An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have dev...

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Main Authors: Massumi, Mohammad, Pourasgari, Farzaneh, Nalla, Amarnadh, Batchuluun, Battsetseg, Nagy, Kristina, Neely, Eric, Gull, Rida, Nagy, Andras, Wheeler, Michael B.
Format: Online
Language:English
Published: Public Library of Science 2016
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068782/
id pubmed-5068782
recordtype oai_dc
spelling pubmed-50687822016-10-27 An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells Massumi, Mohammad Pourasgari, Farzaneh Nalla, Amarnadh Batchuluun, Battsetseg Nagy, Kristina Neely, Eric Gull, Rida Nagy, Andras Wheeler, Michael B. Research Article The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. Public Library of Science 2016-10-18 /pmc/articles/PMC5068782/ /pubmed/27755557 http://dx.doi.org/10.1371/journal.pone.0164457 Text en © 2016 Massumi et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Massumi, Mohammad
Pourasgari, Farzaneh
Nalla, Amarnadh
Batchuluun, Battsetseg
Nagy, Kristina
Neely, Eric
Gull, Rida
Nagy, Andras
Wheeler, Michael B.
spellingShingle Massumi, Mohammad
Pourasgari, Farzaneh
Nalla, Amarnadh
Batchuluun, Battsetseg
Nagy, Kristina
Neely, Eric
Gull, Rida
Nagy, Andras
Wheeler, Michael B.
An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells
author_facet Massumi, Mohammad
Pourasgari, Farzaneh
Nalla, Amarnadh
Batchuluun, Battsetseg
Nagy, Kristina
Neely, Eric
Gull, Rida
Nagy, Andras
Wheeler, Michael B.
author_sort Massumi, Mohammad
title An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells
title_short An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells
title_full An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells
title_fullStr An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells
title_full_unstemmed An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells
title_sort abbreviated protocol for in vitro generation of functional human embryonic stem cell-derived beta-like cells
description The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.
publisher Public Library of Science
publishDate 2016
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068782/
_version_ 1613688052230651904

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