✍🏼 Wanqiu Chen, Chenguang Wang, Zhi-Xue Yang, Feng Zhang, Wei Wen, Christoph Schaniel, Xianqiang Mi, Matthew Bock, Xiao-Bing Zhang, Hongyu Qiu, Charles Wang
🏠 Center for Genomics, School of Medicine, Loma Linda University, Loma Linda, CA, USA
📑 communications biology (2023)
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Abstract
Mesenchymal stromal cells (MSCs) have great value in cell therapies. The MSC therapies have many challenges due to its inconsistent potency and limited quantity. Here, we report a strategy to generate induced MSCs (iMSCs) by directly reprogramming human peripheral blood mononuclear cells (PBMCs) with OCT4, SOX9, MYC, KLF4, and BCL-XL using a nonintegrating episomal vector system. While OCT4 was not required to reprogram PBMCs into iMSCs, omission of OCT4 significantly impaired iMSC functionality. The omission of OCT4 resulted in significantly downregulating MSC lineage specific and mesoderm-regulating genes, including SRPX, COL5A1, SOX4, SALL4, TWIST1. When reprogramming PBMCs in the absence of OCT4, 67 genes were significantly hypermethylated with reduced transcriptional expression. These data indicate that transient expression of OCT4 may serve as a universal reprogramming factor by increasing chromatin accessibility and promoting demethylation. Our findings represent an approach to produce functional MSCs, and aid in identifying putative function associated MSC markers.
How the WOLF was used in this study
The WOLF cell sorter was employed to characterize and isolate specific cell populations during the reprogramming process. After generating induced mesenchymal stromal cells (iMSCs) from peripheral blood mononuclear cells (PBMCs) using episomal vectors, the authors dissociated cultured cells and stained them with antibodies against hematopoietic markers (e.g., CD34, CD45), mesenchymal stromal cell markers (CD73, CD90, CD29, CD166), and pluripotency markers (such as TRA‑1‑60 and NANOG). The WOLF cell sorter was used alongside a BD FACSAria II to analyze these marker profiles, enabling the researchers to assess the efficiency of reprogramming and the expression of surface markers that define iMSC identity versus residual blood cell or pluripotent cell characteristics.
