Background: Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening,\nregenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced\npluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied\nby the risk of insertional mutations in the target cell genome.\nMethods: We demonstrate a novel method using an inactivated viral particle to package and deliver four purified\nrecombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human\nprimary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of\nhuman iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to\nassess the pluripotency of iPMSCs.\nResults: The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human\nfibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and\nconcurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions\nare close to transcription start sites and many of these genes are pluripotent pathway associated. We found that\nDNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an\nepigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate\ninto three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the\ndelivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed\ndifferentiation.\nConclusion: Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel\nhigh-efficiency approach for regenerative medicine from patient-derived cells.
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