High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins

Autor: Sriharsa Pradhan, Guoqiang Zhang, Fanfan Chen, Ling Yu, Zhijun Zhang, Xianghui Li, Yanye Feng, Dapeng Sun, Yongting Wang
Jazyk: angličtina
Předmět:
0301 basic medicine
Cellular differentiation
Genetic Vectors
Induced Pluripotent Stem Cells
Primary Cell Culture
Kruppel-Like Transcription Factors
Whole genome bisulfite sequencing
Medicine (miscellaneous)
Biology
Transfection
Sendai virus
Biochemistry
Genetics and Molecular Biology (miscellaneous)
Epigenesis
Genetic
Proto-Oncogene Proteins c-myc
Kruppel-Like Factor 4
Induced pluripotent mesenchymal stem cell
03 medical and health sciences
SOX2
Humans
Induced pluripotent stem cell
Cell potency
Induced stem cells
Genome
Human
SOXB1 Transcription Factors
Research
Virion
Cell Differentiation
Cell Biology
DNA Methylation
Fibroblasts
Cellular Reprogramming
Molecular biology
Recombinant Proteins
Inactivated viral particle
030104 developmental biology
DNA methylation
Molecular Medicine
CpG Islands
In-vitro differentiation
Stem cell
Octamer Transcription Factor-3
Reprogramming
Germ Layers
Zdroj: Stem Cell Research & Therapy
ISSN: 1757-6512
DOI: 10.1186/s13287-016-0358-4
Popis: Background Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. Methods We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. Results The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Conclusion Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.
Databáze: OpenAIRE
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