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05/28/2022

04/22/2022

A Low Oxygen Atmosphere Supports the Xeno-Free Generation of Human Induced Pluripotent Stem Cells

Rick Cohen, Rutgers University

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We previously demonstrated that using low oxygen tension increased the efficiency of reprogramming human somatic cells to pluripotency. In this study, we extend our findings and further increased the development of this culture paradigm. We are able to observe that post-electroporated fibroblasts cultivated at 4 % O2 grow on synthetic and biological surfaces.

Further, we observed normal spreading of iPSCs and a high level of purity in early iPSC passages. The cells were successfully differentiated into motor neuron and cardiomyocyte lineages emphasizing the efficacy of low oxygen levels during cell cultivation.

Introduction

Reprogramming human somatic cells into the pluripotent state is a subject of thousands of publications since their discovery in 2007. Originally these studies were pioneered with a set of four genes found to be expressed in native human embryonic stem cells; Oct4, Sox2, KLF4, and c-Myc (Lin28 and Nanog), delivered using genetically modifying methods such as retrovirus, and cultured on non-defined matrices.

In the 13 years since these seminal studies many improvements were made such as (1) replacing genetically modifying methods with non-genome altering safer alternatives; (2) replacement of c-Myc oncogene with non-transforming family member, L-Myc; (3) inclusion of small molecules to boost efficiency of reprogramming; (4) optimizing culture conditions which includes the use of low O2 tension (4 - 5 %); and (5) use of clinically relevant defined media and matrices.


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