Paracrine proangiopoietic effects of human umbilical cord blood-derived purified CD133+ cells--implications for stem cell therapies in regenerative medicine

Abstract

CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

FIG. 1.

Purity of isolated from umbilical cord blood (UCB) CD133⁺ cells and fluorescence activated cell sorter (FACS) and transmission electron microscopy (TEM) analysis of UCB CD133⁺ cell-derived microvesicles (MVs). (A) Representative FACS analysis of purity of CD133⁺ UCB cells isolated by paramagnetic beads. Allophycocyanin (APC)-anti-CD133 antibody was designed to a different epitope than that recognized by anti-CD133 Abs on paramagnetic beads. (B) Top panel: Representative dot plot of UCB CD133⁺ cell-derived MVs; bottom panel: representative transmission electron microscopic image of UCB CD133⁺ cell-derived MVs (magnification 5.2×10⁴). (C) Expression of CD34, CXCR4, c-kit receptor (CD117), and FLT3 (CD135) on UCB CD133⁺ cell-derived MVs. Experiment was repeated 2 times. A representative FACS analysis is shown. FSC, forward scatter; SSC, sideward scatter.

FIG. 2.

Expression of selected proangiopoietic factors by human CD133⁺ cells, CD133⁺ cell-derived MVs, and CD133⁺ cell-derived conditioned media (CM). (A) Real-time quantitative reverse transcription–polymerase chain reaction analysis of mRNA in purified CD133⁺ cells and CD133⁺ cell-derived MVs compared to UCB-derived mononuclear cells (MNCs) in which expression has been assumed to be 1.0. The data shown represent the combined results from 4 independent experiments carried out on separate cells. (B) Expression of proangiopoietic factors in CM harvested from CD133⁺ cells and detected by ELISA. The data shown represent the combined results of 4 independent experiments carried out on separate cells. IGF, insulin growth factor; VEGF, vascular endothelial growth factor; FGF-2, basic fibroblast growth factor; HGF, hepatocyte growth factor; SCF, stem cell factor.

FIG. 3.

CD133⁺ cell-derived CM and MVs stimulate signaling pathways and chemoattract human umbilical endothelial cells (HUVECs). (A) Phosphorylation of MAPK^p44/42 and Akt^ser473. Before stimulation, cells were starved overnight in RPMI medium containing 0.5% bovine serum albumin (BSA) in an incubator and subsequently unstimulated (lane 1) or stimulated with CD133⁺ cell-derived MVs (lane 2), CM from CD133⁺ cells (lane 3), CM from CD133⁺ cells w/o MVs, or basic fibroblast growth factor-2 (FGF-2, 50 ng/mL) for 5 min. The experiment was repeated independently 3 times with similar results, and a representative western blot is shown. (B) Chemotactic responsiveness of HUVECs to medium alone (control), CD133⁺ cell-derived MVs, CM from CD133⁺ cells, CM from CD133⁺ cells w/o MVs, and FGF-2 (positive control). The experiment was repeated independently 3 times in quadruplicate, and the data were pooled together. *P<0.01.

FIG. 4.

Tube formation by HUVECs in the presence of CD133⁺ cell-derived CM and MVs. (A) HUVECs were seeded on Matrigel in the presence of medium alone, CD133⁺ cell-derived MVs (30 μg/mL), CM from CD133⁺ cells, CM from CD133⁺ cells w/o MVs, or FGF-2 (50 ng/mL, positive control) in a serum-free medium and incubated for 18 h at 37°C. The experiment was independently repeated 3 times in quadruplicate, and the data were pooled together. *P<0.0001. (B) Representative image of tube formation by HUVECs in the presence of medium alone (negative control), CD133⁺ cell-derived MVs (30 μg/mL), platelet derived microvesicles (30 μg/mL), and FGF-2 (50 ng/mL).