title: Large-scale expansion of Wharton's jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing.
abstract: BACKGROUND
  Successful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing.
  
  
  METHODS
  hWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments.
  
  
  RESULTS
  hWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1-1.30×10(7) cells on 0.5 g of microbeads by days 8-14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5×10(5) cells per well in a 24-well plate by days 6-10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P<0.05) and had a significantly higher proliferation index (P<0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing.
  
  
  CONCLUSIONS
  Thus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. Zhaogf13@mails.jlu.edu.cn.|Department of Toxicology, School of Public Health, Jilin University, Changchun, 130021, P.R. China. Zhaogf13@mails.jlu.edu.cn.
authors.forename: Guifang
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. lfl19920829@163.com.
authors.forename: Feilin
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. lsw.11@163.com.
authors.forename: Shaowei
authors.initials: S
authors.lastname: Lan
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. lipengdongwww@163.com.
authors.forename: Pengdong
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. wangwxl1128@163.com.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. Koujn13@mails.jlu.edu.cn.
authors.forename: Junna
authors.initials: J
authors.lastname: Kou
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. Qixj13@mails.jlu.edu.cn.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Qi
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. Fanrr13@mails.jlu.edu.cn.
authors.forename: Ruirui
authors.initials: R
authors.lastname: Fan
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. haods14@mails.jlu.edu.cn.
authors.forename: Deshun
authors.initials: D
authors.lastname: Hao
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. dlutwcl@163.com.|Harbin Veterinary Research Institute, CAAS - Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, 150001, P R China. dlutwcl@163.com.
authors.forename: Chunling
authors.initials: C
authors.lastname: Wu
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. xiao_bai_1210@163.com.
authors.forename: Tingting
authors.initials: T
authors.lastname: Bai
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. ylli@jlu.edu.cn.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China. jy_liu@jlu.edu.cn.|Department of Toxicology, School of Public Health, Jilin University, Changchun, 130021, P.R. China. jy_liu@jlu.edu.cn.
authors.forename: Jin Yu
authors.initials: JY
authors.lastname: Liu
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D005780:Gelatin / Q000378:metabolism; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D008863:Microspheres; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis; D012867:Skin / Q000293:injuries*; D046509:Tissue Culture Techniques; D059631:Wharton Jelly / Q000166:cytology*; D014945:Wound Healing / Q000502:physiology*
chemical_list: D018398:Homeodomain Proteins; C489427:MYC protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D005780:Gelatin