HUMAN iPS CELL DERIVED ISLET-LIKE ORGANOIDS IMPROVES DIABETES Chandra Ratan, Gautam Buddha University Email- ratan.chandra132000@gmail.com RESULTS
ABSTRACT Insulin independent stem cell therapy. More than 1 million cases per year of type1 diabetes in India, that’s why I study and make you aware of this stem cell therapy. By differentiating human beta cells islets like organoids (HILOs) from induced pluripotent stem cells and it shows non canonical Wnt4 signalling drives metabolic maturation which necessary for glucosestimulated insulin secretion. These functional endocrinal like cell types upon transplantation in diabetic NOD/SCID mice protected from xenograft by over-expression of immune checkpoint protein programmed death-ligand1 (PD-L1) and able to maintain in immune-competent mice for 50 days, and further interferon-gamma doses induced expression of PD-L1 which restricts T-cells activation and graft rejection. These organoids can avoid immune rejection problems so and provides promising results rather than device-dependent and cadaveric therapies.
Transcription analysis show similar results Expression of markers indicates no effects of Wnt and PD-L1 were found on HILOs. Expression of ERRγ improved in vitro insulin secretion in response to glucose. C peptide removal expression and insulin secretion.
Relative expression of key markers in wHILOs and human islets determined by qPCR
WNT expression in human islets
METHOD Differentiate umbilical derived stem cells which mimics the beta cells. Providing differentiation media in HILO cultures to form an organoid like structure. Differentiate into multicellular spheroids islets like by incorporating various transcriptional factor. Induction of PD-L1 expression by treating with IFN2γ Editing by CRISPR-Cas9 to introduce GFP reporter for visualization of insulin promotor activity. Insulin/C-peptide secretion assays. Analysis of organoid by electron microscopy. Compare transcriptional analysis between human islet and human islets like organ (HILOs).
Gene ontology of WNT4-regulated genes in HILOs
Gene expression in sorted insulinexpressing cells (GFP+) from IS, MCS, or human islets
GRAPHICAL REPRESENTATION
Immune profiling of transplanted HILOs.
1.Expression of PD-L1 when treated with IFN.
Schematic showing generation of HILOs. Key factors added to the medium for each developmental step are indicated.
CRISPR–Cas9 knock-in of fluorescent proteins for visualization.
Electron microscopy of wHILOs and human islets.
2. Flow cytometric analysis of insulin expressing and human CD45+ immune cells recovered.
CONCLUSION AND FUTURE STUDIES All indicating astounding results against disease. Further, need to more precise study on PD-L1 protein regarding immune response. This is generally for whose disease in the autoimmune response. It may provide a promising strategy for immuno-competent. Further, need to more effective study in mice and other animal models including primates. REFERENCE https://doi.org/10.1038/s41586-020-2631-z