Endothelial progenitor cells (EPCs) are currently being studied as candidate cell sources for revascularization strategies. Despite these promising results, widespread clinical acceptance of EPCs for clinical therapies remains hampered by several challenges. The challenges and issues surrounding the use of EPCs and the current paradigm being developed to improve the harvest efficiency and functionality of EPCs for application in regenerative medicine are discussed. It has been observed that controversies have emerged regarding the isolation techniques and classification and origin of EPCs. This manuscript attempts to highlight the concept of EPCs in a sequential manner, from the initial discovery to the present (origin, sources of EPCs, isolation, and identification techniques). Human and murine EPC marker diversity is also discussed. Additionally, this manuscript is aimed at summarizing our current and future prospects regarding the crosstalk of EPCs with the biology of hematopoietic cells and culture techniques in the context of regeneration-associated cells (RACs).

The Hedgehog (HH) signaling pathway plays an important role in embryonic and postnatal vascular development and in maintaining the homeostasis of organs. Under physiological conditions, Sonic Hedgehog (SHH), a secreted protein belonging to the HH family, regulates endothelial cell growth, promotes cell migration and stimulates the formation of new blood vessels. The present review highlights recent advances made in the field of SHH signaling in endothelial progenitor cells (EPCs). The canonical and non-canonical SHH signaling pathways in EPCs and endothelial cells (ECs) related to homeostasis, SHH signal transmission by extracellular vesicles (EVs) or exosomes containing single-strand non-coding miRNAs and impaired SHH signaling in cardiovascular diseases are discussed. As a promising therapeutic tool, the possibility of using the SHH signaling pathway for the activation of EPCs in patients suffering from cardiovascular diseases is further explored.

Bone marrow stem cell clonal dysfunction by somatic mutation is suspected to affect post-infarction myocardial regeneration after coronary bypass surgery (CABG). Transcriptome and variant expression analysis was studied in the phase 3 PERFECT trial post myocardial infarction CABG and CD133+ bone marrow derived hematopoetic stem cells showing difference in left ventricular ejection fraction (∆LVEF) myocardial regeneration Responders (n=14; ∆LVEF +16% day 180/0) and Non-responders (n=9; ∆LVEF -1.1% day 180/0). Subsequently, the findings have been validated in an independent patient cohort (n=14) as well as in two preclinical mouse models investigating SH2B3/LNK antisense or knockout deficient conditions. 1. Clinical: R differed from NR in a total of 161 genes in differential expression (n=23, q<0•05) and 872 genes in coexpression analysis (n=23, q<0•05). Machine Learning clustering analysis revealed distinct RvsNR preoperative gene-expression signatures in peripheral blood acorrelated to SH2B3 (p<0.05). Mutation analysis revealed increased specific variants in RvsNR. (R: 48 genes; NR: 224 genes). 2. Preclinical:SH2B3/LNK-silenced hematopoietic stem cell (HSC) clones displayed significant overgrowth of myeloid and immune cells in bone marrow, peripheral blood, and tissue at day 160 after competitive bone-marrow transplantation into mice. SH2B3/LNK−/− mice demonstrated enhanced cardiac repair through augmenting the kinetics of bone marrow-derived endothelial progenitor cells, increased capillary density in ischemic myocardium, and reduced left ventricular fibrosis with preserved cardiac function. 3. Validation: Evaluation analysis in 14 additional patients revealed 85% RvsNR (12/14 patients) prediction accuracy for the identified biomarker signature. Myocardial repair is affected by HSC gene response and somatic mutation. Machine Learning can be utilized to identify and predict pathological HSC response. German Ministry of Research and Education (BMBF): Reference and Translation Center for Cardiac Stem Cell Therapy - FKZ0312138A and FKZ031L0106C, German Ministry of Research and Education (BMBF): Collaborative research center - DFG:SFB738 and Center of Excellence - DFG:EC-REBIRTH), European Social Fonds: ESF/IV-WM-B34-0011/08, ESF/IV-WM-B34-0030/10, and Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany. Japanese Ministry of Health : Health and Labour Sciences Research Grant (H14-trans-001, H17-trans-002) ClinicalTrials.gov NCT00950274

Almost all nucleated cells secrete extracellular vesicles (EVs) that are heterogeneous spheroid patterned or round shape particles ranging from 30 to 200 nm in size. Recent preclinical and clinical studies have shown that endothelial progenitor cell-derived EVs (EPC-EVs) have a beneficial therapeutic effect in various diseases, including cardiovascular diseases and kidney, and lung disorders. Moreover, some animal studies have shown that EPC-EVs selectively accumulate at the injury site with a specific mechanism of binding along with angiogenic and restorative effects that are superior to those of their ancestors. This review article highlights current advances in the biogenesis, delivery route, and long-term storage methods of EPC-EVs and their favorable effects such as anti-inflammatory, angiogenic, and tissue protection in various diseases. Finally, we review the possibility of therapeutic application of EPC-EVs in the clinic.

Considering the impaired function of regenerative cells in myocardial infarction (MI) patients with comorbidities and associated risk factors, cell therapy to enhance the regenerative microenvironment was designed using regeneration-associated cells (RACs), including endothelial progenitor cells (EPCs) and anti-inflammatory cells. RACs were prepared by quality and quantity control culture of blood mononuclear cells (QQMNCs). Peripheral blood mononuclear cells (PBMNCs) were isolated from Lewis rats and conditioned for 5 days using a medium containing stem cell factors, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6 to generate QQMNCs. A 5.3-fold increase in the definitive colony-forming EPCs and vasculogenic EPCs was observed, in comparison to naïve PBMNCs. QQMNCs were enriched with EPCs (28.9-fold, P<0.0019) and M2 macrophages (160.3-fold, P<0.0002). Genes involved in angiogenesis (angpt1, angpt2, and vegfb), stem/progenitors (c-kit and sca-1), and anti-inflammation (arg-1, erg-2, tgfb, and foxp3) were upregulated in QQMNCs. For in vivo experiments, cells were administered into syngeneic rat models of MI. QQMNC-transplanted group (QQ-Tx) preserved cardiac function and fraction shortening 28 days post-MI in comparison with PBMNCs-transplanted (PB-Tx) (P<0.0001) and Control (P<0.0008) groups. QQ-Tx showed enhanced angiogenesis and reduced interstitial left ventricular fibrosis, along with a decrease in neutrophils and an increase in M2 macrophages in the acute phase of MI. Cell tracing studies revealed that intravenously administered QQMNCs preferentially homed to ischemic tissues via blood circulation. QQ-Tx showed markedly upregulated early cardiac transcriptional cofactors (Nkx2-5, 29.8-fold, and Gata-4, 5.2-fold) as well as c-kit (4.5-fold) while these markers were downregulated in PB-Tx. In QQ-Tx animals, de novo blood vessels formed a “Biological Bypass”, observed macroscopically and microscopically, while PB-Tx and Control-Tx groups showed severe fibrotic adhesion to the surrounding tissues, but no epicardial blood vessels. QQMNCs conferred potent angiogenic and anti-inflammatory properties to the regenerative microenvironment, enhancing myocardiogenesis and functional recovery of rat MI hearts.

Under vasculogenic conditioning, pro-inflammatory cell subsets of peripheral blood mononuclear cells (PBMCs) shift their phenotype to pro-regenerative cells such as vasculogenic endothelial progenitor cells, M2 macrophages, and regulatory T cells, collectively designated as regeneration-associated cells (RACs). In this study, we evaluated the therapeutic efficacy of RAC-derived extracellular vesicles (RACev) compared to mesenchymal stem cell-derived EVs (MSCev) in the context of myocardial ischemia reperfusion injury (M-IRI). Human PBMCs were cultured with defined growth factors for seven days to harvest RACs. RACev and MSCev were isolated via serial centrifugation and ultracentrifugation. EV quantity and size were characterized by nanoparticle tracking analysis. In vitro, RACev markedly enhanced the viability, and proliferation of human umbilical vein endothelial cells in a dose-dependent manner compared to MSCev. Notably, systemic injection of RACev improved cardiac functions at 4 weeks, such as fractional shortening, and protection from mitral regurgitation than the MSCev-treated group. Histologically, the RACev-transplanted group showed less interstitial fibrosis and enhanced capillary densities compared to the MSCev group. These beneficial effects were coupled with significant expression of angiogenesis, anti-fibrosis, anti-inflammatory, and cardiomyogenesis-related miRs in RACev, while modestly in MSCev. In vivo bioluminescence analysis showed preferential accumulation of RACev in the IR-injured myocardium, while MSCev accumulation was limited. Immune phenotyping analysis confirmed the immunomodulatory effect of MSCev and RACev. Overall, repetitive systemic transplantation of RACev is superior to MSCev in terms of cardiac function enhancements via crucial angiogenesis, anti-fibrosis, anti-inflammation miR delivery to the ischemic tissue.