Isolation and characterization of mesenchymal stem cells from human tissues

Abstract

Mesenchymal stem cells (MSCs) derived from human adipose tissue and umbilical cord (Wharton’s jelly, UCB) represent a useful source of adult stem cells for cellular therapy and tissue engineering. The biggest concern with the use of MSCs therapeutically relates to their isolation and growth/manipulation ex vivo. This study aimed to establish methods for the routine isolation and characterization of MSCs from human tissues. The objectives were (1) to show that MSCs could be isolated from different human tissues, namely adipose tissue, Wharton’s jelly, and UCB; (2) to confirm the MSC phenotypic profile over at least 10 passages; and (3) to show the multilineage differentiation capacity of the isolated cells. The minimal criteria as defined by the International Society for Cellular Therapy (ISCT) were used to determine whether MSCs were successfully isolated from various human tissues. Two different techniques involving enzymatic digestion or explant cultures were utilized, and compared for isolating MSCs from Wharton’s jelly. Umbilical cord blood has been suggested as another source of MSCs. However, we were unable to grow MSCs from UCB. Proliferation kinetics of isolated MSCs revealed that cords, either from digested cords or cord pieces had a mean PDT from passage 1 to 4 that was approximately 3 fold lower than for the ASCs. Mesenchymal stem cells from adipose tissue and Wharton’s jelly expressed the classical MSC phenotype (CD73+, CD90+, CD105+, CD34-, and CD45-). The cells from Wharton’s jelly showed a more uniform MSC profile over passages, with higher levels of marker expression when compared to ASCs. Variability in phenotype was observed in early ASC passages, whereas WJ-MSCs seemed to attain the MSC phenotype as early as passage 0 for both isolation techniques. Low levels of CD34 positive cells remained in the ASCs. Oil red O staining was used for identifying the lipid droplets in adipogenic differentiation cultures. A colorimetric assay as well as image analysis was used to quantify the differentiation. For the cord samples, both assays produced positive results. Histological examination, however, revealed that the cords did not form lipid droplets. The ASCs showed a statistically significantly greater differentiation capacity into adipocytes compared with the cords (pooled digested and pieces data). Alizarin red S staining was used for identifying calcium deposition during matrix mineralization in osteogenic differentiation cultures. No significant differences in osteogenic differentiation were observed between ASCs and WJ-MSCs. Chondrogenic differentiation was observed for both MSC sources by positive staining of glycosaminoglycans using toluidine blue O. The main findings of the study showed that MSCs, according to the ISCT guidelines, were successfully harvested from adipose tissue. However, due to the lack of adipogenic differentiation of WJ-derived cells, they did not meet the ISCT guidelines to be classified as MSCs, and were referred to as MSC-like cells. Regardless of the isolation technique used, Wharton’s jelly yielded cells with similar proliferation capacity, phenotype, and differentiation capacity. This study did, however, reveal that biological differences do exist between stem cells from different sources.