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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

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Book cover Embryonic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1341))

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.

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Acknowledgements

The authors earlier published the original research article corresponding to this Protocol chapter in Bose et al. (40), where the funding source was duly acknowledged. The authors also greatly acknowledge the contributions of Dr(s) Sudhakar Konda and Pralhad Wangikar, who were also the coinvestigators of the original study. Making of this protocol chapter does not involve any funding from any sources.

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Authors and Affiliations

  1. Level 03, Stem Cell Biology and Tissue Engineering Division, Yenepoya Research Centre, Yenepoya University, University Road, Derlakatte, Mangalore, 575018, Karnataka, India

    Bipasha Bose

  2. 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

    P Shenoy Sudheer

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  1. Bipasha Bose
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  1. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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Bose, B., Sudheer, P.S. (2015). 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. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1341. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_230

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  • DOI: https://doi.org/10.1007/7651_2015_230

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