Novel Evidence for the Presence of Potent, Paracrine, Pro-Angiopoietic Effects of Purified Human Umbilical Cord Blood-Derived CD133⁺ Cells - Implications for Adult Stem Cell Therapies in Regenerative Medicine

As populations of CD34⁺, CD34⁺CXCR4⁺, or CD133⁺ cells that are enriched in stem cells, adult stem and progenitor cells purified from bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (UCB) are currently employed in the clinic to treat damaged organs (e.g., heart after myocardial infarction [AMI] or injured spinal cord or liver). The cell populations expressing these phenotypes are highly enriched primarily for hematopoietic stem/progenitor cells (HSPCs) and small numbers of endothelial progenitors, and for many years it has been wrongly supposed that they can trans-dedifferentiate into tissue-specific cells. However, even when improvement of organ function is observed after employing them in therapy, the lack of a convincing demonstration for the presence of donor-recipient chimerism in treated tissues in most of the studies performed so far indicates that mechanisms other than trans-dedifferentiation of the HSPCs delivered to the damaged organs into tissue-specific cells play a significant role in some positive clinical outcomes. In support of this conclusion, evidence has accumulated that stem cells secrete a variety of growth factors, cytokines, chemokines, and bioactive lipids that interact with the surrounding microenvironment and, when used in therapy, improve cell viability in damaged organs. In particular, more attention is currently being paid to microvesicles (MVs), which are shed from the cell surface or derived from the intracellular membrane compartment as mediators in cell-to-cell communication.

We hypothesized that these positive outcomes in adult stem cell therapies (e.g., by employing CD133⁺ cells) can be explained by the paracrine effects of these cells, involving both soluble factors as well as cell membrane-derived MVs.

CD133⁺ cells were purified from UCB by employing immunomagnetic beads (> 95% purity as checked by FACS) and incubated for 24 hours in RPMI at 37°C in a small volume of medium supplemented with 0.5% albumin. Subsequently, we harvested conditioned media (CM) from these cells and isolated CD133⁺ cell-shed microvesicles (MVs) by high speed centrifugation. We employed sensitive ELISA assays to measure the concentration of important pro-angiopoietic and anti-apoptotic factors in CD133⁺ cell-derived CM and isolated mRNA from both CD133⁺ cells and CD133⁺ cell-derived MVs for RQ-PCR analysis of gene expression. Subsequently, the chemotactic activity of CD133⁺ cell-derived CM and MVs was tested against human umbilical cord blood endothelial cells (HUVECs), and, in parallel, we tested whether CD133⁺ cell-derived CM and MVs induce major signaling pathways in HUVECs. Finally, in in vitro functional assays, we tested the ability of CD133⁺ cell-derived CM and MVs to induce tube formation by HUVECs and the ability of in vivo Matrigel assay implants to induce angiogenesis.

We observed that highly purified UCB-derived CD133⁺ cells express mRNAs and secrete proteins for several pro-angiopoietic factors (e.g. VEGF, KL, FGF-2, and IGF-1) into CM and shed microvesicles (MVs) from the cell surface and endosomal compartment that are enriched for mRNAs encoding VEGF, KL, FGF-2, and IGF-1. Both CD133⁺ cell-derived CM and MVs possessed anti-apoptotic properties, increased the in vitro cell survival of endothelial cells, stimulated phosphorylation of MAPKp42/44 and AKT in HUVECs, induced chemotactic migration, proliferation and tube formation in vitro in HUVECs, as well as stimulated in vivo angiogenesis in Matrigel implants.

These observations suggesting an important role for CD133⁺ cell-derived paracrine signals should be considered when evaluating clinical outcomes using purified CD133⁺ cells in regenerative medicine. Overall, these cell-derived paracrine signals may explain the therapeutic benefits of adult stem cells employed in regeneration of, for example, heart AMI. Finally, we will discuss several possibilities for enhancing secretion and modulating the composition of these paracrine signals that could be explored in the clinic.

No relevant conflicts of interest to declare.