Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue

Abstract

Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor-D (VEGF-D), and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC(CM) resulted in increased tubulogenic efficiency compared with that observed in DPC(CM). Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.

FIG. 1.

Phase-contrast photomicrographs showing morphological characteristics of human mesenchymal stem cells from adipose, bone marrow, dermal sheath, and dermal papilla (ASCs, BMSCs, DSCs, and DPCs) (magnification ×40 and ×100). All MSC populations were cultured under identical conditions to avoid variations incurred by methodology differences.

FIG. 2.

Gene expression of selected paracrine factors in MSC populations. Expression of angiogenic factor mRNAs was comparable between cell populations (A), while expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) was significantly higher in ASCs (B). Stromal-derived factor-1 (SDF-1) expression was comparable in all MSC populations while interleukin-8 (IL-8) was expressed highest in ASCs (C). Expression level of cytoprotective factors, hepatocyte growth factor (HGF), tended to be higher in ASCs and BMSCs, and IGF-1, insulin-like growth factor-1 (IGF-1), expression level was highest in ASCs (D). Values are mean±SEM of n=4–6. *P<0.05, **P<0.01, and ***P<0.001 versus ASCs.

FIG. 3.

Effect of MSC-conditioned medium (CM) on human microvascular endothelial cell proliferation. CM derived from MSC populations showed comparable effects on proliferation of human microvascular endothelial cells (HMECs) in vitro (P=0.49). Pos ctrl, positive control; Neg ctrl, negative control.

FIG. 4.

Paracrine activity of various MSC populations showed similar efficacy at promoting migration and proliferation of HMECs to recover the unpopulated area in the wound-healing assay (P=0.51). HMECs cultured in endothelial growth medium-2 MV (Pos ctrl) recovered the area at a significantly higher percentage as compared with cells cultured in endothelial basal media-2+5% fetal calf serum (Neg ctrl). CM derived from all MSC populations induced an intermediate re-coverage of the “wound,” ranging from 24% to 36%. Magnification ×40. Pos ctrl, positive control; Neg ctrl, negative control.

FIG. 5.

CM derived from MSC populations was able to support the formation of tubular structures in HMECs, which was not evident in the negative control (A). ASC^CM exhibited a comparable ability at supporting vascular network formation as positive control in terms of loops formed (B) and branch points (C) and total length of tubular network (D). ASC^CM was also significantly more efficient than DPC^CM at supporting the tube formation of HMECs in vitro (*P<0.05, **P<0.01). Magnification ×40. Pos ctrl, positive control.

FIG. 6.

ASC^CM supported the formation of tubular structures in HMECs as determined by the number of rings formed (A), branch points (B), and total tube length (C) at 24 h. In the presence of vascular endothelial growth factor-A (VEGF-A), VEGF-D, or both VEGF-A and VEGF-D neutralizing antibodies, the effect of ASC^CM on HMECs was abolished with significantly less complete rings and shorter tube length (*P<0.05 vs. ASC^CM only, #P<0.05 vs. ASC^CM+neutralizing VEGF-D antibody). Open bar, ASC^CM only; closed bar, ASC^CM+neutralizing antibodies.