High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue
Figure 3
UCSCs demonstrate a high level of ex vivo expansion and retain MSC surface marker phenotype for up to 10 passages, or 5 weeks in culture. (a) UCSCs showed a significantly higher level of proliferation in EGM2 growth medium as compared to DMEM (72 hours, P = .038, 96 hours, P = .10). (b) Cells cultured in the different media were distinct morphologically; DMEM-cultured cells were significantly larger in 2D as compared to cells grown in EGM2 (mean ± s.e.m.). (c) We expanded UCSCs for more than 55 population doublings, PDs. Thirteen UCSC populations were expanded for 10 passages, and 3 of these UCSC populations were continued for another 10 passages. The theoretical expansion yield is 1020 cells from an initial 2 104 cells. (d) Mean population doubling time of the UCSCs was 24 ±1.1 hours for the populations at 0–10 PDs, 23 ±1.7 hours at 10–20 PDs, which are significantly faster than the PDT of 28 ±1.3 hours for UCSCs at 20–30 PDs (P < .05, bars represent mean ± s.e.m.). (e) We observed no significant changes in cellular division times which was similar for cells at 0–10 PDs (14.8 ±0.4 hours), 10–20 PDs (15.1 ±0.7 hours), and 20–30 PDs (15.9 ±0.8 hours). (f) Histograms are representative of UCSCs surface marker expression 7 days postisolation. The population is homogeneously positive for mesenchymal cell markers CD44, CD105, CD73, and CD90, and ~50% positive for endothelial cell marker CD146, and negative for hematopoietic cell markers CD34 and CD45, and endothelial cell marker CD144. (g) Surface marker expression levels through 10 passages. UCSCs have steady but moderate CD146 expression levels and remain negative for CD45, CD34, and CD144 expression. The cells are consistently highly positive (≥97%) for all mesenchymal markers analyzed throughout the five-week period and support the reproducibility of the isolation method and stability of the MSC-like phenotype. (h) Representative PCR analysis of low-passage and high-passage UCScs confirmed the presence of mRNA transcripts for MSC genes. Lane 1: UCSCs at passage 2; Lane 2: UCSCs at passage 8; Lane 3: human vascular endothelial cells (HUVEC); Lane 4: no DNA.
