Non-hEPC is almost unified to one kind of endothelial outgrowth cells (EOCs). EOCs-produced from PB ex vivo reported initially by Lin et al. (2000). The concept of adhesive endothelial lineage cell stages using the original culture conditions disclosed clonal colony-forming units of outgrowth ECs cultured from MNCs or blood vessel ECs by Ingram and Yoder et al. (Ingram et al., 2004; Ingram et al., 2005a). These proliferative endothelial lineage cells were termed ECFC, also known in the literature as EOCs or late EPCs. ECFCs do not likely represent the primary “EPC in vivo” phenotype found in our body but rather reflect an EC phenotype emerging from cultured cells (Lin et al., 2000) or vessel wall ECs (Figure 1) (Ingram et al., 2005b). Questions regarding the definition of the primary EPC underlying the ECFC phenotype and in vivo origin are still missing. Initially, ECFCs were identified from the long-term culture of PB (Lin et al., 2000; Hur et al., 2004) or CB-MNCs (Ingram et al., 2004). These were followed by publication of Smadja et al. and Delorme et al., showing ECFCs can be derived from CD34⁺ cells or CD146⁺/CD34⁺/CD45⁺/CD133⁺ or CD177⁺ cells (Delorme et al., 2005; Smadja et al., 2005; Lee et al., 2013; Boisson-Vidal et al., 2018). Additional studies by Case et al. and Timmermans et al. revealed that CD34⁺/CD45⁻ cells could give rise to ECFCs but not a hematopoietic colony (Case et al., 2007; Timmermans et al., 2007). A later publication by Tura et al. confirmed CD34⁺/CD146⁺/CD133⁻ cells are the source of ECFC precursor cells (Tura et al., 2013). They also claimed ECFC precursors might not be derived from BM, because BM-MNCs did not contain CD34⁺/CD146⁺/CD133⁻ cells and did not give rise to any ECFC colony. This discussion was partly supported by a recent publication using fluorescence in situ hybridization analysis of ECFCs derived from allogenic BM transplant patients (Fujisawa et al., 2019). The findings about the fraction of ECFC-producing cells in vivo are somewhat contradicting between the publications regarding CD45 positivity and CD133 positivity, while CD34 positivity is agreed by common consent.

ECFCs have been successfully isolated from not only CB and PB but also from fat tissue (Lin et al., 2013), placenta (Patel et al., 2013b), lungs (Alphonse et al., 2014) and saphenous vein (Green et al., 2017b); these results imply that ECFCs may derived from tissue resident vasculature progenitors. Recent reports indicate a candidate of ECFC origin locates in ECs of blood vessels (Salybekov et al., 2022). The mechanism of non-hEPC mobilization into circulation and physiological functions in kinetics are not defined yet.

Although the proof of BM-derived ECFCs is not approved by the former reports, there are several publication demonstrating that a pluripotent stem cell known as “very small embryonic-like stem cell” (VSEL) is a lineage-negative, CD34⁺, CD133⁺ and CD45⁻ cell with very small size (<6 µm length) located in BM, and differentiate into ECFC-like ECs in human (Havens et al., 2014; Guerin et al., 2015; Lahlil et al., 2018; Domingues et al., 2022). Also other reports disclosed VSELs derived from cord blood CD34⁺ cell differentiated into ECFCs ex vivo (Lahlil et al., 2018) (Domingues et al., 2022). We need further insights and discussions regarding the origin of ECFCs.

Postnatal EPCs was thought closely related to that of HSC populations, both being isolated by cell surface antigens, including CD34, CD133, VEGFR-2, CXCR4, CD105, etc., in human, or c-Kit, Sca-1, Flk-1, etc., in mouse (Peichev et al., 2000; Quirici et al., 2001b; Eggermann et al., 2003; Handgretinger et al., 2003; Alessandri et al., 2004). However, the identification of an exact primary hEPC phenotype was missing, mainly due to the lack of a definitive assay system capable of determining and distinguishing an EPC unambiguously from an HSC.

The initially developed culture assays and colonies (Murohara et al., 2000b; Kalka et al., 2000; Hill et al., 2003) grouped multifarious EPCs into single qualitative class: “adhesive cultured EPCs”, without any hierarchy or proper identification of unwanted cells, while the colonies detected by Hill’s colony assay (Hill et al., 2003) turned out to consist of not colony-forming EPCs, but mainly other hematopoietic cells (Hur et al., 2007; Rohde et al., 2007). Later, an improved EPC-colony forming assay (EPC-CFA) was developed, challenging the field’s traditional views and opening the door to illustrating the developmental hierarchy of hEPCs (Masuda et al., 2011).

EPCs are thought to release autocrine elements for their nutritional support, as well as paracrine factors to develop vasculature and repair of impaired vessels (Figure 2). In vivo EPCs, isolated as CD34⁺ from PB or BM, have been reported to secrete numerous angiogenic elements, such as vascular endothelial growth factor (VEGF), hepatic growth factor (HGF), insulin growth factor-1 (IGF-1), interleukin-8 (IL-8), fibroblast growth factor (FGF) (Majka et al., 2001; Jarajapu et al., 2014) angiopoietin-1, platelet-derived growth factor (PDGF) (Fang et al., 2020), etc. Ex vivo EPCs such as early EPCs as well as ECFCs also represented a similar finding to express a large number of angiogenic factors although the level of VEGF and IL-8 were higher in early EPCs than ECFCs (Table 2) (Cheng et al., 2013).

Collaborative factors with the capacity to promote both immunomodulation and anti-inflammation, such as TGF-β, IL-10, IL-4, IL-6, PGE2, M-CSF, G-CSF, adenosine, etc., have been investigated for years and reported to be expressed by properly stimulated hematopoietic lineage cells (Motz and Coukos, 2011; Rivera and Bergers, 2015). This co-creative property inherently applies to EPCs in regenerative tissues. EPCs secrete multiple factors for both anti-inflammation and immunosuppression, such as VEGF (Jarajapu et al., 2014), PGE2 (Carneiro et al., 2019), TGF-β (Acosta et al., 2019), IL-4, IL-6 (Kado et al., 2018), IL-10 (Zullo et al., 2015) and G-CSF (Wen et al., 2019; Alwjwaj et al., 2021; Yan et al., 2022). Especially, IL-10 and TGF-β are expressed and play a key role in immunomodulation and anti-inflammation in CD34⁺ cells from PB or BM (Majka et al., 2001; Ratajczak et al., 2013; Jarajapu et al., 2014). IL-10 is a strong anti-inflammatory and immunoregulatory cytokine with a variety of direct and indirect effects on innate and adaptive immunity through the induction of M2 macrophages and Treg polarization (Mosser and Zhang, 2008). In acute myocardial infarction (MI) models, IL-10 suppresses in vivo inflammatory responses, which contributes to improved myocardial recovery. TGF-β₁ is a multifunctional cytokine involved in a variety of essential activities, such as embryonic evolution, cellular development, wound recovery, and immunomodulation, which contributes to the anti-inflammatory feedback by encouraging the proliferation of Tregs and inhibiting the differentiation of Th cells (Mantel and Schmidt-Weber, 2011).

MicroRNAs (miRNAs) are a class of non-coding RNAs and have critical functions in the modification of gene expression. Most of them are transcribed from DNA into primary miRNAs, modified into initial miRNAs, and ultimately made into mature miRNAs (Mohr and Mott, 2015). Seminal study showed that CD34⁺ EPC secreted extracellular vesicles (EVs) and activated an angiogenic program in ECs through horizontal mRNA transfer (Table 3) (Deregibus et al., 2007; Sahoo et al., 2011; Xu et al., 2020; Xiong et al., 2022).

According to studies, CB-CD34⁺ EPCs concentrated in the regenerating muscles and improved vasculogenesis and in a murine hindlimb ischemia model, leading to enhanced limb salvage and hemodynamic recovery (Murohara et al., 2000a; Schatteman et al., 2000; Madeddu et al., 2004). Collectively, CD34⁺ EPCs efficiently improve blood flow and vasculogenesis in the hindlimb ischemic models.

According to data from a mouse model of experimental acute myocardial infarction (AMI), human CD34⁺ cells imbedded in the hypoxic/ischemic region and shifted toward cardiomyocytes and ECs, which related to improved heart utility (Kawamoto et al., 2006). Shalaby et al. compared the efficacy of human CB-CD34⁺ and CB-MSCs for the regenerate of heart tissue by stimulation of neoangiogenesis. They reported that the CD34⁺-treated group notably reduced the infarct zone and the amplitude of the T-wave and increased VEGF, Ang-1, HIF-1α, and Tie-2 levels compared to the MSC-treated subjects, suggesting the supremacy of CD34⁺ therapy vs. MSCs in the induction of regenerative pothential (Shalaby et al., 2016). Supportively, human CD34⁺ cells increased angiogenesis and reduced pro-inflammatory cytokines in the infarct zones, leading to improved cardiac function in a rat AMI model (Kim et al., 2016). Moreover, Kawamoto et al. found that PB-derived CD34⁺ EPCs improved regional wall motion, fractional shortening, left ventricle ejection fraction (LVEF), and myocardial neovascularization (Kawamoto et al., 2001). Also, in another study, they showed that CD34⁺ EPCs increased capillary density and decreased the fibrosis rate of the infarct area in the experimental AMI model (Kawamoto et al., 2003). Supportively, Yeh et al. reported that human G-CSF mobilized PB-CD34⁺ cells increased angiogenesis through migration and retention into the myocardium and trans-differentiation into ECs in ischemic tissues (Yeh et al., 2003). Furthermore, transplanting of PB-CD34⁺ cells reduce structural changes in the infarct zone and eliminates any signs of inflammatory cell recruitment seen in the transplantation of total MNCs in a rat MI model (Kawamoto et al., 2006). Also, myocardial injection of BM-CD34⁺ cells improved the vascular network and fractional shortening in the macaque MI model, supporting the efficacy of CD34⁺ cells in a large animal model of MI (Yoshioka et al., 2005). Taken together, records from preclinical studies propose that CD34⁺ cells differentiate into ECs, integrate into vascular system, and secrete angiogenic factors, promoting vascular regeneration in the microcirculation and improving myocardial perfusion in ischemia-induced tissue damage.

The effectiveness of CD34⁺ EPC therapy on the protection of CKD (chronic kidney disease) renal function has only been documented in a small number of experimental investigations. Two previous experimental studies using small and large preclinical experiments of CKD revealed that renal CD34⁺ EPC therapy considerably refurbished kidney function. In detail, Sangidorj et al. demonstrated that the administration of BM-CD34⁺ EPCs immunologically trafficked in the injured kidney site and postponed the collapse of kidney function aside from reduced proteinuria. As well, angiogenic molecules were increased and proinflammatory cytokines and adhesion molecules were decreased, resulting in functional and structural renal preservation in a mouse model of chronic renal failure (Sangidorj et al., 2010). In a large animal model, Chade et al. reported that intrarenal autologous PB-derived KDR⁺/CD34⁺ EPC injection preserved microvascular architecture and function and diminished microvascular remodeling in an experimental stenotic kidney, showing renoprotective effects of CD34⁺ EPC in a pig model of chronic renal artery stenosis (Chade et al., 2009). Also, Ohtake et al. reported the effectiveness of G-CSF mobilized human CD34⁺ therapy in a mice experimental model of ischemic/reperfusion acute kidney injury (AKI), which drastically upgraded renal function and repaired loss of peritubular capillaries because of ischemic condition (Ohtake et al., 2018a). Mechanistically, Vinas et al. reported that delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p through targeting PTEN signaling pathway (Viñas et al., 2016). Recently, Huang et al. demonstrated that PB-CD34⁺ EPC therapy excellently inhibits CKD progression and kidney homeostasis deterioration by means of improvement of neoangiogenesis, blood stream, and potent anti-oxidative, anti-inflammatory, and anti-apoptosis/fibrosis capacities in a rat model (Huang et al., 2015). Taken together, the present knowledge regarding CD34⁺ therapy for kidney-related diseases supports CD34⁺ as a promising therapeutic intervention for preserving kidney functions in renovascular disease.

As above mentioned, CD34⁺ cells supply anti-inflammatory factors via paracrine activities. Comparing the cardioprotective effects of MSCs and CD34⁺ cells, Shalaby et al. demonstrated that CB-CD34⁺ cell therapy significantly decreased recruitment of inflammatory cells and increased angiogenic factors compared to the MSCs-treated group, which discloses the superiority of CD34⁺ therapy over MSCs in therapeutic potential (Shalaby et al., 2016). Of note, CB-derived CD133+/CD34⁺ stem/progenitors exhibit elevated amount of migratory (CXCR4)- and adhesive (LFA-1)-related antigens, allowing them to migrate and home to inflammatory areas that express higher levels of SDF-1α, a CXCR4 ligand (Das et al., 2009). Furthermore, CD34⁺ cell therapy reduces the secretion of inflammatory factors, namely TNF-α, IL-1, IL-6, and NOS2A in the lesion context while overexpression of IL-10. Moreover, human CB-CD34⁺ therapy mitigates NF-κB signaling pathway in dermal fibroblasts by increasing IL-10, providing novel mechanistic evidence of CD34⁺ cell-mediated anti-inflammatory response in a mouse model of wound healing. It worth noting that IL-10 binds to NF-κB and inhibits transcriptional function of this pathway (Kanji et al., 2014). Also, it has been shown that CD34⁺ cells enhanced epicardial and coronary microcirculation via paracrine activity–mediated angiogenic response and secretion of anti-inflammatory factors (Mackie and Losordo, 2011).

Regarding the therapeutic effects of CD34⁺ cells in cerebral ischemia, Taguchi et al. have clarified that CB-derived CD34⁺ cells ameliorated angiogenesis and neurogenesis rates (Taguchi et al., 2004). Of note, injections of PB-derived CD34⁺ cells developed vasculature, neurogenesis, and locomotor activity in a chronic stroke rat experiment (Shyu et al., 2006). Supportively, Tsuji and colleagues reported that CB-CD34⁺ cells increased blood stream to the ischemic zone and decreased missing of ipsilateral hemisphere volume in a neonatal stroke model (Tsuji et al., 2014). Also, CB-CD34⁺ cells alleviates pathological brain injury and improves the neurobehavioral status in a cerebral palsy model (Chang et al., 2021). More recently, Ogawa et al. illustrated that transplantation of PB-CD34⁺ cells improved neurological functions, including grip strength test (to evaluate neuromuscular function), the water maze assay (to evaluate the spatial education skill), and the rotarod test (to evaluate sensorimotor skills) in a murine SCID chronic stroke model, providing another preclinical proof of the CD34⁺ therapy concept in ischemic strokes (Ogawa et al., 2022).

For the first time, Tateishi et al. introduced the clinical utility of PB-CD34⁺ cells in an ischemic limb setting. Based on their results, autologous BM-CD34⁺ therapy ameliorate pain rate and increase walking distance 7 days after transplantation (Tateishi-Yuyama et al., 2002).

In a phase I/IIa trial of CLTI, Kawamoto and coworkers illustrated that G-CSF mobilized CD34⁺ cell transplantation enhanced pain grade, pain-free walking distance, toe brachial pressure index (TBPI), transcutaneous partial pressure of oxygen (TcPO₂), and decreased ulcer size after 3 months of injection (Kawamoto et al., 2009); Supportively, Kinoshita et al. found that TBPI and TcPO₂ parameters improved significantly 4 years after treatment, and no major amputations occurred in this period, indicating the immunity, feasibility, and efficiency of G-CSF mobilized PB-CD34⁺ cell transplantation in CLTI individuals (Kinoshita et al., 2012). Fujita et al. conducted a phase II clinical trial to determine endpoint selection and timing, which revealed significant improvements in perfusion pressure, pain grade, TcPO₂, pain-free walking distance, and TBPI (Fujita et al., 2014). Ohtake et al. reported that G-CSF-mobilized CD34⁺ therapy increased the amputation- and CLTI-free survival index in CLTI individuals without any cell therapy-related side effects (Ohtake et al., 2018b). Tanaka and colleagues studied five dialysis individuals having non-healing diabetic feet in a phase I/IIa trial of G-CSF-mobilized CD34⁺ therapy. 18 weeks after treatment, all of the patients’ wounds had healed completely without serious adverse events (Tanaka et al., 2014). According to the initial randomized and double-blind pilot study regarding CD34⁺ therapy of CLTI, autologous G-CSF mobilized CD34⁺ therapy boosts myogenic and angiogenic activity while decreasing the amputation-free survival rate in a dose-dependent trend at 12 months (Losordo et al., 2012). This finding was later confirmed by a double-blind randomized pilot trial, which suggested that patients with severe intermittent claudication receiving autologous G-CSF mobilized CD34⁺ therapy experienced improved quality of life and longer walking distances (Losordo et al., 2011a). Fang et al. evaluated the long-term efficacy of G-CSF mobilized CD34⁺ cells in CLTI patients. Based on their results, CD34⁺ cells not only increased the amputation‐free survival rate, peak pain‐free walking time (PPFWT), ulcer healing rate, and SF‐36v2 score, but also decreased the Wong‐Baker faces pain rating scale and the recurrence frequency during 5years follow-up period (Fang et al., 2018).

It is worth noting that since 2017, Japan’s ministry of health (MHLW) has permitted the G-CSF-mobilized CD34⁺ cell transplantation for CLTI patients as an advanced type B medical treatment. In addition, a multicenter comparative trial of CLTI subjects aimed at guiding authorization of CD34⁺ therapy as a novel regenerative medicine-made merchandise (Identifier: NCT02501018) was launched in Japan in 2017. The MHLW designated this regenerative medicine product as an applicable pharmaceutical composition for Sakigake Strategy in 2018. The trial was finished in May 2022 with the concluding data still pending.

Coronary microvascular dysfunction leads to angina and poor outcomes in ischemic coronary artery disease, and no specific treatment exists. As mentioned above, CD34⁺ cell transplantation expands microcirculation and improves symptoms, exercise tolerance, and mortality in preclinical studies of refractory angina patients. Nowadays, CD34⁺ transplantation has been assessed in several double-blind and randomized trials in these patients. In phase I/IIa clinical trial using the NOGATM mapping system, autologous G-CSF mobilized PB-CD34⁺ therapy improved the frequency of angina, exercise tolerance, and CCS scale, providing preliminary evidence of the immunity and bioactivity of CD34⁺ cell injection in patients with refractory angina (Losordo et al., 2007). Furthermore, in a prospective, double-blind, randomized, phase II study of refractory angina, autologous G-CSF mobilized CD34⁺ therapy considerably decreased angina frequency and upgraded exercise endurance, angina onset time, and CCS classification without any adverse cardiovascular side effects (Losordo et al., 2011b). A second year of follow-up study shows that autologous G-CSF mobilized CD34⁺ cell transplantation diminishes angina frequency, unwanted heart outcomes, and mortality rates (Henry et al., 2016). These findings are further validated in phase III of the randomized, double-blind study, which shows improved immunity and organ homeostasis in refractory angina patients undergo autologous G-CSF mobilized CD34⁺ therapy (Povsic et al., 2016). Most recently, Henry et al. demonstrated that autologous G-CSF mobilized CD34⁺ cells improves coronary flow reserve, CCS class, and life quality (based on the Seattle Angina Questionnaire (SAQ)) and mitigates angina frequency at 6 months after treatment (Henry et al., 2022). Supportively, Corban et al. documented that autologous G-CSF mobilized CD34⁺ therapy improved microvascular blood flow and SAQ scores and decreased CCS class, nitroglycerin use, and Wilcoxon signed-rank test at 6 months of follow-up (Corban et al., 2022). Johnson et al. reported that autologous G-CSF mobilized CD34⁺ therapy for refractory angina patients is associated with an improved mortality rate and reduced cardiac-related hospitalization in the year following injection compared to the year prior to transplantation (Johnson et al., 2020). In a one more general note, it could be said that CD34⁺ therapy is immune and could be a potent operative regenerative approach for microvascular dysfunction and angina patients.

According to Hofmann et al. injected CD34⁺ cells may effectively accumulate in the area of ischemia and improve cardiac regeneration, While a significant portion of the injected-MNCs cramped in the spleen, liver, and the infarcted myocardium’s center, providing another proof of the superiority of CD34⁺ cells over MNCs (Hofmann et al., 2005). In 2009, Pasquet et al. launched a pilot study using autologous G-CSF-mobilized PB-CD34⁺ cells to treat AMI subjects, which resulted in increased LVEF and sustained structural/functional scar repair (Pasquet et al., 2009). In a phase I dose-dependent investigation of subjects with LVEF≤ 50% after angioplasty for STEMI, Quyyumi et al. found that BM-CD34⁺ cells improved myocardial perfusion and reduced infarct size in a dose-dependent manner (Quyyumi et al., 2011). The PreSERVE-AMI was the largest randomized phase II trial that used autologous BM-CD34⁺ cell transplantation of LV dysfunction post-STEMI. After 12 months, transplanted subjects showed decreased infarct size, hospitalized days, and mortality compared to the non-treated group and increased LVEF in a dose-dependent manner, providing additional proof of the immunity and efficiency of CD34⁺ therapy in STEMI subjects (Quyyumi et al., 2017). Besides, Poglajen et al. demonstrated potential antiarrhythmic benefits of autologous G-CSF mobilized CD34⁺ cells on ventricular arrhythmias subjects (Poglajen et al., 2019). These findings further supported the immunity and efficiency of CD34⁺ cell transplantation in STEMI-suffered individuals.

According to the first report of using autologous G-CSF mobilized CD34⁺ therapy in acute ischemic stroke patients, CD34⁺ cells improved modified rankin score (MRS) and national institutes of health stroke score (NIHSS) in the 6-month follow-up (Banerjee et al., 2014). Similarly, Chen et al. showed in a randomized, single-blind, controlled trial that autologous G-CSF mobilized CD34⁺ therapy improved the NIHSS, the European stroke score (ESS), and the ESS motor score without any serious adverse events in patients with a MCAI (Chen et al., 2014). Furthermore, Sung et al. reported that autologous G-CSF mobilized PB-CD34⁺ cell transplantation increased angiogenesis, the Barthel index, and cognitive ability screening instrument score (CASI) at 6-month follow-up after transplantation without any recurrent stroke or tumorigenesis (Sung et al., 2018). Based on these findings, a randomized double-blind study of CD34⁺ therapy in chronic ischemic stroke (Identifier: jRCT2052200112) began in 2020 and is still recruiting subjects.

Regarding CD34⁺ clinical trials of kidney disease, in a randomized controlled study, Yang et al. recently found that intra-renal transplantation of autologous G-CSF mobilized CD34⁺ cells for CKD patients significantly reduced adverse clinical outcomes (dialysis and death) at 1 year compared to the control group, but did not improve kidney function (Yang et al., 2020). Furthermore, according to Lee et al., G-CSF-mobilized CD34⁺ transplantation into the right renal artery is 100% safe and maintains the renal function in a stationary state, suggesting that CD34⁺ transplantation could mitigate the collapse of renal homeostasis in CKD subjects (Lee et al., 2017). Besides, in phase I/II study, Suzuki and colleagues reported that direct injection of G-CSF-mobilized CD34⁺ cells to both renal arteries of a 36 years old AKI patient significantly improved the serum creatinine level, GFR, angiogenic-related cytokines, and consequently kidney function 23 weeks after cell therapy without major adverse events (Suzuki et al., 2021). Therefore, CD34⁺ cell transplantation could apply renoprotective effects by improved angiogenesis and anti-inflammation capability and could be considered a new therapeutic tool for kidney disorders.