Evaluation of the cell viability of human Wharton's jelly stem cells for use in cell therapy

Abstract

Human umbilical cord Wharton's jelly stem cells (HWJSCs) are gaining attention as a possible clinical source of mesenchymal stem cells for cell therapy and tissue engineering due to their high accessibility, expansion potential, and plasticity. We employed a combination of highly sensitive techniques to determine the average cell viability levels and proliferation capabilities of 10 consecutive cell passages of cultured HWJSCs and then used RNA microarrays to identify genes associated with changes in cell viability levels. We found an initial decrease in cell viability from the first to the third cell passage followed by an increase until the sixth passage and a final decrease from the sixth to tenth cell passages. The highest cell viability levels corresponded to the fifth and sixth passages. The intracellular ionic contents of potassium, sodium, and chlorine suggest that the lower cell viability levels at passages 2, 3, and 8-10 may be associated with apoptotic cell death. In fact, gene expression analysis revealed that the average cell viability was significantly associated with genes with a function in apoptotic cell death, especially pro-apoptotic FASTKD2, BNIP3L genes and anti-apoptotic TNFAIP8 and BCL2L2 genes. This correlation with both pro-apoptotic and anti-apoptotic genes suggests that there may be a complex live-death equilibrium in cultured HWJSCs kept in culture for multiple cell passages. In this study, the highest cell viability levels corresponded to the fifth and sixth HWJSC passages, suggesting that these passages should be preferentially employed in cell therapy or tissue engineering protocols using this cell type.

FIG. 1.

Multi-lineage differentiation capabilities of the cultured HWJSCs at cell passage P1. (A) Undifferentiated control HWJSCs (H&E staining). (B) HWJSCs differentiated to the osteogenic cell type showing positive calcium deposits by alizarin red staining. (C) Differentiation to the chondrogenic phenotype (alcian blue staining). (D) Adipogenic differentiation (oil red O). Scale bars: 50 μm. HWJSCs, Wharton's jelly stem cells. Color images available online at www.liebertonline.com/tec

FIG. 2.

Flow cytometry analysis of CD90 (positive MSC marker) and CD45 (negative MSC marker) in HWJSCs at passages P1, P4, P6, and P9. Cells showing positive expression of each marker are shown in blue; green lines show the expression of cells labeled with the control isotypes. Color images available online at www.liebertonline.com/tec

FIG. 3.

Analysis of cell proliferation and cell viability of 10 successive passages of HWJSC. (A) Cell proliferation as determined by RTCA Real-Time Cell Analysis Cell Index (RTCA CI) and microarray analysis of PCNA and MKI-67. (B) Cell proliferation as determined by electron-probe X-ray microanalysis (potassium/sodium index), trypan blue exclusion analysis, LIVE/DEAD^® Cell Viability Assay, and WST-1. (C) Average cell viability level (ACVL). WST-1, water-soluble tetrazolium salt-1. Color images available online at www.liebertonline.com/tec

FIG. 4.

Histograms representing the mean intracellular concentrations of potassium, sodium, and chlorine and the K/Na ratio for the ten consecutive HWJSC passages (P1 to P10). Statistically significant changes (increases or decreases) are indicated with asterisks. Green bars correspond to the control human fibroblasts, while red bars represent the same cells treated with 10 mM HEMA to induce apoptosis. All measures are expressed as millimoles per kilogram of cell dry weight. Color images available online at www.liebertonline.com/tec

FIG. 5.

Significance Analysis of Microarrays (SAM) results for all genes with a role in apoptotic cell death using multiclass analysis of genes associated with the average cell viability levels (ACVLs). All differentially expressed probe sets are represented in red color, and their names are shown in the plot. We used a δ value that permitted a false discovery rate of 0. Color images available online at www.liebertonline.com/tec