Endothelial progenitor cells (EPCs), have been reported to be cardinal player in the repair of endothelial dysfunction and new blood vessels formation (1). EPCs are mainly located in the bone marrow tissues stem cells and a few of them are found in the peripheral blood as well (2). An injury to peripheral blood vessels or an ischemic stroke leads to EPCs mobilization from bone marrow cells to the blood circulation under chemokine stimulation. EPCs then reside in the endothelium to help repair the damage caused due to injury, ischemia or hypoxia as reported in both patients and animal models (3, 4). Studies have identified EPC contribution in postnatal vasculogenesis, wound healing, postmyocardial infarction, or limb ischemia (5). EPC mobilization and differentiation in ischemic region is mediated by growth factors like vascular endothelial growth factor (VEGF)/KDR, granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor, soluble intercellular adhesion molecule, interleukin-6 (IL-6), and endothelial nitric oxide synthase (eNOS) (6). Low levels of CD34＋ and KDR＋ circulating EPCs have been reported in cardiovascular abnormalities and these studies have indicated these levels as a possible cause of death from such diseases (7). A role of NO in improvement of EPC migration and angiogenesis has also been of interest (8). In humans, number of EPCs is an important factor for evaluation of cardiovascular function. The decrease in EPC numbers and activity may contribute to impaired vascularization in patients with coronary artery disease (CAD) (4). Alzheimer disease (AD) patients have the significantly-lower colony forming units of EPCs (CFUs-EPC). Circulating endothelial cells from AD patients show increased senescence and reduced paracrine angiogenic activity (9). The CFU numbers are significantly reduced in patients with acute stroke. CFU number may indicate a dysfunctional status of EPCs (7).

Animal’s studies show that the progenitor cell mobilization influence the endothelial cell repair after injury (7). A role for paracrine factors from EPC has been observed to have chemotactic and mitogenic effects on keratinocytes and fibroblasts. EPC-conditioned medium injection into diabetic mice promoted wound healing and neovascularization (2). Microvascular endothelial cells follow AKT and ERK signaling pathway in angiogenic functions in rat brain (10). An interesting factor is the reduction of circulating endothelial colony forming cells (ECFCs) with aging, older subjects are found to have fewer ECFCs than their younger counterparts. Reports suggest that bone marrow-derived progenitor cells number remain stable, a reduction in their repairing activity can be due to angiogenic capacity or cellular impairments (11). A characteristic feature of EPCs is the migration and investigators have tried to find an effect of certain factors on their migration activities. The EPCs were used in in vitro migration assays against serum samples of cardiac surgical patients. An effect of macrophage migration inhibitory factor on chemotaxis of EPCs has been observed (3). Studies reported that different molecules like VEGF, stromal derived factor, erythropoietin, NO and extracellular matrix (ECM) cumulatively guide the migration of EPCs. The ECM molecules involved in migration are β₁ integrin and fibronectin. Endostatin, a cleavage product of collagen XVІІІ also increases migration of EPCs (12). In view of increasing significance of EPCs in neovasculogenesis, the current review highlight the reported data form literature for EPC identification, characterization, isolation, storage and transplantation. We suggest that the methods described in this review can be combined with other therapeutic drug targets to support the fast expansion and the effective transplantation of EPCs.

An appreciable amount of data have reported that EPCs can be isolated from human cord blood and peripheral blood and plated on fibronectin coated dishes in culture media supplemented with endothelial growth factors (4). EPCs have been isolated from bone marrow, human umbilical vein, human umbilical cord blood (UCB), peripheral blood circulation by density centrifugation method. Two types of EPCs have been reported from peripheral blood; late EPCs, cobblestone-shaped cells which increase in number and early EPCs which are spindle like cells with low proliferation capacity. It has been reported that EPCs carry out neovascularization through cytokines and matrix metalloproteinase-9 (MMP-9). Vascular damage cause release of cytokines, MMP-9 and growth factors i.e., VEGF and fibroblast growth factor (FGF), in EPCs. Chemokines and growth factors help in EPC mobilization from bone marrow to circulation. eNOS and G-CSF induce mobilization and proliferation of EPCs (13). The morphological characterization of EPCs has been evaluated based on some common factors on which many investigators seems to agree mutually. EPCs were characterized by adhesion to fibronectin, cell surface markers expression, morphologic appearance, acetylated low-density lipoprotein uptake, lectin binding, CFU assay, and ECFCs assay. EPCs show positive expression of endothelial cell markers such as Vwf, Tie2, CD31, VE-cadherin, KDR, and stem cell markers like CD34 or CD133 (4, 14).

Medicinal use of EPCs requires huge numbers of EPCs. Expansion of EPCs in culture is imperative for their therapeutic application because a large amount of EPCs is needed to treat vascular injury (4). Many investigators are after the expansion of EPC in culture through different methods such as addition of growth factors to the culture medium and pre-coating of culture dishes with ECM proteins (15). Interestingly, Lu et al. (16) cultured rat bone marrow cells in high density (26×10⁵ cells/cm²) or regular density (1.66×10⁴ cells/cm²) with the same total number of cells in both. They analyzed that high density cells exhibited smaller size and higher levels of marker expression of EPCs and increased release of pro-angiogenic growth factors as compared to regular density cultured cells. EPCs showed potential recovery of mouse ischemic limbs in vivo by their integration into neo-capillary structure (16). The use of various factors in increasing EPC numbers is increasing by the time and importantly it is giving good results as well. Microgravity (MG) significantly facilitated the proliferation, migration and angiogenesis of human umbilical vein endothelial cells through NO induced activation of FAK/Erk1/2-MAPK signaling pathway. It is suggested that by using MG bioreactor, angiogenic properties of EPCs can be increased (17). Investigators observed sufficient numbers of EPCs from a long term culture of rat adipose derived stem cells in an endothelial basal medium 2 supplemented with endothelial cell growth medium 2 medium. These EPCs showed changes in morphology with time, significant expression of VEGFR-2, strong Dil-ac-LDL uptake and lectin binding (18). Human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes can be used to produce CD34＋ EPCs. hiPSCs–endothelial cells (ECs) show positive expression of CD31 and high VEGF-A and angiopoietin-1 (6). A specific culture method called quality and quantity-control culture (QQ culture) of mononuclear cells (QQMNCs) has been used for whole peripheral blood mononuclear cells (PBMNCs). QQ culture contains a medium containing IL-6, stem cell factor, thrombopoietin, Flt-3 ligand, and VEGF which increases the quality and quantity of EPCs. It upregulates PBMNCs to differentiate into hematopoietic stem cells and reduces the culture time. This culture also enhances the ability of PBMNCs to support regeneration of injured tissue (19). QQ culture improved therapeutic effect of peripheral blood CD34＋ cells in diabetic injury (20) and end-stage renal failure disease patients (21). A summary of expansion method is shown in Table 1. Up till now, the reported data has been able to describe two types of EPCs using different culture environments and their morphological determinants like KDR and CD34 are quite well established. EPC rapid expansion can be achieved through high density bone marrow culture, MG and QQ culture method.

Table 1 . Comparison of expansion methods

EPC expansion method	Effectiveness of method
1. High density (26×105 cells/cm2) culture of rat bone marrow cells	Higher levels of marker expression of EPCs Increased release of pro-angiogenic growth factors
2. Microgravity through nitric oxide induced activation of FAK/Erk1/2-MAPK signaling pathway	Facilitated the proliferation and angiogenesis of human umbilical vein endothelial cells Enhanced angiogenic properties of EPCs
3. hiPSCs produce CD34＋ EPCs	Positive expression of CD31, high VEGF-A and angiopoietin-1 Regeneration of injured tissue
4. Quality and quantity-control culture of MNCs	Increased the quality and quantity of EPCs Reduces the culture time Improves differentiation of PBMNCs to hematopoietic stem cells
5. Long term culture of rat adipose derived stem cells	Numbers of EPCs increases Expression of VEGFR-2

EPC: endothelial progenitor cell, hiPSCs: human induced pluripotent stem cells, MNCs: mononuclear cells, VEGF: vascular endothelial growth factor, PBMNCs: peripheral blood MNCs.

EPC induction with different growth factors has been shown to have effect on its differentiation. Bone marrow derived EPCs can differentiate into endothelial cells and are actively involved in vascular repair (22). Monocytic cells can differentiate into endothelial like cells which indicate a relationship between the endothelial cell system and monocyte/macrophage (23). Nevertheless, EPCs have been induced with various growth factors to enhance differentiation. If induced, EPCs can secrete vasogenic growth factors which activate peripheral mature endothelial cells to accelerate the damaged vascular endothelial cells repair (22). Endothelial differentiation promoting conditions support differentiation of CD34＋ and CD133＋ cells from ECs in in vitro (14). Bone marrow derived EPCs are mobilized and cause an in vitro increase in differentiated EPCs when induced by VEGF (4), β-FGFs and thrombin (6). IL-6 stimulated EPC proliferation and migration (24). eNOS enhances endothelial cells mobilization from the bone marrow, their growth and migration (25). KMUP-1 (7-(2-(4(2-chlorophenyl)piperazinyl)ethyl)-1,3-dimethylxanthine), atorvastatin and simvastatin treated EPCs significantly prevented the hypoxia induced EPCs death and apoptosis (8). It can be concluded that growth factors and NO synthase enhance EPC differentiation and mobilization and these growth factors may make an improvement in therapeutic intervention of EPCs. Studies are needed to evaluate the specificity and most effective treatment dose of these factors in EPC. A specific concentration of growth factors is an important factor for EPC differentiation.

EPCs have been shown to be a potent clinical candidate in wound repair, ischemic stroke and CAD due to their abilities to proliferate, mobilize to ischemic area and involvement in vascular regeneration and angiogenesis (6). Bone marrow derived EPC play roles in diabetes (13), cancer (26), and cardiovascular disorders (11). EPC transplantation effectively promotes angiogenesis after an ischemic stroke in animal models, forming an enriched tubular network which speeds up recovery of nerve function and thus neurogenesis (27). Sufficient amount of EPCs are required for EPC transplantation into vascular grafts surface or injection into ischemic area. Zhou et al. (28) studied the combined effects of EPCs and MMPs inhibitor, BT-94, in diabetic ischemic stroke in in vitro and in vivo experiments. EPCs and BB-94 alleviated cerebral ischemia injury in the MCAO model mice and downregulated the expression of MMPs in OGD/R and MCAO model mice (28). Bone marrow derived EPCs make good candidates for EPC based therapy as they are easily available and have low immunogenicity (29).

Patients with acute cerebral infarct in middle cerebral artery were injected with autologous ex vivo expanded EPCs intravenously without toxicity or allergic reactions (30). Human peripheral blood derived neuronal outgrowth cells were transplanted in the ischemic rat brain and they survived and migrated as well (31). An intracoronary injection of EPCs can improve left ventricular function after acute myocardial infarction (32). EPC administration leads to neuronal regeneration after ischemic stroke in rats (33) and mice (34). Outgrowth of endothelial cells from cultures of blood is derived from transplantable marrow derived cells. These cells have greater proliferative rate and derived from circulating angioblasts (35). Human EPCs were transplanted to athymic nude mice with hindlimb ischemica. Capillary density and blood flow recover were greatly improved in the ischemic hindlimb and rate of lilmb loss was reduced significantly (1). The above reported data demonstrates the fact that EPC transplantation requires its fast availability in sufficient amounts. This transplantation was tested in different mice models where it successfully migrated to injury area, survived well and also resulted in neuronal regeneration.

A promising approach for EPC availability for transplantation purposes is its storage through cryopreservation. Several reports have supported the cryopreservation of EPCs for storage. Many factors such as composition of cells, cell type, cell density freezing and thawing rate, can affect the EPCs cryopreservation efficacy (36, 37). Thawing and freezing can result in a decrease in EPC marker expression, proliferation, differentiation and artery injury recovery which may affect EPCs functions and viability (38, 39). EPC have been stored by freezing peripheral blood, bone marrow and UCB derived MNCs. Interestingly, many studies reported that cryopreserved EPCs do not show changes in proliferation, endothelial functions and viability (1, 36). Investigators have also discussed about number of passages as it was suggested that a limited number of culture passages and cryopreservation do not change EPC phenotype and functions (40). Dimethyl sulfoxide (DMSO) is considered to be the most suitable cryoprotective agent regarding cell recovery after thawing combined with K^＋ modified TiProtec (K^＋ TiP) in vitrification and with DMEM in slow freezing (36). ECFCs can be generated from cryopreserved PBMNCs which can be used as a predictor of alloimmune response in transplantation (40). A summary of comparison of storage methods is shown in Table 2. In summary, thawing and freezing are very important factors which effect the EPC marker expression and their differentiation. It is observed that DMSO is most suitable cryoprotectant for EPC storage.

Table 2 . Comparison of storage methods

1. Cryopreservation	EPCs proliferation, endothelial functions and viability do not change
2. Limited number of culture passages	EPC phenotype and functions do not change
3. DMSO combined with K＋ modified TiProtec (K＋ TiP)	Cell recovery after thawing

EPC: endothelial progenitor cell, DMSO: dimethyl sulfoxide.

It is well established now that EPCs are particularly involved in vasculogenesis. Efficiently increasing informative techniques about EPC isolation, differentiation and expansion are playing an important role in rapid availability of EPC in therapeutics especially in transplantation. Ischemia and tissue injury cause the EPCs production from bone marrow and EPCs are mobilized to circulation. EPC migration from bone marrow into the circultion is guided by signals such as chemokines, growth factors and hypoxia in vivo. ECM molecules are involved in endothelial differentiation process and also influence other paracrine factor released by EPC. Integrins increase growth factor production by EPCs. A cell-cell communication among EPCs govern their differentiation. Thus, in vivo circumstances effect cell fate and function. Recently, the studies are trying to supplement the EPC culture conditions with most of the same factors as in vivo. However, it is quite challanging to optimize in vivo conditions in an in vitro EPC cell culture. In vitro culture enviornment can modulate EPCs characteristics to differentiate differently then in vivo. A schematic diagram of the focus of this review is represensented in Fig. 1. Further studies are required which could find the effect of a specific growth factor or chemokine at specific stage such as division and differentiation during EPC culture. This challenge is quite promising regarding the future of EPC in development of various disease pharmaceutics. Moreover, it is still unresolved which negative factors can potentially inhibit the function of added growth factors or drugs to the EPC culture. It is suggested that more therapeutic targets should be studied which can cause EPC expansion in minimum time while retaining its morphological details.

Figure 1. This schematic diagram shows the summary of endothelial progenitor cell (EPC) isolation, expansion, storage, and transplantation methods. hiPSCs: human induced pluripotent stem cells, MNCs: mononuclear cells, DMSO: dimethyl sulfoxide.

There is no potential conflict of interest to declare.

Conceptualization: ART. Data curation: ART. Formal analysis: ART, ML. MK. Funding acquisition: MK. Investi-gation: MK. Methodology: ART. Project administration: ML, MK. Resources: MK. Software: ART. Supervision: MK. Validation: ART, ML, MK. Visualization: ART. Writing – original draft: ART. Writing – review and editing: ML, MK.

This work was supported by grant from Eisai Korea Inc.