Novel Evidence That Human Umbilical Cord Blood-Purified CD133+ cells Secrete Several Soluble Factors and Microvesicles/Exosomes That Mediate Paracrine, Pro-Angiopoietic Effects Of These Cells – Implications For and Important Role Of Paracrine Effects in stem Cell Therapies In Regenerative Medicine
Authors: Ratajczak Janina , Mierzejewska Kasia , Borkowska Sylwia , Kucia Magdalena , Ratajczak Mariusz Z 
Published: October 2019

Abstract

Background
Populations of CD34+, CD34+CXCR4+, and CD133+ cells are currently employed in the clinic to treat damaged organs (e.g., heart after myocardial infarction [AMI]). These cells are highly enriched, primarily for hematopoietic stem/progenitor cells (HSPCs), and for many years it has been wrongly supposed that HSPCs can trans-dedifferentiate into tissue-specific cells. However, even when improvement of organ function is observed after employing these cells in therapy, the lack of a convincing demonstration of significant donor-recipient chimerism in treated tissues in most of the studies performed indicates that mechanisms other than trans-dedifferentiation play a significant role in positive clinical outcomes. In support of this conclusion, we have already reported that CD34+ cells secrete a variety of growth factors, cytokines, chemokines, and bioactive lipids that interact with the surrounding microenvironment (Blood 2001;97:3075). Furthermore, microvesicles (MVs) or exosomes shed from the cell surface or derived from the intracellular membrane compartment (respectively) are important mediators in cell-to-cell communication and, as we demonstrated, may affect the biology of target cells by horizontal transfer of mRNA and proteins (Leukemia 2006;20:847).

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

Experimental strategies
CD133+ cells were purified from UCB (>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 then 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 UCB 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.

Results
We observed that highly purified UCB-derived CD133+ cells secrete 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 in vitro tube formation in HUVECs as well as stimulated in vivo angiogenesis in Matrigel implants.

Conclusions
Both in vitro and in vivo observations in animal models suggest that an important role for CD133+ cell-derived paracrine signals should be considered when evaluating clinical outcomes following the use of 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 following AMI. Finally, we will discuss several possibilities for enhancing secretion and modulating the composition of these paracrine signals, which could potentially be explored in the clinic.

Disclosures:
No relevant conflicts of interest to declare.