title: In Vitro Differentiation of Pluripotent Stem Cells into Functional β Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets.
abstract: Since the advent of pluripotent stem cells, (embryonic and induced pluripotent stem cells), applications of such pluripotent stem cells are of prime importance. Indeed, scientists are involved in studying the basic biology of pluripotent stem cells, but equal impetus is there to direct the pluripotent stem cells into multiple lineages for cell therapy applications. Scientists across the globe have been successful, to a certain extent, in obtaining cells of definitive endoderm and also pancreatic β islets by differentiating human pluripotent stem cells. Pluripotent stem cell differentiation protocols aim at mimicking in vivo embryonic development. As in vivo embryonic development is a complex process and involves interplay of multiple cytokines, the differentiation protocols also involve a stepwise use of multiple cytokines. Indeed the novel markers for pancreas organogenesis serve as the roadmaps to develop new protocols for pancreatic differentiation from pluripotent stem cells. Earliest developed protocols for pancreas differentiation involved "Nestin selection pathway," a pathway common for both neuronal and pancreatic differentiation lead to the generation of cells that were a combination of cells from neuronal lineage. Eventually with the discovery of hierarchy of β cell transcription factors like Pdx1, Pax4, and Nkx2.2, forced expression of such transcription factors proved successful in converting a pluripotent stem cell into a β cell. Protocols developed almost half a decade ago to the recent ones rather involve stepwise differentiations involving various cytokines and could generate as high as 25 % functional insulin-positive cells in vitro. Most advanced protocols for β islet differentiations from human pluripotent stem cells focused on 3D culture conditions, which reportedly produced 60-65 % functional β islet cells. Here, we describe the protocol for differentiation of human pluripotent stem cells into functional β cells under both 2D and 3D culture conditions.
authors.affiliation: Level 03, Stem Cell Biology and Tissue Engineering Division, Yenepoya Research Centre, Yenepoya University, University Road, Derlakatte, Mangalore, 575018, Karnataka, India. Bipasha.bose@gmail.com.
authors.forename: Bipasha
authors.initials: B
authors.lastname: Bose
authors.affiliation: Molecular Genetics and Cell Biology, School of Biological Sciences, Nanyang Technological University, NTU/SBS Lab location @ Level 2, Singapore Institute for Clinical Sciences Brenner Centre for Molecular Medicine 30 Medical Drive, Singapore, 117609, Singapore.
authors.forename: P Shenoy
authors.initials: PS
authors.lastname: Sudheer
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D004475:Ectoderm / Q000166:cytology; D004707:Endoderm / Q000166:cytology; D005455:Fluorescent Antibody Technique / Q000379:methods; D020869:Gene Expression Profiling; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014157:Transcription Factors
chemical_list: D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; C000715631:NKX2-2 protein, human; C000715627:Nkx2-2 protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors
keywords: 2D differentiation; 3D differentiation; Human embryonic stem cells; Human pluripotent stem cells; Insulin; β islet differentiation