024 8:00AM - 4:35PM

UCLA COVEL COMMONS ● 3RD FLOOR

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

Yukiko Yamashita, Ph.D. (Massachusetts Institute of Technology) Germline immortality and asymmetric stem cell division Moderated by Amander Clark, Ph.D.

9:25-10:05am

Carla Kim, Ph.D. (Harvard University) Cell-cell interactions in regulation of lung homeostasis, disease and cancer Moderated by Brigitte Gomperts, M.D.

10:05-10:30am

Break

10:30-11:10am

Owen Witte, M.D. (University of California, Los Angeles) Finding new targets to treat aggressive epithelial cancers Moderated by Thomas Rando, M.D., Ph.D.

11:10-11:50am

Dino Di Carlo, Ph.D. (University of California, Los Angeles) Secretion encoded single-cell sequencing (SEC-seq) to link cell secretory function to transcriptomes

11:50am-12:30pm

Lunch

12:30-1:30pm

Poster Session

1:30-2:10pm

Kara McKinley, Ph.D. (Harvard University) Regeneration in the uterus Moderated by Amander Clark, Ph.D.

2:10-2:50pm

April Pyle, Ph.D. (University of California, Los Angeles) Human pluripotency to skeletal muscle in development and disease Moderated by Melissa Spencer, Ph.D.\=

2:50-3:15pm

Break

3:15-3:55pm

James Briscoe, Ph.D. (The Francis Crick Institute) Building a spinal cord: the dynamics of neural tube development Moderated by Bennett Novitch, Ph.D.

3:55-4:35pm

Marianne Bronner, Ph.D. (California Institute of Technology) Neural crest stem cell potential along the body axis Moderated by Alvaro Sagasti, Ph.D.

4:35-6:00pm

Reception

Sponsored by a generous endowment from the Margaret M. Bloomfield Family Foundation

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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 funding comes primarily from granting agencies such as the California Institute for Regenerative Medicine (CIRM) and the National Institutes of Health (NIH) with additional funding from generous philanthropy including The Rose Hills Foundation and The Eli and Edythe Broad Foundation.

2024 Stem Cell Symposium Organizing Committee Members: Thomas Rando, M.D., Ph.D. Director, UCLA Broad Stem Cell Research Center Professor, Neurology; Molecular, Cell and Developmental Biology

Amander Clark, Ph.D. Director, UCLA Center for Reproductive Science, Health and Education Professor, Molecular, Cell and Developmental Biology

Dino Di Carlo, Ph.D. Professor, Bioengineering

Caroline Kuo, M.D. Assistant Professor, Pediatrics (Division of Allergy, Immunology, and Rheumatology)

Bennett Novitch, Ph.D. Professor, Neurobiology 5

Speaker Biographies James Briscoe, Ph.D. Principal Group Leader Associate Research Director

The Francis Crick Institute James Briscoe FMedSci FRS is a principal group leader and Associate Research Director at the Francis Crick Institute in London and editor-in-chief of the journal Development. He was educated at the University of Warwick and King's College London where he was awarded a Ph.D. in 1996 for research on JAK/STAT signaling supervised by Ian M. Kerr.

James Briscoe was a postdoctoral researcher at Columbia University with Thomas Jessell. In 2000, he moved to the MRC-National Institute for Medical Research in London to establish his own research group, and in 2001 he was elected an EMBO Young Investigator. He moved to the Francis Crick Institute in 2015. His research interests include the molecular and cellular mechanisms of embryonic development with a particular focus on the developing spinal cord with a particular interest in how the hedgehog signaling pathway regulates the development of this tissue. To address these questions, he uses a range of experimental biology and computational biology techniques with model systems that include laboratory mouse and chick embryos as well as embryonic stem cells. James Briscoe was awarded the EMBO Gold Medal in 2008 and elected a member of the European Molecular Biology Organization (EMBO) in 2009. In 2018, he became editor-in-chief of the journal Development. He is a Fellow of the Academy of Medical Sciences (FMedSci) and was elected a Fellow of the Royal Society (FRS) in 2019. In 2023, he was made an Honorary Foreign member of the American Association of Arts and Sciences.

Marianne Bronner, Ph.D. Professor, Biology Director of the Beckman Institute

California Institute of Technology

Marianne Bronner is a developmental biologist that studies the development and evolution of neural crest cells, a stem cell population that is unique to vertebrates. In particular, her lab has been systematically studying the gene regulatory network responsible for neural crest formation and evolutionary origin, using embryos from diverse animals including lamprey, zebrafish, chick and mouse. She received her ScB in Biophysics from Brown University and Ph.D. in Biophysics from Johns Hopkins University.

Bronner assumed her first faculty position at the University of California, Irvine, before moving to Caltech in 1996. Honors include the Harrison Medal from the International Society for Developmental Biology in 2022, the Conklin Medal from The Society for Developmental Biology in 2013, the Women in Cell Biology Senior Award from the American Society for Cell Biology in 2012, as well as several teaching awards from her institution. She was elected to the American Academy of Arts and Sciences in 2009 and the National Academy of Sciences in 2015.

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Dino Di Carlo, Ph.D. Professor, Bioengineering Vice Chair, Bioengineering

University of California, Los Angeles Dino Di Carlo is the Armond and Elena Hairapetian Professor of Bioengineering at UCLA, serial entrepreneur and inventor. He serves in academic leadership roles as the Vice Chair of the Bioengineering Department and Deputy Director of a National Science Foundation Engineering Research Center. He is an author on >190 peer-reviewed articles and an inventor on >70 issued patents in the U.S. and across the world. He has received numerous awards, including the Presidential Early Career Award for Scientists and Engineers (PECASE), the highest honor bestowed upon young scientists and engineers.

He also has served in business leadership roles. He co-founded several companies in life science research tools, diagnostics, medical device, and pharmaceutical industries over the last 10 years and continues to serve on the board of directors of many of these companies. He is a co-founder and director at Cytovale, an 80+ person company commercializing an FDA-cleared sepsis diagnostic test for rapid triage in the emergency department. He is Scientific Advisor to several life sciences companies, including Cue Health (NASDAQ:HLTH), which developed the first at-home molecular diagnostic test to be cleared by the FDA.

Carla F. Kim, Ph.D. Boston Children’s Hospital Stem Cell Program Professor of Genetics and Pediatrics

Harvard University

Dr. Carla Kim is the Boston Children’s Hospital Professor of Pediatrics in the Field of Regenerative Medicine at Harvard Medical School. Dr. Kim received her PhD in genetics from the University of Wisconsin-Madison in 2002. She joined the Stem Cell Program at BCH and the Department of Genetics at HMS after completing her postdoctoral work in the Tyler Jacks Lab at MIT. Since starting her laboratory in 2006, she has pioneered the use of stem cell biology approaches for the study of adult lung progenitor cells and lung cancer. Her work has contributed to a better understanding of stem cell biology in the lung, development of innovative approaches for examining the cellular and molecular basis of cancer, pulmonary disease and aging, and identification of new therapeutic avenues for lung cancer. A major contribution of Dr. Kim’s lab was the creation of threedimensional co-culture organoid systems to model airway- and alveolar-like structures from lung epithelial progenitor cell functions. The Kim Lab has used this organoid system to model cell-cell communication between epithelial cells and mesenchymal cells in the lung that regulate progenitor cells. They have developed organoid models of the earliest stages of lung cancer and defined techniques to transplant organoid cells into recipient mice for engraftment. Dr. Kim is leading international collaborations to develop pre-clinical models to set the stage for cell delivery for patients with devastating lung diseases. Dr. Kim’s contributions will provide transformative methods to understanding the biology of normal lung, lung disease and lung cancer. Dr. Kim is also the Director of Basic Research Career Development in the BCH Office of Faculty Development. She is creating programming to especially support women and those from groups historically underrepresented in medicine. She received the HMSBiological and Biomedical Sciences Graduate Program Mentoring Award, the NIH R35 Outstanding Investigator Award, and the William Rippe Award for Distinguished Research in Lung Cancer from the Lung Cancer Research Foundation.

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Kara McKinley, Ph.D. Assistant Professor, Stem Cell and Regenerative Biology

Harvard University

Kara McKinley is an Assistant Professor in the Department of Stem Cell and Regenerative Biology at Harvard University and a Freeman Hrabowski Scholar of the Howard Hughes Medical Institute. She started her lab in January 2021. Her lab studies how the uterine lining (endometrium) regenerates after menstruation with the goal of improving care for people with endometrial pathologies and/or menstrual experiences that interfere with their quality of life. She received her Ph.D. from MIT in 2016 under the mentorship of Iain Cheeseman. She performed postdoctoral training in the laboratories of Ron Vale and Ophir Klein at the University of California, San Francisco. She is also the founder of Leading Edge, an initiative to improve the gender diversity of faculty in the biomedical sciences.

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

University of California, Los Angeles

Dr. April Pyle is a Professor and Vice Chair in the Department of Microbiology, Immunology and Molecular Genetics, and a member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research (BSCRC) at UCLA. Dr. Pyle completed her postdoctoral fellowship work with Peter Donovan in 2006 at Johns Hopkins University, and obtained her Ph.D. with Dr. Mary Ann Handel from the University of Tennessee, Knoxville in 2002. Dr. Pyle's lab studies both basic aspects of stem cell biology, reprogramming, and muscle development as well as more translational aspects of human pluripotent stem cell differentiation towards skeletal muscle and muscle stem cells for use in therapeutic approaches for patients with neuromuscular diseases. The lab is also interested in the generation of CRISPR correction strategies for Duchenne Muscular Dystrophy (DMD), a lethal pediatric muscle disease. Dr. Pyle is also co-founder of MyoGene Bio which aims to develop a therapeutic CRISPR/Cas9 platform designed to restore the reading frame of the DMD gene. Her work is funded by NIH, CIRM, BSCRC, DOD and foundation funding. Dr. Pyle is currently a standing member of the NIH Skeletal Muscle Study Section (NIAMS/SMEP), Associate Editor for Stem Cell Reports, and Editorial Board member for Cell Reports Medicine and Cell Stem Cell. Dr. Pyle has received many awards including the UCLA Life Sciences Faculty Excellence Award, the Rose Hills Foundation Scholar, BSCRC/JCCC Ablon Scholars Award and Ayoub Innovation Chair.

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Owen Witte, M.D. Founding Director Emeritus, UCLA Broad Stem Cell Research Center Presidential Chair in Developmental Immunology Professor, Microbiology, Immunology and Molecular Genetics

University of California, Los Angeles

Owen Witte received his undergraduate degree from Cornell and his MD from Stanford University. He completed postdoctoral research at MIT then joined the faculty at UCLA where he presently is a University Professor of Microbiology, Immunology and Molecular Genetics. He is the Founding Director Emeritus of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research (BSCRC) at UCLA. Dr. Witte has made significant contributions to the understanding of human leukemias, immune disorders, and epithelial cancer stem cells. His work includes the discovery of tyrosine kinase activity for the ABL gene and the demonstration of the BCR-ABL oncoproteins in human leukemias. His work also led to the co-discovery of Bruton’s tyrosine kinase (BTK) which is required for normal B-lymphocyte development, and when mutated leads to X-linked agammaglobulinemia, a form of immune deficiency. New inhibitors for BTK are impacting clinical practice for the treatment of certain lymphomas and leukemias. Recent work has concentrated on defining the stem cells for epithelial cancers of the prostate and other organ sites to help define treatments for these diseases, including identifying targets and new methods for immune mediated therapies.

Dr. Witte has extensive experience consulting in the biotech and pharmaceutical industry.

He currently serves on the Board of Directors and Scientific Advisory Board at Allogene Therapeutics, is a Founder and Scientific Advisory Board Chair at Kronos Bio, and Chairs the Scientific Advisory Board at Vida Ventures.

He is a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and the National Academy of Medicine. He has received many awards for his research including recently the Association of American Medical College’s Award for Distinguished Research in Biomedical Sciences and the Stanford University School of Medicine’s Arthur Kornberg and Paul Berg Lifetime Achievement Award in Biomedical Sciences.

Yukiko Yamashita, Ph.D. Professor, Biology Investigator, Howard Hughes Medical Institute

Massachusetts Institute of Technology Yukiko Yamashita obtained her Ph.D. from Kyoto University, Japan, and conducted her postdoctoral fellowship with Minx Fuller at Stanford University (2001-2006). She was a professor at the University of Michigan Ann Arbor (2007-2020), and recently moved to the Whitehead Institute for Biomedical Research/MIT, where she is also an investigator of the Howard Hughes Medical Institute.

She studies the cellular mechanisms of stem cell self-renewal and differentiation, with a particular focus on understanding how adult stem cells divide asymmetrically, giving rise to one stem cell and one differentiating cell, to maintain tissue homeostasis. Another rising interest is the role of satellite DNA, a highly repetitive and non-coding element, in eukaryotic genomes during stem cell divisions and germ line lineages.

Yamashita is a recipient of 2008 Searle Scholar Award, 2009 American Society for Cell Biology WICB junior award, 2011 MacArthur Award, 2016 Tsuneko and Reiji Okazaki Award, 2021 American Society for Cell Biology Fellow, and elected to American Academy of Arts and Sciences in 2023.

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Poster Abstracts 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.

A Meta-Atlas of the Developing Human Cortex Identifies Modules Driving Cell Subtype Specification Patricia Nano 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 a meta-analysis of seven of these published datasets, we have generated 225 meta-modules – gene co-expression networks that can describe mechanisms underlying cortical development. Several meta-modules have potential roles in both establishing and refining cortical cell type identities, and we validated their spatiotemporal expression in primary human cortical tissues. These include meta-module 20, associated with FEZF2+ deep layer neurons. Half of meta-module 20 genes are putative FEZF2 targets, including TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical organoid experiments validated that both factors are necessary for deep layer neuron specification. Importantly, subtle manipulations of these factors drive slight changes in meta-module activity that cascade into strong differences in cell fate – demonstrating how of our meta-atlas can engender further mechanistic analyses of cortical fate specification.

Smad1 and Smad5 Differentially Transduce BMP Signaling During mESC to Dorsal Interneuron in Vitro Differentiation Cristian Rodriguez UCLA - CSUN CIRM Bridges Trainee Lab PI: Samantha Butler, Ph.D. The developing dorsal spinal cord is subject to numerous growth factors that pattern and direct the trajectory of neural progenitor cells to form specific classes of dorsal interneurons (dI). dIs relay and process different sensory modalities, including pain, touch, and proprioception, however little is known on how these distinct populations are generated. Bone morphogenetic proteins (BMPs) are a key factor released from the roof plate during spinal cord development that direct different cell fates. Studies have shown that ablation of the roof plate results in the loss of dI formation. BMP binding triggers a cascade of downstream responses, most notably the activation of receptor activated Smad1 and Smad5, which complex with Smad4 to regulate nuclear transcription. The extent to which Smad1 and Smad5 regulate transcription distinctly or redundantly, during dorsal spinal cord development is unknown. We are assessing this question, by subjecting Smad1 and Smad5 knockout mESC lines, generated using CRISPR, to our in vitro dI differentiation protocols. RT-qPCR, immunohistochemistry, and bulk RNA sequencing have been performed across multiple timepoints along these protocols. Our results indicate that Smad1 and Smad5 mediate BMP signaling both distinctly and redundantly. We are continuing to investigate the specific functional consequences of removing Smad1 and Smad5 on dI identity. Characterizing the downstream BMP signaling mechanisms during dorsal spinal cord development will allow us to identify the novel key mediators that specify distinct dI classes.

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Unraveling the Transcriptional Networks Modulated by Smad1 and Smad5 During Differentiation of Mouse Embryonic Stem Cells into Spinal Dorsal Interneurons Salena Gallardo Lab PI: Samantha Butler, Ph.D. Spinal dorsal interneurons (dIs) mediate somatosensation: our ability to sense the environment, permitting us to react to pain or touch, and move in a coordinated manner. When dIs are damaged, individuals are no longer able to interpret sensory information from the periphery. Spinal dIs are generated when discrete domains of neural progenitor cells arise along the dorsal-ventral axis of the neural tube. Growth factors, including the bone morphogenic protein (BMPs) family, drive the transcriptional changes needed to specify at least six populations of dIs (dI1-dI6). BMP signaling is transduced by two R-Smads, Smad1 and Smad5, during spinal cord development. Our lab has previously demonstrated that Smad1 and Smad5 can direct different cellular functions during dI differentiation, challenging the belief that RSmads function redundantly during embryogenesis. Here we utilize our directed differentiation protocol to derive bona fide dIs from mouse embryonic stem cells (mESCs). We are using this model system to test the hypothesis that Smad1 and Smad5 differentially regulate transcription to drive distinct cellular outcomes during dI specification. We have made Smad1 and Smad5 knock out (∆) mESC lines and using RNA-seq, have profiled gene expression of various in vitro defined developmental time points. Our results have identified multiple candidate genes that appear to be under the transcriptional control of Smad1 or Smad5. Interestingly, a significant number of protocadherin genes appear to be downregulated in Smad1∆ differentiated cell cultures. Future experiments will validate candidate genes in vivo, as well as assess the functional consequences of protocadherin gene silencing during dorsal spinal cord development. We aim to uncover transcriptional networks that are responsible for patterning and dI specification, information that is needed to generate pure populations of dIs for cellular replacement therapies seeking to restore somatosensation.

Atlas of Fetal Metabolism During Mid-to-Late Gestation and Diabetic Pregnancy Cesar Perez-Ramirez BSCRC - CIRM Postdoctoral Trainee Lab PI: Heather Christofk, Ph.D. Mounting evidence suggests metabolism instructs stem cell fate decisions. However, how fetal metabolism changes during development and how altered maternal metabolism shapes fetal metabolism remain unexplored. We present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and liquid chromatography-mass spectrometry (LC-MS), we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Our analysis revealed metabolic features specific to a hyperglycemic environment and signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from hyperglycemic dams. Tracing 13C-glucose revealed disparate fetal nutrient sourcing depending on maternal glycemic states.

Regardless of glycemic state, histidine-derived metabolites accumulated in late-stage fetal tissues. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations due to maternal hyperglycemia.

Characterizing a Novel Self-Instructing Non-Integrated Human Embryo Model Auriana Arabpour BSCRC - Rose Hills Foundation Predoctoral Trainee Lab PI: Amander Clark, Ph.D. Research on in vitro gametogenesis (IVG) aims to reconstitute germ cell development using human pluripotent stem cells (hPSCs) in culture. IVG offers a family planning tool enabling infertile individuals, same-sex couples, and cancer patients to produce biological offspring. The advancement of IVG, however, relies on the faithful recapitulation of human gametogenesis in vitro beginning first with germ cell precursors, known as human primordial germ cell-like cells (hPGCLCs). In this study, we evaluate a novel self-instructing non-integrated embryo model that differentiates hPSCs into hPGCLCs and other key embryonic somatic lineages. Human embryonic stem cells, differentiated in our model undergo morphological changes and migratory events giving rise to specified hPGCLCs identified by the co-expression of human germ-cell specific markers, TFAP2C, SOX17, and NANOG. Single-cell RNA sequencing reveals that our model generates embryonic lineages in addition to hPGCLCs. Immunofluorescence staining with markers for these embryonic somatic lineages suggests a meaningful spatiotemporal relationship between germline cells and somatic progenitors in our culture conditions. We report that our model has the potential for identifying differentiating germ cell precursors in vitro, perhaps in a manner modeling aspects of peri-implantation development.

In Vivo Timeline of Mouse Ovarian Primordial Germ Cell Development Azra Cruz Ordaz UCLA - CIRM COMPASS Scholar Lab PI: Amander Clark, Ph.D. Infertility, the inability to conceive after 12 months or more of attempting, is a highly prevalent issue that affects male and female reproductive systems. Current treatments that exist, such as in vivo fertilization, hormone therapy, and surrogacy, fail to treat infertility at a germ or sex cell level; thus, in vitro oogenesis (IVO) provides a promising alternative. IVO consists of utilizing induced pluripotent stem cells (iPSCs) to produce primordial germ cell-like cells (PGCLCs), the in vitro counterpart of in vivo primordial germ cells (PGCs), which are then combined with fetal ovarian somatic cells (FOSCs) to go on to develop into egg-like cells within a reconstituted Ovary (rOvary). However, PGCLC identity as they develop within the rOvary remains unclear. PGC development in vivo is relatively well understood; therefore, observing the development of PGC identity via a combination of germ cell markers like STELLA, DAZL, and SCP3 is critical to understanding germ cell development in vitro.

Immunofluorescent (IF) staining results show that at embryonic day (E)10.5, germ cells explicitly express STELLA. At E11.5 and E12.5, germ cells mainly express STELLA and DAZL. At E13.5, fewer germ cells express STELLA and instead express DAZL and SCP3, and at E15.5, germ cells express DAZL and SCP3. The IF results demonstrate the varying identity of mouse ovarian germ cells from early to late-stage development and provide insight into the changing identity of germ cells to help improve IVO as an assisted reproductive technology overall.

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Reversing Exhaustion of Surgically Procured Anti-Tumor T Cells Tyler McCaw BSCRC - CIRM Clinical Fellow Lab PI: Joseph G. Crompton, M.D., Ph.D. Surgery is potentially curative for patients with solid tumors, but half of patients will die from disease. Beyond surgery, long-term cures are achieved by engaging the patient’s own immune response.Immunotherapy is a promising (neo)adjuvant treatment strategy that empowers a patient’s anti-tumor immune response, but its efficacy is limited by T cell exhaustion. Exhaustion is an epigenetically enforced progressive terminal differentiation wherein tumor-specific T cells lose stemness and tumor-killing ability. This is irreversible. Moreover, most anti-tumor T cells have become exhausted by the time tumors are clinically detectable, often rendering current immunotherapies ineffective. To make immunotherapy a potentially curative treatment strategy for all patients, we will overcome T cell exhaustion using stem cell technology. We have recovered highly exhausted CD8 T cells from surgically resected soft tissue sarcomas and erased their epigenome by de-differentiating them into induced pluripotent stem cells (iPSC) using Yamanaka factors. We have also demonstrated the ability to re-differentiate iPSCs back into highly functional stem-like CD8 T cells. Interestingly, we have found that the quality—or degree of exhaustion—of the starting CD8 T cells determines the ability to generate these iPSCs. Our findings demonstrate that a patient’s own anti-tumor immune response can be potentiated using stem cell technology—reverting their exhausted, non-functional T cells into iPSCs and then back into highly functional, tumor-specific T cells. Our future work will identify strategies that allow exhausted CD8 T cells to more readily revert into iPSCs for improved therapeutic efficiency. The cellular product created can then be reinfused into the patient and combined with checkpoint blockade antibodies (e.g. anti-PD-1) as a potent immunotherapeutic platform.

Effects of hematopoietic stem cell aging on T cell development Julia Gensheimer Lab PI: Gay Crooks, M.B.B.S 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 the aged progenitor cells and/or changes in the thymic microenvironment. T cells develop from hematopoietic stem cells (HSCs) that enter the thymus, an organ known to atrophy with age. Determining T cell output in vivo has relied on hematopoietic transplantation models and is confounded by the long process of engraftment and thymic recruitment. While some in vitro models have looked at T cell output from aged HSCs, these methods often start with a heterogenous population of cells and produce mixed conclusions about T cell potential. Investigation of intrinsic defects in aged HSCs is further complicated by HSC heterogeneity. 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 and development, we studied phenotypically-defined aged and young HSCs using our in vitro Artificial Thymic Organoid (ATO) system. The ATO fully recapitulates all stages of T cell development from a single HSC. This controlled system allows for detailed dissection and quantification of thymopoiesis 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 inflamed microenvironment restores their T cell potential.

Engineering Stage-Specific Developmental cues to Generate Non-Allogenic, iPSC-derived Chimeric Antigen Receptor Therapy Sang Pil (Alex) Yoo Co-Presenters: Xuegang Yuan, Claire Engstrom, Patrick Chang, Suwen Li, Ralph Crisostomo, Christopher Seet, Donald Kohn, Gay Crooks Lab PI: Gay Crooks, M.B.B.S Chimeric antigen receptor (CAR) T cell is an emerging class of therapy that involves the engineering of patient’s immune cells to treat diseases that are otherwise resistant to traditional treatment regiments. Currently, the process of engineering individualized cellular therapies is costly and difficult to coordinate with clinical care.

Compounding manufacturing challenges, differences in patients’ T cells and stage of disease can contribute to inconsistent clinical responses and oftentimes cancer relapse. Recent advances in stem cell differentiation aim to address the manufacturing and biological challenges of CAR T cell generation. Induced pluripotent stem cells (iPSCs) provide a limit-less source of T cells that can be functionally enhanced with precise genetic modifications. iPSC-engineered T cell therapies would ensure immediate access to treatment that delivers a more durable and consistent clinical response. Unfortunately, there are critical, unsolved biological obstacles to efficiently generating CAR T cells from iPSCs. These obstacles include improper differentiation from iPSCs into unwanted cell types due to the complex biology of CARs. In addition, genetic modifications necessary for stem-cell derived T cells to be compatible with a patient’s immune system can block differentiation altogether. Our work employs a novel gene engineering approach that can deliver the necessary signals and developmental cues at the correct stage development. Our universal platform efficiently generates stem-cell derived, non-allogenic CAR T cells that demonstrate robust tumor killing and control. Our ongoing work will continue to enable the use of stem cell technologies to generate a more abundant and effective source of cancer-specific T cells for immunotherapy.

Defining cell-specific gene regulatory programs in the developing human cortex Celine K. Vuong Lab PI: Luis De La Torre-Ubieta, Ph.D. The human neocortex is responsible for many of our most distinguishing cognitive and social capabilities, often impaired in neuropsychiatric disorders. Previously, we found that genetic variation causing risk for neuropsychiatric disease and influencing cognition, also regulates gene expression programs of the developing human neocortex during neurogenesis. However, the gene regulatory mechanisms governing cortical neurogenesis remain to be characterized. Here we leverage human neural stem cell models, organotypic slice cultures, and transcriptome and epigenome profiling to identify and functionally characterize distal gene regulatory elements (DREs) regulating human corticogenesis. We outline a strategy to generate a cell-type specific functional map of DREs, using a pooled CRISPR interference (CRISPRi) screen in differentiating human neural progenitor cells to modulate the activity of hundreds of DREs, followed by in-depth functional characterization of selected target DREs in organotypic slice cultures. In pilot studies, we demonstrate efficient knockdown of ten transcription factors active during neurogenesis using our pooled CRISPRi strategy, and find significant changes in cell type composition and gene expression patterns upon loss of regulatory proteins enriched in progenitors and developing neurons. Applied to DREs regulating corticogenesis, these studies will provide insight into human-specific gene regulatory mechanisms of corticogenesis, and help translate genetic findings into human disease mechanisms.

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Cell-type-specificity of isoform diversity in the developing human neocortex informs mechanisms of neurodevelopmental disorders Ashok Patowary Co-Presenters: Xuegang Yuan, Claire Engstrom, Patrick Chang, Suwen Li, Ralph Crisostomo, Christopher Seet, Donald Kohn, Gay Crooks Lab PI: Luis De La Torre-Ubieta, Ph.D. Human brain development is under tight molecular genetic control and the recent advent of single-cell genomics has revolutionized our ability to elucidate the diverse underlying cell-types and states. Although RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, previous work has not systematically investigated the role of cell-type-specific splicing or transcript-isoform diversity during human brain development. Here, we leverage single molecule longread sequencing to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution. We identify 214,516 unique isoforms, corresponding to 22,391 genes. Remarkably, we find that 72.6% of these are novel and together with >7,000 novel-spliced exons expands the proteome by 92,422 proteoforms. We uncover myriad novel isoform switches during cortical neurogenesis, implicating previouslyuncharacterized RNA-binding protein-mediated and other regulatory mechanisms in cellular identity and disease. Early-stage excitatory neurons exhibit the greatest isoform diversity and isoform-based single-cell clustering identifies previously uncharacterized cell states. Leveraging this resource, we re-prioritize thousands of rare de novo risk variants associated with neurodevelopmental disorders (NDDs) and reveal that risk genes are strongly associated with the number of unique isoforms observed per gene. Altogether, this work uncovers a substantial contribution of transcript-isoform diversity in cellular identity in the developing neocortex, elucidates novel genetic risk mechanisms for neurodevelopmental and neuropsychiatric disorders, and provides a comprehensive isoform-centric gene annotation for the developing human brain.

Defining molecular 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.1 DS results from trisomy of chromosome 21(hsa21, T21) causing impaired cortical development and altered cell composition and brain architecture.1 However, the molecular mechanisms driving these pathologies remain unclear. We hypothesize that increased dosage of hsa21-encoded transcription factors (TFs) and epigenomic regulators changes global gene expression in neural progenitors, changing neural cell fate specification and differentiation. Here, we profiled ~42K cells from 16 age/sex-matched DS and neurotypical (NT) neocortices via snMulti-ome technology. Using unbiased clustering, we identify 20 cell types in integrated NT and DS samples. In DS tissue, we observe an increase in the proportion of progenitors, interneurons and oligodendrocyte precursor cells, and a decrease in excitatory neurons, microglia, endothelial cells and pericytes. We also identify cell-specific changes in transcriptomic profiles in developing DS brains. Together, these data have identified potential molecular mechanisms that are disrupted during DS development, which may provide a foundation to developing therapeutic targets in the future.

Optimizing Conditions to Improve Differentiation of Pluripotent Cells to Definitive Endoderm For the Purpose of Generating Prostate-Like Cells Samantha Nicole Garcia UCLA - CSUN CIRM Bridges Trainee Lab PI: Andrew Goldstein, Ph.D. Differentiation from pluripotent cells to definitive endoderm cells is the first step prior to initiating organoid formation in our in vitro models. While it is a common practice to differentiate cells, it’s important to optimize the differentiation to definitive endoderm within each individual pluripotent cell line. To add to the improvement on the differentiation we modified the concentration of Activin A, seeding densities, as well as replating dates. We then viewed the results by using flow cytometry, microscopy, and western blots. This was done to improve the common differentiation process to get optimal confluence for cell differentiation to make better organoids and increase the percentage of differentiated cells. While our work is still ongoing, our results will be used to standardize the protocol to generate human prostate like cells.

Metabolic regulation of prostate cancer differentiation and response to treatment Nicholas Nunley Lab PI: Andrew Goldstein, Ph.D. Prostate cancer leads to nearly 35,000 deaths annually in the United States alone. Resistance to androgen receptor (AR) inhibition is a major cause of disease progression and prostate cancer-associated lethality. Independent studies across distinct patient cohorts have established a relationship between the degree of luminal differentiation and responsiveness to therapy in prostate cancer. Understanding the factors that influence luminal differentiation is essential: (1) to predict which patients are likely to respond to AR inhibition or exhibit resistance and (2) to develop new approaches to increase sensitivity to antiandrogens. We recently found that altered pyruvate/lactate metabolism can modulate differentiation in prostate cancer and fuel resistance to therapy. We hypothesize that additional metabolic changes associated with lineage transitions regulate luminal differentiation and treatment resistance in prostate cancer. Using an N-MYC over-expression model to drive the loss of luminal differentiation in prostate cancer cells, we have identified metabolic and transcriptional changes accompanying the lineage transition including altered IDH1-mediated reductive carboxylation. Functional studies will now be used to determine the role of these metabolic changes in luminal differentiation and resistance to AR inhibition in prostate cancer. In summary, we will determine how drivers of lineage transitions influence prostate cancer metabolism and we will define mechanisms by which altered metabolism influences luminal differentiation and response to AR inhibition.

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Mechanisms of airway injury and repair following wildfire smoke exposure Summer Parr UCLA - CIRM COMPASS Scholar Lab PI: Brigitte Gomperts, M.D. Mucociliary clearance protects the airways from the environment by trapping particles in the mucus while ciliated cells beat unilaterally to move the mucus out of the lung. Additionally, innate immunity through Type 1 interferon (IFN) signaling regulates epithelial inflammation and regeneration. Woodfire smoke (WFS) is a component of wildfire smoke that contains toxins that surpass defense mechanisms and damage the airways. Wildfires are a growing global environmental health hazard, but little is known regarding the effects of WFS on the airway epithelium. In addition, the relationship between IFN signaling and airway repair following WFS remains unknown.

Therefore, this project is focused on understanding the mechanisms of lung injury and repair following WFS exposure. Our lab cultures human mucociliary epithelia in air liquid interface (ALI) cell cultures and established a highly reproducible system to expose the cultures to whole WFS. Immunofluorescent stains demonstrated an increase in airway basal stem cells (KRT5) and mucus secreting cells (MUC5B), and a decrease in ciliated cells (Acetylated Tubulin) following WFS exposure. Complete abolition of ciliary beating after exposure to WFS was also observed. These results were confirmed through Real Time Quantitative Polymerase Chain Reaction (RTqPCR) experiments, which displayed an increase in gene expression of Keratin5, MUC5B, and a decrease in FOXJ1. We also identified proteins with significant changes in phosphorylation involved in the IFN innate immunity pathways such as MAVS (p=.0001), LIN9 (p=.0001), and MX2 (p=.0017) following WFS exposure. RTqPCR analysis displayed an upregulation of several genes associated with the IFN pathway, (IFNAR1, IFNAR2, IFNB, MX1). Our data demonstrate that WFS exposure drives IFN signaling and induces a dysregulated injury and repair response. These results will allow for the development of preventative therapies to reduce injury following WFS exposure in at risk individuals.

ERBB family signaling regulates human airway submucosal gland morphogenesis and fate outcomes Andrew Lund BSCRC - Rose Hills Foundation Predoctoral Trainee Lab PI: Brigitte Gomperts, M.D. The airway submucosal glands (SMGs) work in conjunction with the surface airway epithelium as a first line of defense against inhaled environmental insults, producing mucus and antimicrobial secretions that trap particulate matter and combat infection. Hypersecretory disorders, typified by gland hypertrophy and hyperplasia, precipitate chronic inflammation and tissue remodeling, resulting in an untimely decline in airway function. Despite their physiological relevance to airway health and disease, the SMGs remain largely unexplored, especially in humans. To understand the mechanisms governing cell fate decisions and gland remodeling, we leveraged single-cell transcriptomics to infer receptor-ligand interactions targeting the SMG epithelial compartment. Further, we developed a novel human SMG organoid model to functionally assess the predicted interactome. Predicted cell-cell interactions indicate impingement of ERBB family ligands upon SMG epithelial stem/progenitor populations.

Moreover, treatment of human SMG organoids with ERBB ligands, namely HBEGF and NRG1, promote branching and budding morphogenesis during organoid expansion, whereas AREG seemingly drives tubuloacinar fate commitment during organoid differentiation. Taken together, our data suggest a mechanism by which ERBB signaling may drive glandular hyperplasticity.

Mechanisms of airway injury and repair following wildfire smoke exposure Summer Parr UCLA - CIRM COMPASS Scholar Lab PI: Brigitte Gomperts, M.D. Mucociliary clearance protects the airways from the environment by trapping particles in the mucus while ciliated cells beat unilaterally to move the mucus out of the lung. Additionally, innate immunity through Type 1 interferon (IFN) signaling regulates epithelial inflammation and regeneration. Woodfire smoke (WFS) is a component of wildfire smoke that contains toxins that surpass defense mechanisms and damage the airways. Wildfires are a growing global environmental health hazard, but little is known regarding the effects of WFS on the airway epithelium. In addition, the relationship between IFN signaling and airway repair following WFS remains unknown.

Therefore, this project is focused on understanding the mechanisms of lung injury and repair following WFS exposure. Our lab cultures human mucociliary epithelia in air liquid interface (ALI) cell cultures and established a highly reproducible system to expose the cultures to whole WFS. Immunofluorescent stains demonstrated an increase in airway basal stem cells (KRT5) and mucus secreting cells (MUC5B), and a decrease in ciliated cells (Acetylated Tubulin) following WFS exposure. Complete abolition of ciliary beating after exposure to WFS was also observed. These results were confirmed through Real Time Quantitative Polymerase Chain Reaction (RTqPCR) experiments, which displayed an increase in gene expression of Keratin5, MUC5B, and a decrease in FOXJ1. We also identified proteins with significant changes in phosphorylation involved in the IFN innate immunity pathways such as MAVS (p=.0001), LIN9 (p=.0001), and MX2 (p=.0017) following WFS exposure. RTqPCR analysis displayed an upregulation of several genes associated with the IFN pathway, (IFNAR1, IFNAR2, IFNB, MX1). Our data demonstrate that WFS exposure drives IFN signaling and induces a dysregulated injury and repair response. These results will allow for the development of preventative therapies to reduce injury following WFS exposure in at risk individuals.

Modeling cell population dynamics in early hematopoiesis reveals mechanisms of myeloid bias mediated by NFκB-dysregulation Apeksha Singh Lab PI: Alexander Hoffmann, Ph.D. Alterations in hematopoiesis are associated with perturbations in NFκB signaling. Aging, for example, is correlated with increased myeloid and decreased lymphoid output, as well as elevated NFκB activity in hematopoietic stem and progenitor cells (HSPC) and inflammatory cytokines in bone marrow. Further, patients with myelodysplastic syndrome (MDS) show increased expression of NFκB signaling pathway components in HSPC and mesenchymal cells. Despite being implicated in these contexts, it remains unknown how elevated NFκB signaling might affect developmental dynamics of HSPC. To interrogate these effects, we leveraged data from the IκB– mouse (Nfkbia+/- Nfkbib-/- Nfkbie-/-). HSPC counts demonstrated myeloid biased multipotent progenitor (MPP) abundances. Math modeling suggested that accelerated ST-HSC differentiation with skewed fate commitment is insufficient for the increase in MPP2/3 cells. To explore additional mechanisms for myeloid bias, we utilized single-cell RNA sequencing (scRNAseq) data from IκB– and control HSPC. We assigned cells to a differentiation trajectory from HSC to erythrocytemegakaryocyte, myeloid, and lymphoid primed progenitors and fit these data to a population dynamics model. This analysis suggested not only less lymphoid specification in HSC but also increased expansion of early myeloid primed cells in the IκB–. Differentially expressed genes along the HSPC lineages support the model predictions. We finally used scRNAseq of HSPC isolated from MDS patients and healthy controls to see if features of NFκB-mediated immune dysregulation were observed. These data demonstrated myeloid bias in progenitors and elevated NFκB signaling in MDS HSPC. Applying the same analyses developed for the murine datasets revealed commonalties between developmental dynamics of MDS and IκB– HSPC. These analyses motivate further study of consequences of immune dysregulation on hematopoiesis and identification of NFκB signaling perturbations in pathological contexts.

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NFkB Dysregulation in the Bone Marrow Niche Drives Epigenomic Reprogramming of Hematopoietic Stem Cells and Myeloid Bias Jennifer Chia Lab PI: Alexander Hoffmann, Ph.D. Aging and chronic inflammation are tightly linked, resulting in hematopoietic bias toward myeloid cell fates and away from lymphoid lineages. However, it remains unclear whether chronic inflammation is sufficient to drive myeloid bias. In addition, the bone marrow niche plays critical roles in supporting steady-state hematopoiesis, and alterations in the niche may similarly influence disordered hematopoiesis. The NFkB system is a central, pleiotropic regulator of inflammatory signaling in many cell types, and we thus reasoned that experimental perturbations of NFkB signaling may be useful to dissect the causal role of persistent inflammation in myeloid bias. We generated a mouse model of inflammatory NFkB dysregulation (“IkB–“ Nfkbia+/- Nfkbib-/- Nfkbie-/-), which shows an increase in bone marrow inflammatory cytokines, as well as myeloid bias in the peripheral blood and among hematopoietic stem and progenitor cells (HPSC), similar to aged mice. In bone marrow transplantation assays, an IκB– niche was sufficient to drive myeloid-biased hematopoiesis. However, myeloid bias was lost upon secondary transplantation into a WT recipient. We performed ATAC-seq and scRNA-seq on HSPC sorted from IkB– or WT control recipients, which revealed an increase in accessibility for myeloid-priming transcription factor motifs in HSC, with corresponding increase in target gene expression and transcriptional myeloid bias. We also detected similar transcriptional changes in published murine and human aged HSPC datasets. Our results indicate that an inflamed niche is sufficient to induce myeloid bias in HSPC, and is associated with epigenomic reprogramming at the stem cell level. However, inflammatory-associated myeloid bias can be also reversed by re-introduction into a non-dysregulated niche. Together, these data suggest that targeting the bone marrow niche in chronic inflammatory states such as aging may be a viable strategy for improving disordered hematopoiesis.

CARGO-AGNOSTIC LIPID NANOPARTICLES TO CORRECT CYSTIC FIBROSIS IN AIRWAY STEM CELLS Ruth Foley Co-Presenters: Alicia Sanoyca Lab PI: Steven J. Jonas, M.D., Ph.D. Cystic fibrosis (CF) is a disorder arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that leads to chronic lung infections and respiratory failure. Diseases like CF are appealing targets for gene therapies, but existing gene editing approaches that enable the site-specific correction of diseasecausing defects rely on large double-stranded DNA (dsDNA) constructs that are inefficient to package and deliver to targeted airway basal stem cells (ABSCs) lining the CF airway. To address this challenge, we designed and tested a gene therapy strategy that applies lipid nanoparticle (LNP) vehicles that facilitate transport of CFTRdirected CRISPR/Cas9 gene editing tools to ABSCs. Our preliminary studies show that hypotonic EGTA carrier solutions clear a path to ABSCs in air liquid interface (ALI) culture models of the human airway for LNPs loaded with green fluorescent protein (GFP)-encoding mRNA cargoes. Confocal microscopy analysis of these ALI cultures exhibit expression of the GFP reporter. The capabilities of LNPs packaged with CRISPR/Cas9 cargoes configured to direct editing within the 5’ untranslated region of the endogenous CFTR locus were tested using a human bronchial epithelial cell line (16HBE14o-). Packaging these gene editing reagents into a single LNP resulted in ca. 14% integration compared to ca. 10% when the dsDNA donor was packaged in a separate nanoparticle. These studies informed experiments applying the LNPs for correction of CFTR in a CF mutant cell line (16HBEge-G542x). LNPs carrying a CFTR dsDNA correction template achieve up to 3.5% integration.

Thesenanotechnology-enabled tools overcome limitations in the cargo-carrying capacity of existing viral vector and nanoparticle delivery vehicles. This work sets a new precedent for packaging and delivering large dsDNA templates in nanocarriers, which can be leveraged to accelerate the clinical deployment of gene therapies for CF and other genetic diseases.

Tools for in vivo CAR T cell generation: Antibody-Lipid Nanoparticle mediated, targeted activation and transfection of human T cells.

Diego Sergio Banuet Gonzalez Co-Presenters: Anya Yakimenko Lab PI: Steven J. Jonas, M.D., Ph.D. Cell-based immunotherapies have revolutionized the treatment of pediatric cancers, with next generation approaches exploring the engineering of modified immune cell populations in vivo. The packaging and delivery of chimeric antigen receptor (CAR)- expression constructs in customized lipid nanoparticles has enabled non-viral generation of CAR T cells. While LNPs are a promising technology to mediate CAR expression in T cells, a key barrier to clinical translation of LNPs for in vivo CAR T cell generation lies in their non-specific delivery. Here we report the design of α-CD3 antibody conjugated LNPs for direct targeting and engineering of T cells in vivo. Our current, non-targeted optimization efforts produce consistently monodispersed LNPs with diameters less than 200 nm and mRNA encapsulation efficiencies above 80%.

We observed expression of Green Fluorescence Protein (GFP)-encoding mRNA cargoes in as little as 4 h post LNP treatment of activated T cells, with at least 80% of treated cells expressing GFP for up to 72 h, which occurred during active T cell proliferation and before visible exhaustion. Our click chemistry-based antibody conjugation strategy relies on a biorthogonal tetrazine - trans cyclooctene-mediated conjugation of αCD3 monoclonal antibodies to the surface of LNPs. Addition of the αCD3 monoclonal antibodies has shown equal transfection to our non-specific LNP platform, with our preliminary studies indicating the potential of our αCD3-modified LNPs to both stimulate and transfect naïve T cells. Our ongoing studies explore the cell-type specificity of our targeting platform, and the demonstration of functional, CD19 CAR T expression. Deployment of such nanotechnologies in good manufacturing practice-compliant settings will empower research focused on cellular immunotherapies and have the potential to revolutionize both the application, and accessibility of cellular based therapies.

Molecular Effect of Sars-Cov2 Spike Protein on DPSC Potency Alique Kurdian UCLA - CSUN CIRM Bridges Trainee Lab PI: Yong Kim, Ph.D. SARS-CoV-2 is an infectious disease that is categorized under biosafety-level-3 due to being potentially lethal. This research project centers on the interaction between Dental Pulp Stem Cells (DPSCs) and SARS-CoV-2. Existing studies have highlighted the potential of DPSCs to alleviate the severity of Covid-19 through their regenerative capabilities; however, there are very few studies concerning the changes in DPSC potency induced by the virus. The immediate aim of the project is to determine any cellular and molecular effects of the Spike protein on stem cell potency and differentiation capacity of DPSCs. My PI's lab has focused on genomic and epigenomic studies on hESCs and DPSCs and has sufficient expertise and model systems to investigate this. My project will stand independently from the ongoing research in my lab, distinct from the focus of my Principal Investigator. The experimental procedures I intend to undertake include streaking bacteria, colony selection, DNA purification and prepare plasmid constructs, and the generation of pseudotyped lentiviral particles by transfecting them into 293T cells. Specifically, I will be producing SARS-CoV-2 Spike pseudotyped lentiviruses to investigate how the virus impacts DPSCs at the molecular level. Furthermore, I will be centering my attention on the change in stem cell characteristics of DPSCs and how infection with SARS-CoV-2 affects the differentiation potency of DPSCs. Differentiating Dental Pulp Stem Cells into osteogenic lineages will provide valuable insights toward understanding the molecular mechanisms that regulate their stem cell potency.

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Lentiviral Stem Cell Gene Therapy for Alpha Thalassemia Eva Segura BSCRC - Rose Hills Foundation Predoctoral Trainee Lab PI: Donald B. Kohn, M.D. α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes resulting in the reduction of α-globin chains, one the two subunits forming the adult hemoglobin (HbA) responsible for oxygen transport. Severe anemia, requiring lifelong blood transfusions and chelation therapy, arises when α/β-globin chain ratio falls below 0.3 resulting from the absence of, or defects in, three or all four α-globin genes. While an allogeneic hematopoietic stem cell transplant (HSCT) is a potentially curable option, it is drastically limited by both the small percentage of patients having an immunologically matched donor, as well as, by the potentially dangerous complications from graft-versus-host disease. To overcome these limitations, our research project developed a one-time autologous stem cell gene addition therapy in which functional copies of α-globin gene integrate into the genome of patient’s hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the α/β-globin chain imbalance and restore hemoglobin production.

The design of the α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia, which has achieved transfusion independence in patients with transfusion-dependent β-thalassemia major. Top two AGLVs were assessed for the correction of alpha thalassemia major (ATM), which is characterized by the lethal deletion of all α-globin genes. Both vectors possessed high CD34+ infectivity α-globin chain expression levels reaching that of healthy donor levels, overall restoring hemoglobin function in all three ATM donors. Specifically, A2-Globe LV replaced the production of nonfunctional β-globin tetramers with the production of HbA, reaching 80% of that of healthy donors, while maintaining a low vector copy number (VCN). Furthermore, both AGLVs showed no off-lineage α-globin expression in myeloid cells. Overall, this project developed an efficient LV for a gene therapy for α-thalassemia.

Optimizing Repair Template for an Autologous Stem Cell Therapy Approach to Treat X-linked Agammaglobulinemia Karen Li Lab PI: Donald B. Kohn, M.D. X-linked Agammaglobulinemia (XLA) is a rare x-linked immune deficiency caused by mutations in the Bruton’s Tyrosine Kinase (BTK) gene, which lead to severely decreased B-cell production and protective antibodies. Currently, the only curative treatment for XLA is an allogeneic stem cell transplant, which is limited by graftversus-host disease, graft rejection, and donor availability. We propose an autologous stem cell therapy approach to treat XLA, where a functional copy of BTK is delivered into patient hematopoietic stem cells and integrated via CRISPR-Cas9 mediated homology directed repair. Previously, a donor template named APEX, which is an AAV carrying the full-length BTK cDNA with regulatory elements, was created and shown to restore BTK expression to near-physiologic levels. However, this donor resulted in ectopic T-cell expression, so further optimization must be done to improve lineage specificity. Due to AAV capacity limitations, other delivery methods will be explored to increase flexibility in the design of the repair cassette. Thus, the APEX template was synthesized as plasmid and double-stranded DNA (dsDNA) and transfected in a BTK knockout cell line. Upon transfection, the dsDNA template shows correct transgenic integration and BTK expression. Overcoming this limitation, new donor templates can be tested as dsDNA. The donor template was modified to integrate additional endogenous intronic sequences into the btk cDNA to restore physiologic regulation. Those constructs will be tested further to optimize on-target specificity. This work can lead to the creation of a non-viral donor with limited offtargets that can restore a correct BTK expression pattern in XLA patients.

Novel Lentiviral Vector Design for Alpha Thalassemia Andrea Gutierrez UCLA - CSUN CIRM Bridges Trainee Lab PI: Donald B. Kohn, M.D. Alpha thalassemia is a genetic blood disorder caused by a mutation(s) in the α-globin genes, (HBA2 and HBA1) and results in the reduction of the hemoglobin protein complex constituted of 2 α- and β-globin chains. The severity of the condition is proportional to the number of defective α-globin genes and varies greatly – from asymptomatic to blood transfusion-dependent, and in the most severe case, to fetal demise.This project aims to develop a stem cell gene addition therapy through the development of a lentiviral-mediated delivery of HBA2 gene in the genome of hematopoietic stem and progenitor cells (HSPCs) of alpha thalassemia patients. Previously developed lentiviral vectors (LVs) optimized to express α-globin chains have resulted in HbA restoration in alpha thalassemia differentiated red blood cells (RBCs). My goal is to investigate the effects of integrating a truncated erythropoietin (Epo) receptor (tEpoR) to the candidate vectors. Because the binding of EPO in early hematopoietic erythroid progenitor cells drives erythropoiesis, we hypothesize that integrating tEPOR in our vectors will lead to an increase of corrected RBCs. Newly designed vectors will be assessed and compared to parental vectors (engineered to solely carry HBA2) in healthy and alpha thalassemia donor HSPCs. Following transduction, HSPCs will undergo RBC differentiation where gene transfer efficiency, gene expression levels, and RBC count will be evaluated. Overall, this project aims to optimize α-globin LVs and to advance the field providing a one-time curable gene therapy for alpha-thalassemia.

Effect of Matrix Stiffness on the Epigenetic State of Mesenchymal Stem Cells Johnathan Horn UCLA - CIRM COMPASS Scholar Lab PI: Song Li, Ph.D. Human Mesenchymal stem cells (hMSCs) are multipotent stem cells that have the innate ability to turn into other various cell types within the human body. This makes MSCs very critical to the field of regenerative medicine. While MSCs have been extensively utilized in research, the role of biophysical cues, such as matrix elasticity, on the epigenetic state of MSCs is not well understood. To better understand the influence of matrix stiffness on the epigenetic state of MSCs, MSCs were grown on polyacrylamide gels with varying elasticities (40kPa, 20kPa, and 1kPa) and analyzed for distinct euchromatin (Ach3, H3K4me3, H3K4me1, H3K27ac) and heterochromatin marks (H3K9me3, H3K27me3 ) via immunofluorescence analysis. Our preliminary findings indicate that soft matrices (20 kPa and 1 kPa) increased most of the histone marks examined compared to 40 kPa gels. Interestingly, an intermediate surface stiffness of 20 kPa promoted higher levels of H3K4me3 compared to 1 kPa gels and stiff substrates. In contrast, H3K27me3 marks were more pronounced on relatively soft (1 kPa) gels. With the data all pooled together, our findings indicate that there is some underlying change to the epigenetic state of the MSCs, implying that there is an inner mechanical change within the MSCs in response to the matrix stiffnesses. These observed responses could affect downstream MSC behavior and differentiation. The data from the project allows us to better understand the biophysical regulation of the epigenetic state of MSCs and potentially increase the efficiency and effectiveness of MSC research and therapies.

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In situ formation of long-lasting tumor-specific T memory stem cells using biomimetic nanoparticles Mahdi Hasani Lab PI: Song Li, Ph.D. Solid tumors account for more than 90% of cancer deaths, indicating the need for new and effective therapies. Personalized cancer immunotherapy, particularly cancer vaccines, offers a promising approach to preventing cancer recurrence by creating a long-term memory of immune cells against cancer. In this study, we developed a nanobiomaterial-based cancer vaccine (nanoVax) to delivers cancer neo-antigens and a cocktail of immune cell modulators to the tumor draining lymph node, which can promote the formation of cancer-specific T memory stem cells (TMSCs) to help fight cancer.

We found that treatment with nanoVax significantly increased the activation of dendric cells (DCs) compared to control samples. Moreover, nanoVax induced the formation of antigen-specific TMSCs in vitro, and significantly shrank tumors in vivo following repeated injections. Treatment with nanoVax also supported the long-term survival of mice, with 30% of mice becoming tumor-free 100 days after treatment. Similar to in vitro observations, nanoVax can significantly affect the activation of DCs and T cells withing the tumor tissue as explored using flow cytometry.

Our study demonstrated that our nanoparticle vaccine's design could engineer in vivo formation of TMSCs, providing long-term immunity against primary tumors and preventing tumor recurrence. Our findings indicate that these immunomodulatory nanoparticles can interact effectively with immune cells in vitro and in vivo, presenting tumor neoantigens and adjuvant to activate antigen-presenting cells and promote the formation of cancer antigen-specific TMSCs. Our engineered nanoparticles offer a promising platform for DC and T cell activation and in vivo clearance of tumors. Our approach lays the groundwork for the practical steps of translation toward personalized immunotherapy, potentially offering a new and effective therapy for cancer patients.

Synthetic Biology Platform for User-Defined Multilineage Cell Reprogramming Tyler Hoffman Lab PI: Song Li, Ph.D. During development and maintenance of skeletal muscle, multipotent progenitors serve to form the distinct cellularity and extracellular matrix structure required for function. Following large injury, muscle tissue is incapable of self-repair due to lack of endogenous cues to guide regeneration. Volumetric muscle loss injuries result in fibrosis at the site of injury causing lack of mobility and reducing quality of life. There lacks an understanding of the influence of co-differentiation on tissue maturation and repair, due to limited strategies to control multi fate differentiation within the same culture. Synthetic notch (synNotch) receptors have emerged as powerful tools for user-defined control over cell differentiation and morphogenesis through intracellular orthogonal signaling. In this work, we develop synNotch engineered cells and tunable microenvironments to spatially coordinate skeletal muscle and endothelial differentiation.

Mouse embryonic fibroblasts were co-transduced with designed plasmids using lentiviruses to generate stable cell lines. We demonstrate that green fluorescent protein (GFP) and mCherry can induce the expression of master transcription factors to control multi-fate differentiation. Transgene activation and the extent of differentiation are evaluated via inducible fluorescent reporters, morphological changes, and lineage-specific markers. Synthetic ligands patterned on a 2D substrate lead to spatially defined muscular and endothelial differentiation within the same culture. The influence of substrate stiffness and viscoelasticity on synthetic gene activation and differentiation were evaluated. In 3D matrix, conjugation of ligands to polymers enables robust synthetic gene activation of engineered cells encapsulated within gelatin methacryloyl (GelMA) hydrogels. Our demonstration explores a new approach to control cell differentiation via synthetic material inputs to multiplex control over cell states for the development of complex tissues.

Immunomodulatory vascular graft for organ transplantation Zeyang Liu Co-Presenters: Xinyang Ge; Youcheng Yang Lab PI: Song Li, Ph.D. Organ transplantation is the only cure for end-stage organ failure, but recipients face challenges like organ rejection, medication side effects, and infection. The effectiveness of oral immunosuppressive drugs is compromised, with only about 20% reaching the bloodstream, necessitating high doses that can harm the liver and other organs. Furthermore, patient nonadherence to multiple daily drug regimens increases graft failure risk, impacting patient health and healthcare resources.

Addressing these issues, we propose a novel double-layered drug-delivering vascular graft (DDVG) for local, sustained delivery of immunosuppressants to the transplanted organ. This approach could reduce immunosuppressant dosage, enhance transplantation success, and alleviate patient noncompliance burdens. Our DDVG, featuring an inner blend of poly(lactic-co-glycolic acid) (PLGA) and poly(L-lactide-co-ε-caprolactone) (PLCL) for drug release and an outer polycarbonate urethane (PCU) layer for mechanical support, is designed for high patency and a minimum 6-month drug release. We used tacrolimus in liver transplantation as a model, with in vitro and in vivo studies demonstrating feasibility. Our findings suggest that DDVG could significantly advance organ transplantation outcomes, offering a more effective, patient-friendly treatment approach with broader implications for transplantation medicine.

Immunocytochemistry assessment of vocal fold regeneration after cell-based implant in rabbits Larissa Nicolas Lab PI: Jennifer L. Long, M.D., Ph.D. The human vocal folds are vibrating structures arranged as a specialized lamina propria (LP) and stratified squamous epithelium. In patients who have undergone oncologic resection of their vocal cords or patients with chronic vocal fold injury due to trauma or surgery, the scarring of the LP impairs vibration and phonation for which there is currently suboptimal therapy.

Cell-based outer vocal fold replacement (COVR) offers a potential treatment for severe vocal fold scarring or cancer reconstruction. Previous work in rabbits using human adipose-derived stem cells (ASC) in fibrin suggested that a hybrid structure emerged by 2 months, containing both implanted and host cells. This project uses immunocytochemistry to better define the phenotypic fate of implanted cells and features of the extracellular environment. New Zealand white rabbits underwent COVR implantation or vocal fold resection and were allowed to heal for 2 months. Larynges were harvested and immunocytochemistry performed. Cellular targets included Human Leukocyte Antigen (HLA), CD31, E-cadherin, and Smooth Muscle Actin (SMA). Extracellular targets included laminin and decorin. HLA was present in all implanted sections and was used to identify human cells. In adjacent sections, HLA-positive cells were identified expressing E-cadherin, SMA, or CD31. These 3 markers were also present in injured vocal folds not receiving COVR. Decorin and laminin were also present. From this, we conclude that implanted human ASC persist and express markers of epithelial, mesenchymal, and endothelial phenotypes after COVR in rabbits. Host cells also infiltrate the structure, producing a hybrid host-graft vocal fold. Mature extracellular matrix also develops. This information provides greater insight that can aid us in understanding the mechanism of wound healing and vocal fold recovery due to COVR implantation.

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Defining metabolic flexibility in hair follicle stem cell induced squamous cell carcinoma Carlos Galvan BSCRC - Rose Hills Foundation Predoctoral Trainee Lab PI: William Lowry, Ph.D. Among the numerous changes associated with the transformation to cancer, cellular metabolism is one of the first discovered and most prominent. However, despite the knowledge that nearly every cancer is associated with the strong upregulation of various metabolic pathways, there has yet to be much clinical progress on the treatment of cancer by targeting a single metabolic enzyme directly. We previously showed that inhibition of glycolysis through lactate dehydrogenase (LDHA) deletion in cancer cells of origin had no effect on the initiation or progression of cutaneous squamous cell carcinoma, suggesting that these cancers are metabolically flexible enough to produce the necessary metabolites required for sustained growth in the absence of glycolysis. Here we focused on glutaminolysis, another metabolic pathway frequently implicated as important for tumorigenesis in correlative studies. We genetically blocked glutaminolysis through glutaminase (GLS) deletion in cancer cells of origin, and found that this had little effect on tumorigenesis, similar to what we previously showed for blocking glycolysis. Tumors with genetic deletion of glutaminolysis instead upregulated lactate consumption and utilization for the TCA cycle, providing further evidence of metabolic flexibility. We also found that the metabolic flexibility observed upon inhibition of glycolysis or glutaminolysis is due to post-transcriptional changes in the levels of membrane lactate and glutamine transporters. To define the limits of metabolic flexibility in cancer initiating hair follicle stem cells, we genetically blocked both glycolysis and glutaminolysis simultaneously and found that frank carcinoma was not compatible with abrogation of both of these carbon utilization pathways. These data point towards metabolic flexibility mediated by regulation of nutrient consumption, and suggest that treatment of cancer through metabolic manipulation will require multiple interventions on distinct pathways.

Unlocking the Potential of UK5099, Addressing Aging Related Tissue Decline and Cancer Susceptibility.

Laura Carmona UCLA - CSUN CIRM Bridges Trainee Lab PI: William Lowry, Ph.D. Hair follicle stem cells (HFSCs) are crucial for hair regeneration, wound healing, and skin homeostasis. Studies have demonstrated that altering the metabolism of HFSCs influences their activation. Specifically, the use of UK5099, a small molecule inhibitor targeting the mitochondrial pyruvate carrier, increases lactate dehydrogenase (Ldh) activity to activate HFSCs and accelerate hair growth in young mice. Interestingly, UK5099 has the same effect in aged mice. Although during aging there is a decline in tissue function and regenerative capacity, UK5099 has the ability to enhance hair regeneration. Preliminary studies have also revealed aged mice treated with UK5099 significantly decrease the number of tumors formed with age. These findings propose the rejuvenation of other tissues during aging and the prevention of age related tumorigenesis. In this study, we aim to explore the anti-aging and anti-cancer effects of UK5099 in multiple tissues and overall physiology. Mice will be treated with chow containing UK5099 or Vehicle (control) to quantify age related phenotypes such as tumors and problems with eyes, skin, and body condition.

We will also harvest tissues that are known to change with age including skin, muscle, fat, and liver to characterize aging in each condition. Aging and cancer has become a significant public health issue due to increasing disease susceptibility, diminishing quality of life and rising health care costs, making strategies to combat these diseases of foremost importance

Smooth muscle cell (SMC) Piezo1 supports intestinal epithelial growth and adaptation in a murine model Ivett Perez Co-Presenters: Allison Flores, Nicole Kushnir, Ariana Infante, Margaret Kim, Evelyn Arambula, Elmira Tokhtaeva, RS Solorzano-Vargas, Geoanna Bautista, Martín G. Martín UCLA - CSUN CIRM Bridges Trainee Lab PI: Martín Martín, M.D. Piezo-1/2 are mechanosensitive channels responsive to stretch and lead to the release of Ca++. Enteric smooth muscle cell (SMC) contraction results from Ca++ influx.

Evidence suggests the role of SMCs in maintaining the intestinal stem cell (ISC) niche essential for epithelial growth and regeneration. We hypothesized that loss of Piezo1 in the SMCs will lead to altered epithelial growth and differentiation. To assess Piezo1's role in SMCs in mediating intestinal growth, we used a Piezo1/mTmG/Myh11-ERT2/Cre-LoxP model, generating a model with Piezo1 control (Piezo1WT) and knockout (Piezo1ΔSMC) in the SMCs of the gut muscularis.

Tamoxifen was given to 6-week-old mice and assessed 10 and 21 days later. In Piezo1ΔSMC mice, villus and crypt length were longer than in Piezo1WT mice. The populations of ISC (Olfm4), goblet (PAS), and Tuft (Dclk1) cells were more abundant in Piezo1ΔSMC mice. Paneth (Lys), EEC (Chga), and ECC (5-HT) populations of cells were significantly depleted in Piezo1ΔSMC mice compared to Piezo1WT. Ki67 confirmed marked proliferation and a decline in crypt-based β-catenin nuclearization consistent with non-canonical Wnt signaling in Piezo1ΔSMC verses to Piezo1WT mice. CC3 staining found that epithelial cell apoptosis was more abundant along the crypt-villus axis in Piezo1WT. In Myh11+ peri-cryptal cells, Piezo-1/2 staining is absent in Piezo1WT and Piezo1ΔSMC mice. Piezo1+ clusters on the basolateral membrane of the epithelium in the crypt base were larger in Piezo1WT. At the same time, both groups had a few cells within the epithelium with an abundance of cytoplasmic Piezo1. In contrast, Piezo2 distribution differed significantly, with Piezo1WT mice having more abundant cytoplasmic staining, whereas Piezo1ΔSMC mice had larger Piezo2 clusters on the basolateral membrane of the epithelium. In summary, Piezo1 in the SMCs maintains the intestinal epithelial architecture and cellular components, including the ISCs and Piezo-1/2 in crypt base cells.

Novel multisystemic disorder associated with mutations of a non-clinical gene, CEP192 Adriana Infante Co-Presenters: Sergio Solorzano Lab PI: Martín Martín, M.D. Ciliopathies compromise a group of systemic disorders caused by monogenetic mutations resulting in abnormalities of cilia structure and function in all cells, including stem cells. Cilia, a microtubule cell structure originating from the centrosome, is codified by highly conserved genes and mediates multiple intra and extracellular signals, including proliferation, polarity, and tissue maintenance. The centrosomal protein of 192 (CEP192) is required for mitotic centrosome maturation and bipolar spindle assembly and has a critical role in pericentriolar material recruitment, centrosome maturation, centriole duplication, and ciliogenesis. Whole exome and Sanger sequencing were performed on two siblings with an undiagnosed multisystemic disorder and detected a compound heterozygous mutation in the CEP192 gene. We identified seven additional probands and collected comprehensive genotype and phenotype data, with additional functional analysis done on the CEP192 variants to examine cell cycle function. We hypothesize that a mutation in CEP192 negatively affects cell structure and division, resulting in irregularities in the cell cycle. Live cell imaging was conducted on synchronized HeLa Kyoto cells containing endogenous H2B-mCherry and alpha-tubulin mEGFP labels that were transfected with wild-type or mutant forms of CEP192. Additionally, siRNA was used to knock down endogenous CEP192. These functional studies revealed the formation of disorganized, multipolar, and abnormally shaped bipolar cellular phenotypes during mitosis in HeLa cells transfected with mutant forms of CEP192 when compared to controls. Thus, the CEP192 protein is crucial in cell division, impacting the rate of mitosis and cellular morphology. This research has brought insight into the clinical and cellular phenotypic changes caused by the bi-allelic mutation of CEP192 and the relevance of this gene in cell division, marking biallelic mutations of CEP192 as a novel multisystemic clinical disorder.

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Characterization and targeting of uterine carcinosarcomas, an aggressive subtype of endometrial cancer Christopher Ochoa BSCRC - Rose Hills Foundation Predoctoral Trainee Lab PI: Sanaz Memarzadeh, M.D., Ph.D. Endometrial cancer (EC) poses a significant health concern as it is the most common gynecologic disease affecting women today. Despite efforts to better understand and treat EC, there has been an increasing trend in the number of cases diagnosed each year. One of the most aggressive subtypes of EC are uterine carcinosarcomas (UCS). Although UCS cases are rare, patients diagnosed with UCS are often resistant to conventional therapies and have poor treatment outcomes. UCS cases are unique and particularly aggressive partially because they are comprised of not only an epithelial malignant component, but also a stromal one. Unfortunately, not much is known about the origin of the cellular duality seen in UCS and therapies specifically targeting this disease are lacking. As such, our goal is to provide a comprehensive understanding of the tumor cells and the immune landscape within UCS. To accomplish this, we have profiled a cohort of EC patient samples across multiple platforms, including, cytometry by time of flight (CyTOF), flow cytometry, and single-cell RNA sequencing to better characterize EC and UCS from multiple angles. Additionally, we’ve begun efforts to find effective targeting strategies using UCS patient samples in vitro and are actively working to generate multiple patient-derived xenograft models of UCS for in vivo analyses. Our current analysis shows that UCS samples have a decreased presence of tumor-infiltrating lymphocytes, a higher expression of stem cell markers (e.g., SOX2), and are susceptible to targeting via immune cell therapeutic strategies in conjunction with anti-HER2 monoclonal antibody treatment.

These preliminary results reveal the stemness of UCS tumors and potential targeting strategies by utilizing a dual-therapeutic method by leveraging the immune system.

Strategies to target recurrent ovarian cancers Gabriella DiBernardo Lab PI: Sanaz Memarzadeh, M.D., Ph.D. Ovarian cancers (OC) constitute ~5% of cancer deaths in women, yet there have been few treatment advancements for women afflicted by this disease. Despite adherence to standard of care for treatment patients often experience disease recurrence and drug resistance. Investigating mechanisms fueling recurrence and drug resistance is imperative to elicit more effective treatments and improve patient outcomes. To address this, we have taken advantage of using primary patient tissues, a critical resource in OC research. With IRB approval, our lab has created a repository of primary tissues collected from OC patients of varying diagnoses, disease states, and platinum sensitivities. To expand on available materials, we have created and validated physiologically relevant patient derived xenograft models that preserve the original landscape of the primary tumor.

Our lab, in collaboration with many UCLA investigators, has taken a multifaceted approach to fully harness the information that can be uncovered within these primary tissues. Our team has utilized primary recurrent and longitudinally collected high grade serous ovarian cancer (HGSOC) samples to profile subpopulations of therapy resistant cells and their interaction with the immunosuppressive microenvironment. Flow cytometry and CyTOF analysis revealed recurrent samples had increased SOX2, Oct3/4, and Nanog indicating enriched stemness. HGSOC samples with enriched neuroendocrine-like features in HGSOC samples providing a rationale for utilizing therapies from other NE-enriched cancers for OC. Several novel cell based immune therapies are being utilized to target these aggressive tumors such as iNKT cells, supercharged NK cells, and exploration of unique t-cell receptors to target antigens expressed in OC (WT1). Our ultimate goal is to make impactful discoveries that will lead to more efficacious treatment options and improved prognosis for women with gynecologic cancers.

MYCT1 MODERATES ENVIRONMENTAL SENSING VIA ENDOCYTOSIS TO PRESERVE HUMAN HSC SELFRENEWAL Julia Aguade-Gorgorio Lab PI: Hanna Mikkola, M.D., Ph.D. The different types of blood cells in the body arise from a few specific cells called hematopoietic stem cells (HSC). HSCs are important therapeutically, since they can be transplanted from healthy individuals to regenerate the blood of patients with cancer or inherited blood diseases. To produce blood, HSCs need to accurately sense their environment and decide between dormancy, producing more HSCs, or producing mature blood cells. However, the balance between these options becomes altered when cells are cultured outside the body. Growing human HSCs in the laboratory for therapeutic means is of utmost difficulty.

To overcome this challenge, we identified genes responsible for the function of human HSCs that are not properly active in culture. We discovered that MYCT1 gene becomes inactivated during culture, and causes HSCs to lose their ability to multiply. Additionally, HSCs become hyper-sensitive to the signals from their environment and have exaggerated responses. Importantly, we have also discovered that rescuing MYCT1 activity in HSCs can restore some of the defects that occur during culture and improve the capacity of HSCs to produce blood after transplantation. These studies may lead to broadening the therapeutic use of HSCs in the treatment of blood cancers and other blood diseases.

Spontaneous Emergence of Tissue-scale Chirality Decoupled from Left-Right Signaling in the Heart Naofumi Kawahira Lab PI: Atsushi (Austin) Nakano, M.D., Ph.D. The proper functioning of the heart is closely linked to its 3D shape and tissue organization. Beyond the asymmetric shape of the organ, tissue-scale chiral structures are observed at the apex (vortex cordis) and within the heart walls, contributing to the contraction motion. Despite the significance of these chiral structures, the mechanisms underlying their emergence and their interrelation remain largely unexplored.

Establishing the origins of chirality involves exploring several possibilities: 1) under the influence of Left-Right signals, 2) depending on morphological constraints of organs, or 3) generated autonomously by cells. Based on the insights from autopsy studies of heterotaxy patients, we hypothesize that tissue-scale chiral structures develop autonomously at the cellular level, independent of left-right signaling or macroscopic organ shape. To test this hypothesis, we utilized a heterotaxy mouse model.

Three-dimensional imaging of the mouse embryonic heart revealed that specific fiber orientations emerge between E12.5 and E15.5. A rapid increase in twisting angle coincided with the thickening of the heart walls, suggesting a narrow time window for the establishment of the global fiber pattern during embryonic development. Our data also indicate that helical chirality is maintained in heterotaxy mice, irrespective of the inverted organ shape. These results support our hypothesis that tissue-scale chirality originates cell-autonomously, rather than being influenced by extracellular cues, including left-right signaling or macroscopic boundary conditions.

Our findings suggest that distinct mechanisms govern tissue-scale and organ-scale cardiac morphogenesis. At the tissue-scale, cardiac fiber patterns emerge cellautonomously. In contrast, organ-scale morphogenesis is guided by left-right signals. This study unveiled the intricate layers of organ morphogenesis, contributing to future research in this field.

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Nucleotide imbalance causes impaired cardiomyocyte maturation and mitochondrial dysfunction via AMPK in the embryonic heart of diabetic pregnancy Haruko Nakano Co-Presenters: Cesar Perez, Richard Law, Naofumi Kawahira, Jennifer Thompson, Jun Park, Heather Christofk, Atsushi Nakano Lab PI: Atsushi (Austin) Nakano, M.D., Ph.D. Glucose plays a central role in the metabolic environment of the fetal heart, and maternal hyperglycemia is the most common non-genetic contributor to heart defects.

While previous research using in vitro methods has revealed how high glucose impedes heart muscle cell maturation, the in vivo effects on organ development remain largely unexplored. In this study, we used a multi-omics approach: One is single-cell RNA sequencing of fetal mouse hearts from normal and diabetic pregnancies. The other is metabolomics, the first comprehensive metabolic profiling of fetal organs, which revealed a critical signaling mechanism that disrupts cardiomyocyte maturation in diabetic pregnancies.

The transcriptomic study revealed that cardiomyocyte maturation is delayed in diabetic pregnancy compared to normal pregnancy. In addition, the heart muscle cells contain mitochondria with underdeveloped ultrastructure.

The metabolomics study used over 500 mouse embryonic tissues from four organs at four stages of normal and diabetic pregnancy. Metabolomics data revealed increased glycolysis as expected, but unexpectedly limited coupling to the TCA cycle and imbalanced nucleotide synthesis, which is consistent with our previous findings.

Our investigation of AMPK signaling, a pathway critical for sensing adenine nucleoside balance and maintaining cellular homeostasis, revealed decreased phosphorylation of the AMPKɑ subunit in the fetal heart from diabetic pregnancies. Further experiments showed that forced activation of AMPK could enhance cardiomyocyte maturation even under high glucose conditions.

This suggests a critical role for AMPK signaling in the impaired maturation of the fetal heart under hyperglycemic conditions.

In conclusion, our findings suggest that an unbalanced increase in nucleotides, coupled with AMPK suppression, is an important pathology contributing to fetal heart immaturity in diabetic pregnancy.

Human Formin FHOD3 is Formin’ Sarcomeres Primarily by Elongation Dylan Valencia Lab PI: Atsushi (Austin) Nakano, M.D., Ph.D. Contractility depends on accurately controlled assembly of the actin cytoskeleton. Formins are a group of actin assembly proteins. One family of formins known as Formin HOmology Domain-containing protein (FHOD) is critical to the formation of the fundamental contractile unit, the sarcomere, in cardiomyocytes. Mutations in the FHOD3 gene are estimated to account for 1-2% of hypertrophic cardiomyopathy (HCM). Despite our knowledge of FHOD3L’s importance in vivo, its biochemical and cellular activities remain poorly understood. How does FHOD3L’s ability to initiate formation of filaments (nucleation) and control their length (elongation) contribute to the structural integrity of the sarcomere in cardiomyocytes? We are correlating in vitro biochemical analysis with cellular assays, using neonatal rat cardiomyocytes (NRCs) as a model system. We identified a mutation in the FH2 domain that reduces nucleation strength by 70%, but maintains elongation ability compared to wild-type.

We also determined that mutation of the prolines in the FH1 domain diminishes elongation while maintaining nucleation activity. In NRCs, FHOD3L knockdown results in disassembly of the sarcomere structure and reduced contractions. Sarcomeres and contractility are rescued by wild-type FHOD3L and the nucleation-hindering mutant, but not the elongation-hindering mutant, indicating that FHOD3L’s elongation ability is necessary for sarcomere formation whereas decreased nucleation is tolerated. We found that an HCM-linked FHOD3L mutant within a regulatory domain is degraded. The results of this work will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere integrity and provide insight into the mechanisms by which formins build specific structures in vivo.

Modeling the Emergence of Neural Network Dysfunction in Rett Syndrome Using Mosaic Organoids Natella Baliaouri BSCRC - CIRM 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 Xchromosome 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.

Organoid production and characterization of Dup15q hIPSC’s via Immunohistochemistry Negien Shalmani UCLA - CSUN CIRM Bridges Trainee Lab PI: Bennett Novitch, Ph.D. Cerebral brain organoids are an emerging platform for studying neurodevelopmental disorders. Human derived brain organoids can be used as a model to study Dup15q syndrome, a genetic anomaly on chromosome 15q that exhibits duplications on the maternal allele. This genetic alteration has been implicated with developmental delays in motor and language skills, epilepsy, difficulties in daily living skills, and autism with a range of differing addition symptoms such as central hypotonia. The Novitch lab will investigate the role of Dup15q on cells and circuits in different regions of the brain by generating cortical, ganglionic eminence, and hippocampal organoids effectively resembling in vivo conditions. To study this role four distinct cell lines will be used: the Dup 1-8 cell line is an isodicentric chromosomal abnormality resulting in an extra chromosome with duplications on regions 15q11-q13, the Dup 1-8 corr. cell line which is a CRISPR-corrected wild-type Dup 1-8 cell line, the DupCB/Rx68i cell line is another isodicentric chromosomal abnormality derived from a different patient compared to the Dup 1-8 line, and the SCC115 line which has triplication of the gene on the maternal chromosome. Immunohistochemical analyses with a range of developmental markers, mutant organoid size, laminar structure, and cellular composition will be performed on organoids of different ages to verify structural characteristics and correct protein expression compared to the neurotypical control. Single-cell transcriptomics of different condition organoids will help us identify changes in gene expression and signaling pathways between the control and mutant organoid samples. We also plan to conduct Ca2+ imaging to study the activity of networks in more mature organoids (days 100-120), followed by extracellular recordings for a deeper analysis. Using these organoids as a model will allow us to properly recapitulate the neural profile of Dup15q through early network events. 20

Role of PRC2 in the Erosion of the Inactive X Chromosome in Human Pluripotent Stem Cells Sophia Peavy UCLA - CIRM COMPASS Scholar Lab PI: Kathrin Plath, Ph.D. Reprogramming of female human fibroblasts to induced pluripotent stem cells (hiPSCs) ushered in a new area in stem cell research, has enabled new disease studies using regenerative approaches due to the derivation of patient specific pluripotent stem cell lines. However, all female hiPSCs display a culture-induced erosion of X chromosome inactivation (XCI). In this process, Xist RNA which is responsible for silencing the inactive X chromosome (Xi) is lost and this chromosome re-exhibits gene expression resulting in double dosage. The cause of Xi erosion and XIST loss is still unknown. The Polycomb Repressive Complex 2 (PRC2), is responsible for trimethylating histone H3 at lysine (K) 27, which is recruited to the X chromosome in hiPSCs but not somatic cells, albeit H3K27me3 accumulates on the Xi in both cells.

Since PRC2 and H3K27me3 facilitate silencing of the Xi via Xist RNA, we hypothesize that changes in gene expression of PRC2 contributes to the Xi-erosion process.

We hypothesize that chemical inhibition of PRC2 will consequently delay erosion. To test this hypothesis, we cultured hiPSCS exhibiting minimal erosion with two PRC2 inhibitors and one PRC1 inhibitor which prevents accumulation of PRC2 to the Xi. We quantified rates of erosion via RNA FISH to observe Xist loss and immunofluorescence to quantify other proteins associated with XCI. We did not find a significant decrease in erosion rates as a result of the PRC2 inhibitors. While there is evidence of a slight decrease in the rates of erosion, we cannot confidently conclude that the inhibition of PRC2 leads to delayed erosion of female hiPSCs. Although this evidence is inconclusive, it provides insight into the role of PRC2 and the erosion process. Moving forward we plan to repeat and expand these experiments to better understand the role of PRC2 in relation to erosion and additionally quantify the effects of other chemical inhibitors relating to XCI to analyze the consequences on erosion.

Deciphering the Role of Dhx9 in the Formation of Nuclear Compartments and Gene Expression Clara Cano Co-Presenters: Iris Dror, Yu Sun, Andrea Garcia Angulo, Alexander Lee, Kathrin Plath BSCRC - CIRM Predoctoral Trainee Lab PI: Kathrin Plath, Ph.D. It has long been proposed that RNA plays important regulatory and structural roles in the nucleus as individual RNAs are known to establish and demarcate specific nuclear compartments.1 A classic example is the inactive X-chromosome (Xi) compartment in female mammalian cells. Transcripts of the long non-coding RNA (lncRNA) Xist seed dynamic supramolecular complexes (SMACs) through RNA-protein and transient protein-protein interactions, which yields high concentrations of silencing proteins in the Xi compartment.2 Despite the importance of RNAs in forming nuclear compartments, it is still unclear how the assembly and disassembly of these RNA-based compartments is regulated. Interestingly, the RNA helicase, Dhx9, which is known to remodel RNA and ribonucleoprotein complexes in an energydependent manner, biochemically purifies with Xist.3 Using the degron tag (dTAG), we acutely reduced Dhx9 levels in mouse embryonic stem cells (ESCs) to understand the role of Dhx9 in the X-inactivation process as well as its global nuclear role in ESCs. We show that loss of Dhx9 disrupts Xist enrichment and the accumulation of the repressive H3K27me3 mark on the Xi. Surprisingly, we observed increased cell death upon prolonged depletion of Dhx9. Consistent with this finding, transcriptomics studies show that cell cycle and DNA repair genes are downregulated immediately upon Dhx9 depletion. Inspecting the RNA-sed data, we found that Dhx9 protects transcripts from Adenosine-to-Inosine editing, particularly within 3’ untranslated regions. Our work suggests a critical function of Dhx9 in the regulation of Xist dynamics, facultative heterochromatin, the cell cycle and RNA processing in embryonic stem cells.

Vascularizing Innervated Skeletal Muscle in vitro Gabriella Cox UCLA - CSUN CIRM Bridges Trainee Lab PI: April D. Pyle, Ph.D. During development, signals such as Wnt and bone morphogenetic proteins (BMP) secreted by the neural tube, the lateral mesoderm and surrounding tissues allow the formation of the somites, which in turn develop the myotome. Then, through vasculogenesis, endothelial cells (ECs) enhance the proliferation of skeletal muscle progenitor cells (SMPCs) during myogenesis while the innervation of muscle fibers play a role in their maturation. It has been shown that the angiogenesis and the interaction between ECs and the satellite cells (SCs) play a role in the formation of a mature SC-niche. Over the last decade, creation of efficient cell-based therapies that aim to regenerate skeletal muscle have been impaired by the quality and the maturity of the SMPCs. Here, we hypothesized that adding ECs to a co-culture of muscle fibers innervated by motor neurons could improve the proliferation of SMPCs, allow the formation of a satellite cell niche-like, and eventually improve the maturation of the SMPCs in vitro and in vivo. Using a protocol optimized in our laboratory, we are able to co-differentiate human embryonic stem cells (hESCs) into myofibers innervated by motor neurons. We have developed approaches to enable the production of endothelial cells and the formation of a large capillary network by altering the concentrations of small molecules and growth factors to form a co-culture from hESCs. These results lay the foundation to explore the role of the ECs in modulating SMPC proliferation and maturation.

The Role of Lipid Droplet Dynamics in Muscle Stem Cells Differentiation and Fusion Yutzil Herrera-Rebolledo UCLA - CIRM COMPASS Scholar Lab PI: Thomas A. Rando, M.D., Ph.D. Lipid Droplets (LDs) are organelles present in the cytoplasm of various cell types. Their primary role involves storing free fatty acids (FFA) for subsequent use in metabolic pathways, as a source of energy, and for membrane synthesis. LDs play a vital role in regulating harmful polar lipids within cells, serving as a defense mechanism against lipotoxicity. The mechanisms by which age affects the functional capacities of muscle stem cells, called satellite cells (SCs), are not fully understood.

Our data show that levels of triglycerides (TAGs) and LDs are decreased in SCs isolated from old mice when compared to young. We hypothesize that changes in the functional dynamics of LDs are important contributors to the loss of function of SCs in aged tissue. 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 using specific inhibitors: Atglistatin (ATGLin) to increase the number of LDs in cells isolated from old mice, and inhibitors A922500 and PF-06424439 (DGATin) to inhibit LD formation in young cells. We found that 50 uM of ATGLin completely blocked the differentiation process, and 25 uM significantly decreased myotube fusion and growth. Incubation of SCs with DGATin resulted in a significant increase of myosin heavy chain (MHC) positive area when compared to the control. DGATin also reversed the negative effects of ATGLin. The data indicate that LD dynamics plays a vital role in SC differentiation, as well as myotube fusion and growth. The observed results likely arise from augmented cellular demand for FFA during periods of heightened growth. The exact mechanism underlying this relationship and its potential implications for the diminished regenerative capacity of aging muscles require further research. This data might help improve therapies targeting those affected by age-related conditions such as reduced muscle strength and regeneration.

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How to control the fate of stem cells after transplantation? Bioinstructive Scaffolds Enhance Stem Cell Engraftment for Functional Tissue Regeneration after Severe Trauma Di Wu Lab PI: Thomas A. Rando, M.D., Ph.D. Stem cell therapy is a promising approach for tissue regeneration after traumatic injury, yet current applications are limited by inadequate control over the fate of stem cells after transplantation. Here we introduce a bioinstructive material engineered to replicate the native temporal dynamics of growth factors across different stages of muscle regeneration. This biomaterial orchestrated the behavior of transplanted muscle stem cells (MuSCs), guiding them through proliferation and differentiation stages by programmable release of basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-1), respectively. When applied to a volumetric muscle loss (VML) defect, the bioconstruct significantly enhanced myofiber formation, alongside notable improvements in angiogenesis, innervation, and muscle function restoration. In addition, this bioconstruct demonstrated remarkable efficacy in promoting functional muscle formation when employing aged or human MuSCs, highlighting the translation potential of this approach. These results represent a promising strategy for MuSC delivery as VML therapy and a versatile platform to direct the fate of stem cells via orchestrated growth factor release.

Atglistatin maintains skeletal muscle stem cells in a quiescent state in vitro independently of lipid droplets formation Agnieszka Czyzowska-Froemling Lab PI: Thomas A. Rando, M.D., Ph.D. Muscle stem cells (MuSCs) play a crucial role in muscle regeneration. In response to injury or exercise, they quickly activate, enter the cell cycle, proliferate, differentiate, and fuse to repair damaged myofibers. A subset of activated MuSCs can undergo self-renewal, ensuring the maintenance of a MuSCs reservoir for future regeneration.

This regenerative capacity of muscles may decline in some pathological conditions and with age. Even though stem cell transplantation holds promise for those patients, isolating MuSCs for therapeutic purposes is challenging, as they rapidly exit quiescence, diminishing their transplantation potency. In this study, we found that 25 μM of Atglistatin (ATGLin), an Adipose Triglyceride Lipase inhibitor, significantly reduced EdU incorporation compared to the control (58.1% vs. 34.6%, p < 0.001) and improved MuSC survival in vitro (16.6% apoptotic cells vs. 12.6%, p=0.570). 50 μM of ATGLin entirely prevented cells from exiting quiescence, increased survival by 16% (p=0.002), and was able to induce a return to quiescence of cycling MuSCs. ATGLin-treated MuSCs were also much smaller than control cells, which is typical for a quiescent state. Moreover, release from ATGLin allowed the cells to activate similarly to the control. ATGLin is a well-known substance that causes accumulation of lipid droplets (LDs) within cells. To further investigate the mechanism by which ATGLin keeps cells quiescent, we used FFAs to promote LDs formation independently of ATGLin, and Diglyceride Acyltransferases inhibitors to prevent the LDs formation. We found that the effect of ATGLin is independent of LDs generation. More research is needed to reveal the exact mechanism, the role of lipid metabolism in the activation of MuSCs, and in vivo application of ATGLin-induced quiescence. The ability of ATGLin to prevent MuSC activation and induce a return to quiescence in cycling MuSCs constitutes a potentially valuable and effective tool for MuSCs therapeutics.

Targeting IGF2BP3 enhances anti-leukemic effects of menin-MLL inhibition in MLL-AF4 leukemia Tasha Lin BSCRC - CIRM Clinical Fellow Lab PI: Dinesh S. Rao, M.D., Ph.D. Despite the development and success of therapies for leukemias, there are still patients with particularly challenging subtypes of leukemia, including leukemia with MLLAF4, and who experience poor outcomes of early relapse and death. Our work aims to understand the biology of MLL-AF4 leukemia and leverage that understanding into novel therapies. Our group has previously shown that the RNA binding protein, insulin-like growth factor 2 binding protein 3 (IGF2BP3), is critical to supporting MLLAF4 leukemia and represents a promising therapeutic target. RNA binding proteins are emerging as a novel class of therapeutic targets in cancer and have unexplored potential to be combined with existing therapies. Here, we combined the targeting of IGF2BP3 with commercially available menin-MLL inhibitors and studied their combined effects on suppressing leukemia in the lab and in mice. Targeting IGF2BP3 sensitized MLL-AF4 leukemia to the negative effects of menin-MLL inhibitors on cell growth and leukemic initiating cells, a subset of leukemia cells that are often treatment-resistant, and therefore, thought to be responsible for relapsed or refractory disease. We found that inhibition of IGF2BP3 and menin-MLL helped the leukemia cells differentiate into normal white blood cells, a process that is thought to help control and kill leukemia. Our work has shown that IGF2BP3 is a potent therapeutic target and demonstrates a new combinatorial approach for targeting leukemia.

Optimizing iPSC-Derived RPE Delivery to the Subretinal Space: Innovations in Robotic Surgical Systems and High-Precision Imaging Methods Aya Barzelay BSCRC - CIRM Clinical Fellow Lab PI: Steven Schwartz, M.D. The application of stem cell treatment for patients experiencing blindness due to retinal degeneration holds significant therapeutic promise but presents substantial challenges. This study aims to optimize the delivery of iPSC-derived stem cells, addressing the complexity of transferring cells through microneedles as small as 36 gauge and achieving accurate delivery via microsurgery into a subretinal space measuring only a few microns.

We evaluated the viability, survivability, and functionality of iPSC-derived RPE cells using the Constellation surgical machine and a micro delivery system. Additionally, we developed a robotic arm to perform subretinal injections, ensuring accuracy and precision in the delivery process.

Passing through the micro delivery system, comprised of 36-gauge microneedle, the cells demonstrated high viability and functionality up to 72 hours post-delivery. An image processing algorithm was developed to detect the subretinal space, guiding the robotic arm through an automated trajectory we established, resulting in successful delivery to the subretinal space.

Our iPSC-derived RPE cells exhibited robust viability and functionality despite passing through the micro delivery system. The developed robotic arm accurately detected and injected into the subretinal space, significantly enhancing the delivery of stem cells and thereby maximizing their therapeutic potential.

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Generating pluripotent stem cell lines with defined MHC expression for TCR discovery Nicki Calvert UCLA - CSUN CIRM Bridges Trainee Lab PI: Christopher S. Seet, M.D., Ph.D. The study explores the augmentation of T cell antigen presentation through the manipulation of MHC gene expression in a B2M-knockout human pluripotent stem cell (PSC) line and Artificial Thymic Organoids (ATOs). We will test the hypothesis that modification of antigen presentation during T cell development from PSCs will positively select for T cells expressing T cell receptors (TCRs) specific to the modified MHC or peptide-MHC (pMHC). The overall goal is to develop an MHC-modular system for the rapid discovery of antigen-specific TCRS for cancer immunotherapy.

The methodology involves CRISPR Cas9-mediated B2M knockout in the H1 PSC line, cloning and lentiviral transduction of pMHC gene constructs, and subsequent assays to assess T cell antigen specificity. The process described here includes the establishment of a homogeneous population of B2M-knockout PSCs through single cell cloning, with validation confirming the success of the knockout. Flow cytometry is employed to characterize antigen expression. The results demonstrate the efficiency of CRISPR/Cas9 B2M knockout cells, which sets the foundation for further investigations into the specificity and functionality of T cells in the ATO system.

Artificial Thymic Organoids (ATOs), utilizing RB27 media and cytokines (SCF, FLT3, IL7), will be used as a system to mimic thymic T cell development. The aim is to generate diverse T cells specifically restricted to the introduced pMHC allele. Future goals include validating transduced pMHC molecule expression in the B2M knockout cell line and integrating transduced cells into ATO cultures. Specific TCR assessment for both specificity and functionality will be conducted using pMHC tetramers. The research provides the potential of adjusting MHC expression for influencing T cell development, offering implications for personalized medicine and immunotherapies.

Identifying Muscle Stem Cell Tropic AAV Variants through Directed Evolution.

Harry Chen Lab PI: Melissa Spencer, Ph.D. Gene therapy holds great promise in treating a large number of previously untreatable diseases. Delivery, however, remains one of the most challenging hurdles to overcome. Recombinant Adeno-Associated Viruses (rAAVs) have become a powerful delivery platform in gene therapy due to its safety and efficacy. Nonetheless, certain cell and tissue types elude targeting with rAAVs. Here, we have established a strategy to selectively enrich for muscle stem cell (MuSC) tropic rAAV variants through directed evolution of a 7 amino acid sequence between amino acids 589A and 590 Q in surface region VIII (VR-VIII) of the AAV9. Through this, we have identified 8 MuSC targeting rAAVs. Preliminary results have demonstrated that these variants significantly outperform wildtype AAV9 in targeting quiescent MuSCs. While further characterization must be done, the targeting of MuSCs may open the door to gene therapies of diseases like Duchenne Muscular Dystrophy and other forms of muscular dystrophy.

Metabolic Shifts in Glutamine-Derived Nitrogen Contributes to the Acquisition of the Earliest Cell Fates Mary T. Doan BSCRC - CIRM Predoctoral Trainee Lab PI: Michael Teitell, M.D., Ph.D. Shifts in metabolite levels and fluxes help determine whether human pluripotent stem cells (hPSCs) self-renew or undergo differentiation. We previously identified that upon exit from pluripotency, each lineage handles exogenous glutamine (Gln) differently to support their requirements for fate acquisition. Glutaminase (encoded by GLS1 and GLS2), converts Gln into glutamate, which can be processed for tricarboxylic acid (TCA) cycle anaplerosis. All lineage types repressed GLS2 expression, whereas GLS1 is upregulated only in mesoderm and endoderm and these lineage types perished in GLS1-inhibited conditions. This lineage-specific GLS1 sensitivity suggests mesoderm and endoderm could favor exogenous Gln processing for anaplerotic reactions to satisfy fate requirements, whereas ectoderm commitment is less dependent upon this route of Gln usage. How ectoderm is using exogenous Gln during its identity programming and the relation between the fate of Gln and dynamic hPSC cell state transitions has yet been fully elucidated. Here, we show that the metabolic regulation of Gln nitrogen (Gln-N), in the form of amine and amide groups, is distinctly altered during the progression of hPSC fate transitions. Gln amine fluxes revealed ectoderm uniquely skews incorporation of Gln-N away from TCA cycle anaplerosis. Instead, ectoderm shifts Gln-N towards nucleotide biosynthesis, demonstrated by high Gln amide flux into nucleotide precursors during early differentiation which resulted in a significantly higher abundance of nucleotides and their derivatives at the end of differentiation to possibly support the high proliferation rate observed in ectoderm. These data provide insight into how differential Gln-N disposition and utilization during hPSC differentiation may contribute to different hPSC fate transitions, with modulation of Gln-N being a potential approach to produce higher quality hPSC derivatives for studying early development and eventual therapeutic usage.

Rewiring Nucleotide Metabolism to Support Pluripotent Stem Cell Fate Transitions in Early Development Michelle H. Zhang Lab PI: Michael Teitell, M.D., Ph.D. Successful embryogenesis requires temporal shifts in the use of nutrients that fuel anabolic and bioenergetic pathways to support distinct cell needs for specific fate acquisitions. One of the earliest decisions in embryonic development is modeled by a primed-state human pluripotent stem cell (hPSC) to self-renew or transition into ectoderm, endoderm, or mesoderm lineages. Each lineage has unique patterns of growth and proliferation, with an hPSC shift to ectoderm accompanied by higher growth rates and mass accumulation than transitions into the other two lineages. Nucleotide synthesis is known to support proliferation by providing substrates for DNA and RNA, yet the role of nucleotide metabolism in hPSC trilineage differentiation is not fully understood. To assess shifts in nucleotide metabolism during hPSC fate transitions, we directed hPSCs into specific lineage types under nutrient-balanced media conditions. RNA-seq, immunoblots, and metabolomics displayed upregulated de novo nucleotide synthesis enzyme expression and nucleotide levels in the shift from hPSC to ectoderm, but a decrease in these components for transitions to endoor mesoderm. Distinct to only ectoderm, media footprint analysis showed a high uptake of aspartate (Asp), an essential nitrogen donor in de novo nucleotide biosynthesis. Yet, hPSCs directed into ectoderm under exogenous Asp withdrawal did not impede cell viability or fate commitment, suggesting sufficient intracellular Asp levels satisfy the requirements to progress ectoderm fate acquisition. These data suggest that, unlike endo- or mesoderm, as hPSCs transition to ectoderm, there is elevated de novo nucleotide synthesis activity to support high proliferative needs as cells progress to ectoderm fate. Further probing this metabolic vulnerability can provide insight into how nucleotide metabolism can be targeted to understand hPSC fate transitions in early development and enhance hPSC differentiation protocols for biomedical applications.

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Rosiglitazone-dependent Remodeling of Translational Selectivity in Adipose Progenitors Mirian Krystel De Siqueira Lab PI: Claudio Villanueva, Ph.D. Adipose tissue constitutes a dynamic hub integral to energy homeostasis and metabolic regulation. Despite its remarkable ability to adapt and transform in response to various physiological and environmental stimuli, adipose tissue plasticity is impaired in obesity. The mechanisms responsible for the limited adaptability of adipose tissue in obesity have not been fully characterized. Here we have explored a novel layer of regulation, which involves the translatome modulation of the adipose stromal vascular fraction in response to obesity and an acute PPARg agonist treatment, rosiglitazone. First, scRNASeq analysis of obese inguinal and epididymal tissue provided a transcriptional profile atlas of the cellular remodeling from an obese to a lean-like state. Remarkably, in addition to the downregulation of inflammation-related transcripts, both epididymal and inguinal stromal fractions exhibited an upregulation of lipid-related metabolism and ribosomal transcripts. In particular, adipocyte progenitor and preadipocyte populations presented an enhanced ability to differentiate in adipocytes ex-vivo and an upregulation in the ribosome and, peptide chain elongation pathways. Such ribosomal remodeling was directly driven by PPARγ binding to the promoter of several small and large ribosome genes. Finally, we characterized the translation selectively in the stromal epididymal fraction, highlighting a buffering response and a fat-exclusive preferential translation after rosiglitazone treatment. Moreover, enhanced translation efficiency in rosiglitazone-elicited polysomes promoted the translation of transcripts containing G-rich sequences in their 5’ untranslated regions. These findings provide insights into how rosiglitazone remodels the stromal vascular fraction of the adipose tissue in a PPARγ-depended and independent manner. Furthermore, we have uncovered that translatome remodeling is a major mechanism to keep translation homeostasis and to maintain adipose tissue health.

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer transdifferentiation Chia-Chu Olga Chen BSCRC Predoctoral Trainee Lab PI: Owen Witte, M.D. Small cell neuroendocrine carcinoma is a rare and aggressive subtype seen in many advanced stem-like cancers. Typically, early-stage cancers (adenocarcinoma) develop into this highly malignant form through resistance to therapy. This complex process is known as small cell neuroendocrine differentiation and involves the alteration of cancer cell identities to evade various forms of treatment. To better understand this development in prostate cancer specifically, we developed a highly effective and reproducible model that allows us to drive human prostate cells into a small cell neuroendocrine state. By using the model, we discovered that tumor cells chose one or the other way to become neuroendocrine. These two pathways are associated with two proteins, ASCL1 and ASCL2, respectively. We found that not only prostate cancer, but also lung cancer, demonstrated the same pattern and pathology suggesting a pan-cancer significance. This study will provide more insights into how small cell neuroendocrine cancer develops and expand the possibility of future therapeutic targets.

Generation of off-the-shelf CAR-NKT cells for cancer immunotherapy with genetically engineered HSPCs and feeder-free differentiation culture Yanruide Li BSCRC - CIRM Postdoctoral Trainee Lab PI: Lili Yang, Ph.D. Current CAR-T cell therapy's clinical potential is hampered by its autologous nature that poses significant challenges in manufacturing, costs, and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSPCs into allogeneic NKT cells and their CAR-armed derivatives (AlloCAR-NKT cells). The AlloCAR-NKT cells are generated at high yield, purity, and robustness at the research scale used in our studies. These cells exhibit potent antitumor efficacy, showing effective tumor homing, expansion, and persistence in vivo. Impressively, AlloCAR-NKT cells can alter the tumor microenvironment by selectively depleting immunosuppressive TAMs and MDSCs, and can antagonize tumor immune evasion by deploying CAR/TCR/NKR tripletargeting mechanisms. AlloCAR-NKT cells also demonstrate an appealing safety profile with low GvHD and CRS risks, and exhibit a stable “hypoimmunogenic” phenotype attributed to epigenetic and signaling regulations. The reported technology presents a scalable strategy for diverse allogeneic CAR-NKT cell products, with potential for clinical translation and commercialization against various cancers.

MESOTHELIN CAR-ENGINEERED MUCOSAL ASSOCAITED INVARIANT T (MAIT) CELLS FOR CANCER IMMUNOTHERAPY Kuangyi Zhou Lab PI: Lili Yang, Ph.D. Mucosal-associated invariant T (MAIT) cells are an innate-like T cell characterized by their expression of an invariant TCR that recognize the highly conserved MHC class I-like molecule MR1. Upon activation, MAIT cells rapidly proliferate, produce a variety of cytokines and cytotoxic molecules, and trigger efficient antitumor immunity.

MAIT cells possess unique promise for the development of cancer immunotherapy because of a potent cytotoxic capacity, long-term in vivo persistence, and a MR1restricted TCR that does not induce graft-versus-host disease (GvHD). However, healthy donor peripheral blood mononuclear cells (PBMCs) contain relatively low numbers of MAIT cells, making it difficult to mass produce therapeutic doses of allogeneic MAIT cells. Therefore, this project aimed to overcome these challenges to engineer PBMC-derived MAIT cells with a mesothelin-targeting chimeric antigen receptor for mesothelin expressing cancers.

We have developed a human PBMC-derived MCAR-MAIT for cancer immunotherapy. MCAR-MAIT cells show potent cytotoxicity when compared to conventional MCAR-T cells in vitro. They also display several cytotoxic pathways as described in our mechanistic studies of NKG2D and DNAM-1. Overall, our data suggest a promising product for cancer immunotherapy and offer new understanding of how this project functions.

Although this project developed a MCAR-MAIT cell product with comparable cytotoxicity in vitro to conventional MCAR-T. However, irradiated PBMC-5-OP-RU stimulated MCAR-MAIT suffer from relatively low fold expansion and persistence. Further studies should investigate new methods of MAIT stimulation and expansion to increase quantity and quality of CAR-MAIT product. In addition, new CAR antigens such as GPC-3 and CD70 could be used for CARs targeting a wider variety of solid tumors. Lastly, MCAR-MAIT cells efficacy, safety, and bio-distribution should be tested in vivo to progress to translational applications.

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SAVE THE DATE 21st Annual Stem Cell Symposium FEBRUARY 7, 2025

FOR MORE INFORMATION, VISIT STEMCELL.UCLA.EDU Photo: Neural sphere of motor neurons and glial cells derived from hIPSCs Source: BSCRC Microscopy Core