Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy

Abstract

Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy.

Figures

Figure 1

Characterization of mesenchymal stem cells (MSCs) from bone marrow (BM), adipose tissue (AT), and umbilical cord blood (UCB) in Passage 5. (a) Morphology of cultured BM-, AT-, and UCB-MSCs. All MSCs exhibited spindle-shaped morphology. Scale bar = 50 μm; and (b) During incubation in specialized induction media, multi-lineage differentiation was measured by staining for typical lineage markers. In MSCs from three sources, osteogenic cells were evaluated by bone type alkaline phosphatase staining (top). Chondrogenic cells accumulated sulfated proteoglycan that stained with safranin O (middle). Adipogenic cells accumulated lipid vacuoles within the cytoplasm that stained with oil red O (bottom). Scale bar = 50 μm (Magnificiation: 100×).

Figure 2

Growth kinetics of BM-, AT-, and UCB-MSCs. (a) UCB-MSCs showed more population-doubling (PD) than other tissues in all passages; (b) Clonogenetic capacity was measured by colony forming unit-fibroblast (CFU-F) assay. UCB-MSCs formed more colonies than BM- or AT-MSCs in Passage 3; (c) Comparison of final PD indicates maximal expansion potential. (a–c) Error bars represent the means ± SD. BM (n = 12), AT (n = 7), UCB (n = 12); *p < 0.05, **p < 0.01; and (d) Each population doubling time (PDT) was analyzed in Passage 5 to 6 and in Passage 11 to 12, respectively. In both passages, the PDT of UCB-MSCs was significantly lower. Error bars represent the means ± SD, n = 5; **p < 0.01; P, passage.

Figure 3

Cellular senescence of BM-, AT-, and UCB-MSCs. (a) Senescence stage evidenced by positive Gal staining in P6, P9 and P12. Staining was quantified by positive cell count. Error bars represent the means ± SD, n = 5; *p < 0.05, **p < 0.01; (b) Expression of senescence-associated proteins (p53, p21, and p16) in each MSC was gradually increased during cell expansion (P5, P8 and P11); and (c) In Passage 12, protein expression was measured by Western blotting. (b–c) β-actin was used as an internal control. Results were quantified by densitometry with signals normalized to the standard signal (BM), set as 100%. UCB-MSCs expressed significantly lower levels of senescence-associated proteins. Error bars represent the means ± SD, n = 3; *p < 0.05, **p < 0.01; P, passage.

Figure 4

Anti-inflammatory effects of BM-, AT-, and UCB-MSCs in an LPS-induced inflammatory model. (a) Rat alveolar macrophages were stimulated with LPS and co-cultured with MSCs. The co-cultures were maintained for 3 days and the supernatants were analyzed for inflammatory cytokines (rat IL-1α, IL-6 and IL-8) by ELISA. UCB-MSCs exhibited significantly lower levels of inflammatory cytokines. Error bars represent means ± SD, n = 5; **p < 0.01; (b) Ang-1 secretion. UCB-MSCs secreted a significantly higher level of Ang-1; (c) Human recombinant Ang-1 (hrAng-1) significantly reduced expression of inflammatory cytokines in LPS-treated macrophages; and (d) Pretreatment of siRNA for Ang-1 significantly reduced anti-inflammatory effect of UCB-MSC in a co-culture system; (b–d) Error bars represent means ± SD, n = 3 per group; *p < 0.05, **p < 0.01; MΦ, macrophage.