title: In vitro differentiation of human umbilical cord Wharton's jelly mesenchymal stromal cells to insulin producing clusters.
abstract: OBJECTIVE
  To investigate the differentiation of human Wharton's jelly derived mesenchymal stromal cells (WJ-MSCs) to insulin producing clusters (IPC) this study was conducted.
  
  
  METHODS
  The umbilical cords samples were collected from full term caesarian section mothers and the WJ-MSCS were cultured from tissue explants in High glucose-Dulbecco's Modified Eagle Medium (H-DMEM); H-DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. The expression of CD90, CD44, CD105, CD34 and CD133 as well as osteogenic and adipogenic differentiation of cells in appropriate medium were also evaluated. The cells were differentiated toward IPC with changing the culture medium and adding the small molecules such as nicotinic acid, epidermal growth factor, and exendin-4 during 3 wk period. The gene expression of PDX1, NGN3, Glut2, insulin was monitored by reveres transcription polymerase chain reaction method. The differentiated clusters were stained with Dithizone (DTZ) which confirms the presence of insulin granules. The insulin challenge test (low and high glucose concentration in Krebs-Ringer HEPES buffer) was also used to evaluate the functional properties of differentiated clusters.
  
  
  RESULTS
  WJ-MSCS were positive for mesenchymal surface markers (CD90, CD44, CD105), and negative for CD34 and CD133. The accumulation of lipid vacuoles and deposition of calcium mineral in cells were considered as adipogenic and osteogenic potential of WJ-MSCS. The cells also expressed the transcriptional factors such as Nanog and OCT4. During this three step differentiation, the WJ-MSCS morphology was gradually changed from spindle shaped cells in to epithelioid cells and eventually to three dimensional clusters. The clusters expressed PDX1, NGN3, Glut2, and insulin. The cells became bright red color when stained with DTZ and the insulin secretion was also confirmed. In glucose challenge test a significant increase in insulin secretion from 0.91 ± 0.04 μIu/mL (2.8 mmol/L glucose) to to 8.34 ± 0.45 μIu/mL (16.7 mmol/L glucose) was recorded (P < 0.05). The insulin secretion of undifferentiated WJ-MSCS was not changed in this challenge test.
  
  
  CONCLUSIONS
  WJ-MSCs have the ability to differentiate in to islet-like cells in vitro. However, this process needs further optimization in order to generate efficient and functional IPCs.
authors.affiliation: Seideh Masoomeh Nekoei, Negar Azarpira, Transplant Research Center, Shiraz University of Medical Science, Shiraz 7193711351, Iran.
authors.forename: Seideh Masoomeh
authors.initials: SM
authors.lastname: Nekoei
authors.affiliation: Seideh Masoomeh Nekoei, Negar Azarpira, Transplant Research Center, Shiraz University of Medical Science, Shiraz 7193711351, Iran.
authors.forename: Negar
authors.initials: N
authors.lastname: Azarpira
authors.affiliation: Seideh Masoomeh Nekoei, Negar Azarpira, Transplant Research Center, Shiraz University of Medical Science, Shiraz 7193711351, Iran.
authors.forename: Ladan
authors.initials: L
authors.lastname: Sadeghi
authors.affiliation: Seideh Masoomeh Nekoei, Negar Azarpira, Transplant Research Center, Shiraz University of Medical Science, Shiraz 7193711351, Iran.
authors.forename: Sulmaz
authors.initials: S
authors.lastname: Kamalifar
keywords: Beta cell; Islet; Mesenchymal stromal cells; Umbilical cord