title: High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.
abstract: 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.
  
  
  CONCLUSIONS
  Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.
authors.affiliation: Shanghai R&D Center, New England Biolabs, Shanghai, 201203, China.
authors.forename: Fanfan
authors.initials: F
authors.lastname: Chen
authors.affiliation: New England Biolabs, Ipswich, MA, 01938, USA.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Shanghai R&D Center, New England Biolabs, Shanghai, 201203, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Yu
authors.affiliation: Shanghai R&D Center, New England Biolabs, Shanghai, 201203, China.
authors.forename: Yanye
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Shanghai R&D Center, New England Biolabs, Shanghai, 201203, China.
authors.forename: Xianghui
authors.initials: X
authors.lastname: Li
authors.affiliation: Med-X Research Institute of Shanghai Jiao Tong University, Shanghai, 200030, China.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Med-X Research Institute of Shanghai Jiao Tong University, Shanghai, 200030, China.
authors.forename: Yongting
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Shanghai R&D Center, New England Biolabs, Shanghai, 201203, China. dapeng_sun@163.com.|New England Biolabs, Ipswich, MA, 01938, USA. dapeng_sun@163.com.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Sun
authors.affiliation: New England Biolabs, Ipswich, MA, 01938, USA. pradhan@neb.com.
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan
mesh_terms: D002454:Cell Differentiation; D065150:Cellular Reprogramming; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D015894:Genome, Human; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors
keywords: In-vitro differentiation; Inactivated viral particle; Induced pluripotent mesenchymal stem cell; Induced pluripotent stem cell; Whole genome bisulfite sequencing