Evolution of autologous endothelial progenitor cell therapy for tissue regeneration and vasculogenesis

Personalized Medicine Universe 5 (2016) 8e15

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Personalized Medicine Universe journal homepage: www.elsevier.com/locate/pmu

Review

Evolution of autologous endothelial progenitor cell therapy for tissue regeneration and vasculogenesis Hiroko Hagiwara, Rica Tanaka* Departments of Plastic and Reconstructive Surgery, School of Medicine, Juntendo University, Tokyo, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 January 2016 Received in revised form 19 April 2016 Accepted 21 April 2016

Endothelial progenitor cell (EPC) transplantation therapy is a promising method in the field of regenerative therapy. EPCs, which were identified in adult peripheral blood, have the ability to differentiate into endothelial cells (ECs). Because of the potential for EPCs to promote tissue regeneration, transplantation of autologous EPCs is now recognized as a novel therapeutic option for revascularization and blood vessel repair in ischemic diseases, diabetic refractory limb ulcers, and other conditions. However, aging and various conditions and disease states, such as diabetes and arteriosclerosis, are associated with reduced numbers and dysfunction of EPCs, which may result in decreased efficacy of autologous therapies. Therefore, in order to achieve better clinical outcomes in the treatment of a variety of conditions and diseases, further advances in regenerative therapies utilizing EPCs are required. In this review, we describe the potential roles of EPCs in vasculogenesis and tissue regeneration. We focus on the development of EPC-based treatments and provide insights into the future of regenerative therapies utilizing EPCs for tailor-made medicine. © 2016 International Society of Personalized Medicine. Published by Elsevier B.V. All rights reserved.

Keywords: Endothelial progenitor cells Regenerative therapy Ischemia Transplantation Cell culture optimization

1. Introduction Blood vessels are distributed throughout the body and are the largest organ in the human body; they play an important role in supplying oxygen and nutrients to all tissues. The functions of blood vessels are indispensable for life; however, aging and various disease states, including diabetes and atherosclerosis, damage vessels, leading to delayed tissue repair and serious ischemic disease. Once all therapeutic modalities are exhausted, many ischemic diseases are difficult to cure, leading to low quality of life or even death. Endothelial progenitor cells (EPCs), which have the potential to differentiate into endothelial cells (ECs) and vascular structures [1], make up the bulk of adult vascular stem cells, and researchers have investigated the potential application of EPCs as a novel therapeutic option for incurable ischemic disease [2e7]. Many preclinical studies have demonstrated the efficacy of EPCs in tissue regeneration and vasculogenesis for the treatment of various diseases. Moreover, because EPCs may be effective in the

* Corresponding author. Department of Plastic and Reconstructive Surgery, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: þ81 03 8313 3111; fax: þ81 03 5812 1225. E-mail address: [email protected] (R. Tanaka).

treatment of many ischemic diseases, EPC therapy has been applied clinically to patients with limb ischemia [7,8]. The first large clinical trial of autologous EPC therapy for limb ischemia demonstrated the safety and efficacy of EPCs in vascular perfusion and limb salvage. The Therapeutic Angiogenesis by Cell Transplantation (TACT) trial was the first multicenter collaborative study of autologous bone marrow-derived mononuclear cell (MNC) implantation in patients with critical limb ischemia. The TACT trial demonstrated the high angiogenic potential of EPCs, which increased blood flow and alleviated symptoms in patients, and confirmed the efficacy and safety of EPC transplantation therapy for critical limb ischemia [7,8]. After publication of the TACT study results, EPC therapy was investigated in various ischemic diseases, including intractable ulcers, bone fractures, spinal cord injuries, and other incurable diseases; positive results were achieved for regenerating tissue through the induction of angiogenesis and vasculogenesis [4,6,9]. Notably, EPCs account for only a very small percentage of the MNC population within the bone marrow, and they are mobilized to the peripheral blood [10,11]. Therefore, collection of a large quantity of bone marrow aspirate or peripheral blood is necessary to obtain sufficient amounts of EPCs, representing a major limitation, particularly for patients with debilitating diseases [12]. Thus, methods for collecting a sufficient number of EPCs with high

http://dx.doi.org/10.1016/j.pmu.2016.04.002 2186-4950/© 2016 International Society of Personalized Medicine. Published by Elsevier B.V. All rights reserved.

H. Hagiwara, R. Tanaka / Personalized Medicine Universe 5 (2016) 8e15

angiogenic potential must be improved to facilitate EPC transplantation therapy in patients with incurable ischemia resulting from various underlying diseases, such as diabetes. For example, several studies have described novel methods, such as ex vivo expansion culture, for better EPC yields from patients with ischemia [3,13e17]. In this review, we will focus on the therapeutic potential and limitations of current EPC therapies and discuss the development of more practical and effective EPC-based therapies for future applications. 2. Basis of EPC therapy Blood vessel formation can be achieved through vasculogenesis and angiogenesis. Vasculogenesis is the process through which new blood vessels are formed by the differentiation and migration of mesodermally derived EPCs to form interconnecting capillaries in early stages of embryonic growth. In contrast, angiogenesis is defined as neovascularization occurring postnatally through the proliferation and migration of pre-existing ECs. However, after elucidating the role of EPCs in vasculogenesis [1,18], researchers concluded that this process also occurs in adults. Thus, vessel formation in adults is now known to occur through the interacting processes of vasculogenesis and angiogenesis [18e21]. EPCs can be identified and characterized through detection of surface markers, such as CD34, CD133, Flk-1/kinase insert domain receptor (KDR), CXCR4, and CD105 in humans, and receptors such as c-kit, sca-1, and CD34 þ Flk-1 (vascular endothelial growth factor receptor 2 [VEGFR2]) in mice [1,18,22e26]. For example, when a part of the body (e.g., the skin) receives an injury stimulus, some chemokines, including stromal cell-derived factor (SDF)-1, angiopoietin (Ang)-1, and granulocyte-colony stimulating factor (G-CSF), respond to the event, and EPCs are mobilized from the bone marrow to the peripheral blood for tissue and vessel repair [27,28]. Blood vessel formation by EPCs is also promoted by indirect effects. The EPCs that migrate to impaired tissue produce various proangiogenic cytokines and growth factors, promoting proliferation and migration of pre-existing ECs, activating angiogenesis, and contributing indirectly to vascular regeneration [27,28]. Urbich et al. analyzed the expression profiles of cytokines in human peripheral blood-derived EPCs by using microarrays [28]. A gene tree analysis revealed high levels of mRNA encoding angiogenic growth factors, such as VEGF-A, VEGF-B, SDF-1, and insulin-like growth factor (IGF)-1, in EPCs. Additionally, significant levels of VEGF, SDF1, and IGF-1 proteins were released into cell culture supernatants of EPCs, consistent with the enhanced mRNA expression. Immunohistological analysis of ischemic limbs from nude rats revealed that VEGF is also released from recruited human EPCs in vivo. Therefore, these previous studies have shown that EPCs exhibit both direct angiogenic effects and autocrine and paracrine effects, including the release of various growth factors and cytokines in ischemic tissues, thereby promoting tissue regeneration. Although the previous reports mention the multifunctional activity of EPCs in promoting tissue regeneration by vasculogenesis and angiogenesis, direct EPC markers have not been identified. Clinically, both CD34positive cells and CD133-positive cells are applied as EPCs; however, not all CD34-positive cells and CD133-positive cells show EPC function. We and other researchers believe that vasculogenic EPCs exist within these cell populations, and that defining these populations is an important topic for further investigation [29]. 3. Preclinical studies of EPC-based therapy To determine the origin and role of EPCs contributing to postnatal vasculogenesis, Asahara et al. performed a preclinical study

9

involving mouse bone marrow transplantation in transgenic mice constitutively expressing b-galactosidase under the transcriptional regulation of an EC-specific promoter, such as Flk-1/LacZ (LZ) or Tie-2/LZ, using wound healing, murine ischemic hindlimb, and myocardial ischemia models [1,18]. They reported that cutaneous wounds examined at 4 and 7 days after skin removal by punch biopsy showed incorporation of EPCs into foci of neovascularization at high frequency. One week after the onset of hindlimb ischemia, LZ-positive EPCs were found to be incorporated into capillaries among skeletal myocytes [18]. After permanent ligation of the left anterior descending coronary artery, histological samples from sites of myocardial infarction demonstrated incorporation of EPCs into foci of neovascularization at the border of the infarct. These results indicated that postnatal neovascularization does not rely exclusively on sprouting from pre-existing blood vessels (angiogenesis); instead, EPCs enter circulation from the bone marrow to contribute to postnatal physiological and pathological neovascularization, which is consistent with postnatal vasculogenesis [18]. Murohara et al. examined whether EPCs could be isolated from umbilical cord blood, a rich source for hematopoietic progenitors, and whether in vivo transplantation of EPCs could modulate postnatal neovascularization in a model of hindlimb ischemia [31]. Numerous cell clusters, spindle-shaped and attaching (AT) cells, and cord-like structures were found to develop from cultures of cord blood MNCs. Fluorescence-trace experiments revealed that cell clusters, AT cells, and cord-like structures were primarily derived from CD34-positive MNCs. AT cells incorporated acetylated low-density lipoprotein (LDL), released nitric oxide, and expressed KDR, VE-cadherin, CD31, and von Willebrand factor but not CD45. Locally transplanted AT cells survived and participated in capillary networks in the ischemic tissues of immunodeficient nude rats in vivo. Moreover, AT cells were shown to exhibit multiple endothelial phenotypes and were defined as a major population of EPCs. Therefore, umbilical cord blood is a valuable source of EPCs, and transplantation of cord blood-derived EPCs represents a promising strategy for modulating postnatal neovascularization. In turn, several studies of CD34-positive cell transplantation have been performed to verify whether transplantation with purified CD34-positive cells has higher vasculogenic potential than transplantation with MNCs, including CD34-negative cells. Schattman et al. investigated the possibility that CD34-expressing leukocytes enriched for angioblasts could be used to accelerate the rate of blood flow restoration in nondiabetic and streptozotocin (STZ)induced diabetic HFh11nu mice undergoing neovascularization due to hindlimb ischemia [32]. Indeed, diabetic mice treated with CD34-positive cells showed significantly greater restoration of blood flow by 2 days after surgery compared with control mice, further suggesting the high angiogenic efficacy of CD34-positive cells. Furthermore, Iwasaki et al. reported a correlation between the amount of CD34-positive cells and vasculogenic function [30]. In their study, peripheral blood-derived CD34-positive cells were isolated from the total MNCs of patients with limb ischemia by apheresis after 5 days of G-CSF administration. Additionally, different amounts of CD34-positive cells were intramyocardially transplanted after ligation of the left anterior descending coronary artery of nude rats. Functional assessments using echocardiography and a microtip conductance catheter at day 28 revealed dosedependent preservation of left ventricular function following CD34-positive cell transplantation. Immunohistochemistry for human-specific brain natriuretic peptide (BNP) demonstrated that human cardiomyocytes were present in the ischemic myocardium at day 28 in a dose-dependent manner. Reverse transcription polymerase chain reaction indicated that human-specific gene expression of cardiomyocyte markers such as BNP, cardiac

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troponin-I, myosin heavy chain, Nkx 2.5, smooth muscle actin, and sm22, as well as EC markers such as CD31 and KDR, was enhanced in a dose-dependent manner in myocardial infarction tissue. This study showed that transplantation of human CD34-positive cells may have significant and dose-dependent effects on vasculogenesis and cardiomyogenesis with functional recovery from myocardial infarction. Taken together, these results of animal experiments revealed that EPC transplantation is effective for ischemic diseases, and that CD34-positive cells have an important role in EPC therapy. Thus, EPC therapies can be applied clinically. 4. Clinical applications of EPC transplantation therapy Current EPC-based therapies can be divided into nonselective EPC therapies and selective EPC therapies. Nonselective EPC therapies are performed by transplanting bone marrow-derived MNCs, including both the EPC fraction and the non-EPC fraction [7,8]. Selective EPC therapies are performed by transplanting purified EPCs from peripheral blood or bone marrow-derived MNCs. The purified EPCs include CD34 þ and CD133 þ cells, which have the potential for vasculogenesis [7,8] (See Table 1). 4.1. Nonselective EPC therapies Based on promising outcomes of preclinical studies, clinical studies of EPCs for the treatment of ischemic diseases and intractable ulcers have begun. A multicenter clinical trial in Japan, the TACT trial, examined the use of EPCs to treat arteriosclerosis obliterans and thromboangiitis obliterans (TAO, Buerger's disease) [7,8,33]. From the data collected during the TACT trial, TateishiYuyama et al. reported that autologous bone marrow-derived MNCs injected into patients with limb ischemia improved patients' walking time on a treadmill and alleviated pain [7]. Moreover, Matoba et al., who described the long-term outcomes of the TACT trial after intramuscular implantation of bone marrowderived MNCs in patients with chronic limb ischemia [8], reported 3-year survival rates of 80% in patients with atherosclerotic peripheral arterial disease (PAD) and 100% in patients with TAO. Importantly, the 3-year amputation-free rates were 60% in patients with PAD and 91% in patients with TAO, and significant improvements measured by leg pain scale, ulcer size, and pain-free walking distance were maintained for at least 2 years after MNC transplantation therapy. Later, in the TACT-NAGOYA-HEART study, Izawa et al. studied intramyocardial injection of autologous bone marrowderived MNCs in patients with severe coronary artery disease [33]. This was the first study to demonstrate an improvement in left ventricular regional myocardial diastolic function as a result of increasing regional myocardial perfusion after intramyocardial injection of autologous bone marrow-derived MNCs. Due to the success of the TACT trial, EPC transplantation has become a promising therapy for intractable ischemic diseases. Thus, because autologous nonselective EPC therapy can be performed rapidly and at low cost, many institutions have begun to apply nonselective EPC therapy in a variety of contexts. 4.2. Selective EPC therapy Selective EPC therapy, performed by transplanting purified CD133-or CD34-positive cells from MNCs, was developed to improve therapeutic efficacy beyond that of nonselective EPC therapy. Because of the scarcity of CD133-or CD34-positive cells in the bone marrow and peripheral blood, MNCs are collected after administration of G-CSF, which stimulates the production of CD133-or CD34-positive cells. These cells are amplified within the

bone marrow and then mobilized to the peripheral blood. Burt et al. reported a small cohort study in which autologous CD133-positive cells were implanted into muscles of the lower extremities of patients with critical limb ischemia, whose only other option was limb amputation [34]. Their results showed that application of an enriched EPC population might improve the safety and efficiency of EPC transplantation for limb salvage in patients with clinical limb ischemia [8]. Kawamoto et al. reported the first phase I/IIa clinical trial of transplantation of autologous peripheral blood CD34positive cells, representing the endothelial and hematopoietic progenitor-enriched fraction, in no-option patients with atherosclerotic PAD or TAO with critical limb ischemia (CLI) [35]. During the 12-week observation period following cell therapy, all patients exhibited improvements in Wong-Baker FACES pain ratings, toe brachial pressure index, transcutaneous partial oxygen pressure, total or pain-free walking distance, and ulcer size. No death or major amputation occurred, and severe adverse events were rare, although mild to moderate events relating to G-CSF administration and leukapheresis were frequent during the 12-week follow-up period. Because this first clinical study of implantation in patients with critical limb ischemia (CLI) was successful, selective EPC therapy has now been applied as a novel treatment for various conditions, such as diabetic refractory limb ulcers, bone fractures, and other ischemic diseases [34e36]. The first phase I/II clinical trial using autologous G-CSF-mobilized peripheral blood CD34-positive cell therapy for nonhealing diabetic ulcers was performed by Tanaka et al. [5]. In this study, nonhealing diabetic ulcers were treated with 2  107 G-CSF-mobilized peripheral blood CD34-positive cells as an EPC-enriched population in five patients. Although a minor amputation was required and recurrence was observed in three out of five patients, no major amputations, deaths, or other serious adverse events were observed following transplantation. Interestingly, patients who were treated with cells having higher numbers of vasculogenic colonies and higher percentages of CD34/KDR double-positive cells showed better clinical outcomes, as demonstrated by faster wound healing and fewer recurrences or heterotopic ulcers. These results suggested that the vasculogenic potential of EPCs and the numbers of EPCs transplanted directly affect the efficacy of EPC cell transplantation therapy [5]. 4.3. Comparison of selective and nonselective EPC therapies Because nonselective therapy does not require isolation of specific subsets of cells and is therefore more cost-effective than selective EPC therapy, many institutions have chosen to apply this type of therapy. However, recent preclinical and clinical studies have shown that the vasculogenic effects of selective EPC therapy are superior to those of nonselective therapy, and that selective EPC therapy results in fewer adverse events than nonselective therapy. For example, Hendrikx et al. showed that the number and percentage of CD34-positive cells in the nonselective therapy responder group were higher than those in the nonresponder group in patients with abnormalities in their global and regional ventricular ejection fractions [37]. Moreover, Hoffman et al. reported that the regional EPC homing rate with selective therapy was higher than that with nonselective therapy in patients with acute myocardial infarction [38]. Kawamoto et al. and Sekiguchi et al. demonstrated that nonselective therapy with samples that included many non-EPCs resulted in severe hemorrhagic infarction and tissue inflammation resulting from the inflammatory cell population within the CD34-negative cell population [22,36]. Because CD34-or CD133-positive cells represents a very small percentage of cells in the bone marrow and peripheral blood, stimulation to increase the number of EPCs, such as by G-CSF

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Table 1 Clinical studies of EPC therapy. Author (Ref.)

Diseases

Number of subject

BM-MNC or PBTateishiYuyama et al. MNC [7]

Transplanted cells

Limb ischemia

Matoba et al. [8]

BM-MNC

Limb ischemia (PAD, TAO)

Izawa et al. [34]

BM-MNC

Coronary artery disease

Burt et al. [35]

CD133þ cell (PB)

Limb ischemia (ASO,TAO,TE)

All endpoint significantly improved in ischemia legs injected MNC. Furthermore, BM-MNC has higher clinical effect compared with PB-MNC. 3years-cohort study of BMRandomized controlled ABI, TcO2(mmHg), Rest PAD: Male¼ 56, MNC transplantation in limb trial pain,Ulcer size, Pain-free Female¼18 TAO: ischemia. Clinical effect of the walkingdistance Male¼34, study was observed 3 years Female¼7 after transplantation. n¼15 unblinded Catheterization, Scintigraphy Intramyocardial injection of (Resting and autologous BM-MNC in exercise),Echocardiography: coronary artery diseases Tissue Doppler imaging and patients improved LV regional Exercise myocardial myocardial diastolic function, contrastecho cardiography as a result of increasing regional ,Treadmill myocardial perfusion. unblinded Amputation , Rest pain SF-36; Transplantation of ASO: Male¼ 4, CD133positive cells physical component score, Female¼3 TAO: significantly improved limb Pain-free treadmill walking Male¼1 TE: ischemia and patients' QOL at time,ABI, Maximum VO2 Male¼1 post six months after the therapy. However, the results werenot seen after one year. (Low dose)PAD: Single blinded TWD,TBPI,Rest pain, TWD at week 12 after PB n¼4, BD: n¼2 (Mild TcO2(mmHg), CD34þ cell therapy significantly increased in a dos)PAD: n¼1, BD: dose-dependent manner in n¼7 (High dose) limb ischemia patients. PAD: n¼0, BD: n¼3 ASO: Male¼ 4 BD: unblinded Rest pain, ABI, TcO2, Among the responders, high Male¼1 Female¼2 Thermography, Angiography numbers of circulating PB CD34þ and CD133þcells persisted for 1 month after the treatment, but not in nonresponders. 4-months follow-up study of Randomized Cardiac MRI,Thallium Control group Scintigraphy, Flow Cytometric intramyocardial Male: n¼10 MBCaracterization of Transplanted transplantation of BM-MNC to MNC group Male: patients with acute myocardial cells n¼7 Female: n¼3 infarction. At 4 months, there was no significant difference in global LVEF, but recovery of regional contractile function in nonviable scar was observed in the transplanted group. Randomized 18F-FDG Radiolabeling BMC-MNC therapy to acute Protocol1:n¼3 myocardial infarction improved Protocol2:n¼3 cardiac functional and the Protocol3:n¼3 homing of the cells to affected area were confirmedby 3D PET. Male:n¼4 Patients treated with cells unblinded Rest pain, Ulcer size, Time having higher numbers of Female:n¼1 ofcomplete wound closure, vasculogenic colonies and Amputation, Adverse effect, higher percentages of CD34/ Angiography, EPC colonyKDR double-positive cells Forming Assay showed better clinical outcomes, as demonstrated by faster wound healing and positive prognosis withourecurrence or heterotopic ulcers.

Kawamoto et al. CD34þ cell (PB) [36]

Limb ischemia (PAD,BD)

Kajiguchi et al. CD34þ,133þ cell [37] (BM,PB(n¼1))

Limb ischemia

Hendrix et al. [38]

Myocardial infarction

BM-MNC

Hofmann et al. BM-MNC CD34þ [39] cell (BM)

Myocardial infarction

Tanaka et al. [5]

Diabetic ulcer

CD34þ cell (PB)

Study design

Male¼ 38 Female¼ GroupA: n¼25, 7 unblended GroupB: n¼20, randomized double-blind

Primary endpoint

Results

ABI, TcO2(mmHg), Rest pain, New collateral, Pain-free walking time

Abbreviations: BM ¼ Bone marrow; PB ¼ Peripheral blood; MNC ¼ Mononuclear cell; CD34þ cells ¼ CD34-positive cells; CD133þ cells ¼ CD133-positive cells; PAD ¼ Peripheral arterial disease; TAO ¼ Thromboangiitis obliterans; TE ¼ Thromboembolic disorder; ASO ¼ Arteriosclerosis obliterans; BD ¼ Buerger's disease; ABI ¼ Ankleebrachial index; Max VO2 ¼ Maximal oxygen consumption/aerobic capacity; SF-36 ¼ Short form-36; TWD ¼ Total walking distance; TBPI ¼ Toe brachial pressure index; TCO2 ¼ Transcutaneous oxygen pressure; MRI ¼ Magnetic resonance imaging; FDG ¼ Fluorodeoxyglucose; PET ¼ Positron emission tomography; KDR ¼ Kinase insert domain receptor; LV ¼ Left ventricular; LVEF ¼ Left ventricular ejection fraction.

injection, is needed for selective EPC therapy. However, G-CSF injection can be an invasive procedure in patients with potentially debilitating diseases. Therefore, new methods are needed to generate large amounts of functional EPCs while limiting the physical burden to patients [5,22,34].

5. Limitations of the present EPC therapy The efficacy and safety of EPC therapy in various diseases, such as ischemic diseases and intractable ulcers, have been established through various clinical studies. However, conventional EPC

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therapies have two major problems. First, the collection of EPCs is currently highly invasive. Presently, EPC therapy requires bone marrow aspiration and apheresis to collect a large number of EPCs; thus, the process represents a large burden for patients [5,22,34]. Second, the number of EPCs is low and the cells can be dysfunctional for various reasons, such as participant age and co-morbidity with diabetes. Therefore, the efficacy of autologous EPC therapy can be limited if applied directly after isolation without any enhancement of its function. Edelberg et al. have shown that aging is associated with a decline in neovascular activity in animal models [39]. Specifically, EPCs isolated from young animals exhibit high expression levels of angiogenic growth factors in vitro, whereas EPCs from aged mice do not. Furthermore, transplantation of bone marrow from young mice into aged mice restores cardiac angiogenesis. Similar to aged individuals, individuals with diabetes have an increased risk of impaired neovascularization in both humans and animal models. Tepper et al. demonstrated that EPCs from patients with diabetes exhibit impairments in processes critical for neovascularization, such as blood vessel growth and tubulization [40]. Moreover, Tanaka et al. reported that impaired mobilization and function of diabetic EPCs represent major limitations to autologous EPC therapy for patients with diabetes having intractable ulcers [5]. Because the mechanism by which EPC function is reduced in geriatric patients and diabetic patients is still unknown, many studies are now attempting to elucidate the mechanisms of EPC dysfunction in aged individuals and patients with diabetes. For example, signaling through pathways such as CCL5/CCR5 and SDF1a/CXCR4 are important for EPC homing, and dysfunctions in these signaling pathways have been reported in patients with diabetes [41,42]. Recent studies have shown that various proteins are modified with reducing sugar, which can lead to deterioration of function. Bhatwadekar et al. demonstrated that advanced glycation of vascular substrates such as fibronectin impairs EPC adhesion, spreading, and migration. Proteins modified with advanced glycation end products are expressed in aged individuals and patients with diabetes [43]. Additionally, Kim et al. recently showed that

high glucose conditions induce autophagy, a cellular pathway involved in protein and organelle degradation, in EPCs [44]. Indeed, conversion of microtubule-associated light chain 3 (LC3)-I to LC3-II is increased in EPCs under high glucose conditions; generation of oxidative stress and disruption of mitochondrial permeability are also observed in this context. Taken together, these studies demonstrate that many mechanisms are involved in impaired vasculogenic potential and reduced numbers of EPCs with aging and in the context of diabetes. Therefore, further improvements in EPC-based therapies need to be tailored for different conditions, such as diabetes, hypertension, or heart failure, as well as the stage of the disease. 6. Ex vivo expansion culture New approaches that facilitate the recovery of EPC function and improve the bioactivity of EPCs for optimal treatment of ischemic diseases and intractable ulcers in patients with diabetes should be considered. In order to enhance the efficacy of EPC transplantation therapy, several groups have developed ex vivo expansion culture systems [3,13e17]. For example, Lippross et al. demonstrated that culture media enriched with bFGF and platelet growth factor (PRGF) boost the expansion of cultured EPCs [14]. Additionally, Ott et al. cultured EPCs in medium enriched with stem cell factor (SCF), VEGF, and stem cell growth factor (SCGF)-b as a method for ex vivo expansion, resulting in improvement of left ventricular function after myocardial infarction in a model of myocardial ischemia [15]. However, these new approaches are still limited by the insufficient quality and quantity of EPCs for transplantation. Accordingly, to improve the quality and quantity of EPCs, Masuda et al. [3] developed a breakthrough culture method called Quality and Quantity culture (QQc), which can expand the number of EPCs and improve the vasculogenic potential of EPCs (Fig. 1). This medium is serum free and enriched with optimal cytokines and growth factors, such as VEGF, thrombopoietin (TPO), SCF, Flit3, and interleukin (IL)-6, and only 7 days of floating culture is needed. Seven days after culture in QQc, human CD133 cells showed

Serum-free expansion culture

Quality and Quantity Culture: QQc Drawing peripheral blood or cord blood

EPCs transplanta on therapy

EPCs isola on (CD34+/CD133+ cells)

Enhance therapeu c poten al of EPC Quan ty : increase cell number Quality : enhance EPC differen a on

5G(VEGF,SCF,TPO,IL-6,Flt-3) Serum-free medium 7days floa ng culture

Fig. 1. Quality and Quantity Culture (QQc) system. Masuda et al. developed a breakthrough culture method, the Quality and Quantity culture system, which can expand the number of EPCs and improve their vasculogenic potential. This system may overcome difficulties with existing EPCs therapies. Abbreviations: CD34þ cells ¼ CD34-positive cells; CD133þ cells ¼ CD133-positive cells.

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Post-QQc diabetic progenitor cell therapy accelerates wound closure. Isola on of c-kit+Sca-1+lin2 (KSL) cells as EPCs from STZ induced diabe c mouse or control mouse femurs and bias .

DAY0

DAY7

DAY14

DAY21

FC Freshly isolated cells

7days serum-free QQc

QC

FD Transplanta on of healthy EPCs (FC, QC) vs diabe c EPC s (FD,QD) vs PBS control in wounded euglycemic mice with dorsal stented ulcer

QD PBS (from Tanaka R et al.,Diabetes, 2014)

Fig. 2. Post-QQc diabetic progenitor cell therapy accelerates wound closure. Freshly isolated diabetic KSL-treated wounds exhibited significantly lower wound closure rates compared to freshly isolated control KSL-treated wounds. In addition, the efficacy of post-QQc diabetic c-kit þ Sca-1 þ lin2 (KSL) cells used in EPC therapy was demonstrated. The percent wound closure achieved with adoptive transfer of diabetic QQc KSL cells was not significantly different from the percent wound closure achieved with adoptive transfer of control QQc KSL on day 14. Post-QQc diabetic EPC therapy effectively improved euglycemic wound closure. Abbreviations: KSL ¼ c-kit þ Sca-1þlin2 cells; STZ ¼ Streptozocin; PBS ¼ Phosphate-buffered saline; FC ¼ Freshly isolated control cells; QC ¼ post-QQc control cells; FD ¼ Freshly isolated diabetic cells; QD ¼ post-QQc diabetic cells.

significantly higher capacity to differentiate into EC lineages with enhanced expression of endothelial markers, such as VEGFR-2, CD146, and von Willebrand factor, compared to that of cells not cultured in QQc. Cells cultured in QQc also exhibited enhanced capacity to form tube-like structures and production of proangiogenic growth factors, such as VEGF, compared with cells not cultured in QQc. Indeed, a 7-day culture of umbilical cord blood-derived CD133-positive cells with QQc produced a 52.9-fold

increase in total cell numbers and a 3.28-fold increase in the frequency of definitive EPC colony forming units. This resulted in a 203.9-fold increase in the estimated total definitive EPC colony forming units in vitro; thus, QQc improved the differentiation potential of EPCs. Additionally, cells precultured with or without QQc were intramyocardially transplanted into nude rats in a model of myocardial infarction. Dose-dependent improvements in global left ventricular contractility were observed with transplantation of

Development of EPCs transplantation therapy

Selec ve

Expansion culture

Bone marrow total MNCs

EPCs- enriched cells

MNC in BM or PB

CD34+/CD133+ cells

Quality and Quan ty enhancement EPCs

Non-selec ve

CD133 cells Cultured CD34+/CD133+

More Effec ve EPC thearpy

M Vascular progenitor cells

Tissue cells

Expanded/improved G-CSF stumula on Purified EPCs

Angiogenesis ( +) Vasculogenesis ( ) Isola on technique ( - ) Medical cost ( -)

Progenitor cells Ex-vivo culture Enriched cytokines & growth factors

Angiogenesis ( ++ ) Vasculogenesis (+) Isola on technique ( + ) Medical cost (+)

Angiogenesis ( ++) Vasculogenesis ( ++) Isola on technique ( ++) Medical cost ( ++ )

Fig. 3. Evolution of autologous endothelial progenitor cell therapies for tissue regeneration and vasculogenesis. Although the technique for cell isolation can be difficult and the cost can be high, EPC-based therapies have been developed to increase the number and bioactive function of EPCs for vascular regeneration. Abbreviations: MNC ¼ Mononuclear cell; BM ¼ Bone marrow; PB ¼ Peripheral blood; EPC ¼ Endothelial progenitor cells; G-CSF ¼ Granulocyte colony-stimulating factor; CD34þ cells ¼ CD34-positive cells; CD133þ cells ¼ CD133-positive cells.

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QQc-cultured cells, further suggesting the superiority of QQc as a culture medium for enhancing vasculogenesis in cell populations containing CD133-positive cells. Furthermore, immunohistochemistry showed more abundant human and rat ECs and cardiomyocytes in the infarcted myocardium following transplantation of cells precultured with QQc compared with those observed after transplantation of cells not precultured with QQc [3]. In a separate model, Tanaka et al. hypothesized that QQcdependent restoration of diabetic EPC function may improve wound closure [6]. To test this hypothesis, they used diabetic bone marrow-derived c-Kit (þ)/Sca-1 (þ)/Lin() (BM-KSL), a cell population that included murine bone marrow-derived EPCs, to investigate cell activity and wound closure. This QQc system significantly increased the number of BM-KSL cells in both diabetic and control groups and increased the vasculogenic potential of diabetic KSL cells compared with that of control KSL cells [6] (Fig. 2). Thus, these results demonstrated that QQc culture could be used to restore murine diabetic EPC dysfunction and expand the number of EPCs in diabetic patients, therefore representing a novel solution to overcome the limitations of existing EPC therapies. 7. Future tasks and perspectives Despite the wide range of outcomes, stem cell therapies are moving rapidly toward clinical application worldwide. Both experimental and clinical results have indicated the promise of EPC transplantation therapies for the treatment of various diseases such as ischemic diseases and intractable ulcers, which are presently difficult to cure. Although new strategies to enhance the survival and longevity of EPCs prior to delivery are being developed, establishing less invasive and more effective therapies for patients with various diseases is still a priority. QQc can generate large amounts of functional EPCs from a small number of cells after only 7 days of serum-free culture; thus, the development of QQc systems may provide qualitative and quantitative advantages in the treatment of ischemic diseases (Fig. 3). Therefore, the goal in EPC therapy is to obtain a sufficient number of functional cells from a small volume of peripheral blood for autologous application of EPCs to induce vasculogenesis and tissue regeneration. We are now modifying the QQc method for outpatient EPC therapies using just a small amount of blood, which can be collected quickly and easily from patients in various clinical states.

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

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[17]

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Funding Our lab is supported by the Research Project for Practical Applications of Regenerative Medicine from the Japan Agency for Medical Research and Development (16bk0104037h0002), AMED, and JSPS KAKENHI (Grant Number 26713051). Conflict of interests

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