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Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation.

ABSTRACT: Definitive endoderm (DE) is a vital precursor for internal organs such as liver and pancreas. Efficient protocol to differentiate human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DE is essential for regenerative medicine and for modeling diseases; yet, poor cell survival during DE differentiation remains unsolved. In this study, our use of B27 supplement in modified differentiation protocols has led to a substantial improvement. We used an SOX17-enhanced green fluorescent protein (eGFP) reporter hESC line to compare and modify established DE differentiation protocols. Both total live cell numbers and the percentages of eGFP-positive cells were used to assess differentiation efficiency. Among tested protocols, three modified protocols with serum-free B27 supplement were developed to generate a high number of DE cells. Massive cell death was avoided during DE differentiation and the percentage of DE cells remained high. When the resulting DE cells were further differentiated toward the pancreatic lineage, the expression of pancreatic-specific markers was significantly increased. Similar high DE differentiation efficiency was observed in H1?hESCs and iPSCs through the modified protocols. In B27 components, bovine serum albumin was found to facilitate DE differentiation and cell survival. Using our modified DE differentiation protocols, satisfactory quantities of quality DE can be produced as primary material for further endoderm lineage differentiation.

SUBMITTER: Wang H

PROVIDER: S-EPMC4620527 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation.

Wang Han H Luo Xie X Yao Li L Lehman Donna M DM Wang Pei P

Stem cells and development 20150803 21

Definitive endoderm (DE) is a vital precursor for internal organs such as liver and pancreas. Efficient protocol to differentiate human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DE is essential for regenerative medicine and for modeling diseases; yet, poor cell survival during DE differentiation remains unsolved. In this study, our use of B27 supplement in modified differentiation protocols has led to a substantial improvement. We used an SOX17-enhanced green fluo ...[more]

PMID: 26132288

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Project description:Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.

Dataset Information

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation.

Publications

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation.

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