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Highly efficient reprogramming and characterization of induced pluripotent stem cells by using a microwell array.

ABSTRACT: Reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) enables the possibility of generating patient-specific cells. However, the low efficiency issue associated with iPSCs generation has limited iPSCs usage in research and clinical applications. In this study, we developed a high efficiency system to generate iPSCs by using a polydimethylsiloxane stencil. This device could be applied to the localization and reprogramming of human fibroblasts. Herein, a well-defined culture system based on a stencil, which supported efficient reprogramming of fibroblasts into iPSCs with 2-4 fold increase in efficacy over conventional methods, is presented. Subsequently, we prepared a multiple analysis system, which used a micro-patterned scissile microarray to characterize iPSCs. The results showed that iPSCs could be cultured into micro-patterns in a precisely controlled manner on the scissile poly(ethylene terephthalate) sheet, which was cut into pieces for subsequent analyses, indicating that this method allows multiple analyses to establish iPSC pluripotency in the same sample. Our approach provides a simple, cost-effective, but highly efficient system for the generation and characterization of iPSCs, and will serve as a powerful tool for establishing patient- and disease-specific pluripotent stem cells.

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Supplementary material is available for this article at 10.1007/s13770-016-0015-0 and is accessible for authorized users.

SUBMITTER: Lee H

PROVIDER: S-EPMC6170868 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Highly efficient reprogramming and characterization of induced pluripotent stem cells by using a microwell array.

Lee Hyun H Kim Gyu Man GM Choi Jin Ho JH Park Min Hee MH Bae Jae-Sung JS Jin Hee Kyung HK

Tissue engineering and regenerative medicine 20161217 6

Reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) enables the possibility of generating patient-specific cells. However, the low efficiency issue associated with iPSCs generation has limited iPSCs usage in research and clinical applications. In this study, we developed a high efficiency system to generate iPSCs by using a polydimethylsiloxane stencil. This device could be applied to the localization and reprogramming of human fibroblasts. Herein, a well-defined cultu ...[more]

PMID: 30603450

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Project description:Reprogramming of somatic cells to the phenotypic state termed "induced pluripotency" is thought to occur through three consecutive stages: initiation, maturation, and stabilisation. The initiation phase is stochastic but nevertheless very important as it sets the gene expression pattern that permits completion of reprogramming; hence a better understanding of this phase and how this is regulated may provide the molecular cues for improving the reprogramming process. c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPKs) are stress activated MAPK kinases that play an essential role in several processes known to be important for successful completion of the initiation phase such as cellular proliferation, mesenchymal to epithelial transition (MET) and cell cycle regulation. In view of this, we postulated that manipulation of this pathway would have significant impacts on reprogramming of human fibroblasts to induced pluripotent stem cells. Accordingly, we found that key components of the JNK/SAPK signaling pathway increase expression as early as day 3 of the reprogramming process and continue to rise in reprogrammed cells throughout the initiation and maturation stages. Using both chemical inhibitors and RNA interference of MKK4, MKK7 and JNK1, we tested the role of JNK/SAPK signaling during the initiation stage of neonatal and adult fibroblast reprogramming. These resulted in complete abrogation of fully reprogrammed colonies and the emergence of partially reprogrammed colonies which disaggregated and were lost from culture during the maturation stage. Inhibition of JNK/SAPK signaling resulted in reduced cell proliferation, disruption of MET and loss of the pluripotent phenotype, which either singly or in combination prevented establishment of pluripotent colonies. Together these data provide new evidence for an indispensable role for JNK/SAPK signaling to overcome the well-established molecular barriers in human somatic cell induced reprogramming. Stem Cells 2016;34:1198-1212.

Dataset Information

Highly efficient reprogramming and characterization of induced pluripotent stem cells by using a microwell array.

Electronic supplementary material

Publications

Highly efficient reprogramming and characterization of induced pluripotent stem cells by using a microwell array.

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