Hiroto Iwasaki

CD34-Positive Cells Exhibit Increased Potency and Safety for Therapeutic Neovascularization After Myocardial Infarction Compared With Total Mononuclear Cells

Background— We compared the therapeutic potential of purified mobilized human CD34+ cells with that of mobilized total mononuclear cells (tMNCs) for the preservation/recovery of myocardial tissue integrity and function after myocardial infarction (MI). Methods and Results— CD34+ cells were purified from peripheral blood tMNCs of healthy volunteers by magnetic cell sorting after a 5-day administration of granulocyte colony-stimulating factor. Phosphate-buffered saline (PBS), 5×105 CD34+ cells/kg, 5×105 tMNCs/kg (low-dose MNCs [loMNCs]), or a higher dose of tMNCs (hiMNCs) containing 5×105 CD34+ cells/kg was transplanted intramyocardially 10 minutes after the induction of MI in athymic nude rats. Hematoxylin and eosin staining revealed that moderate to severe hemorrhagic MI on day 3 was more frequent in the hiMNC group than in the PBS and CD34+ cell groups. Immunostaining for human-specific CD45 revealed abundant distribution of hematopoietic/inflammatory cells derived from transplanted cells in the ischemic myocardium of the hiMNC group. Capillary density on day 28 was significantly greater in the CD34+ cell group (721.1±19.9 per 1 mm2) than in the PBS, loMNC, and hiMNC groups (384.7±11.0, 372.5±14.1, and 497.5±24.0 per 1 mm2) (P<0.01). Percent fibrosis area on day 28 was less in the CD34+ cell group (15.6±0.9%) than in the PBS, loMNC, and hiMNC groups (26.3±1.2%, 27.5±1.8%, and 22.2±1.8%) (P<0.05). Echocardiographic fractional shortening on day 28 was significantly higher in the CD34+ cell group (30.3±0.9%) than in the PBS, loMNC, and hiMNC groups (22.7±1.5%, 23.4±1.1%, and 24.9±1.7%; P<0.05). Echocardiographic regional wall motion score was better preserved in the CD34+ cell group (21.8±0.5) than in the PBS, loMNC, and hiMNC groups (25.4±0.4, 24.9±0.4, and 24.1±0.6; P<0.05). Conclusions— CD34+ cells exhibit superior efficacy for preserving myocardial integrity and function after MI than unselected circulating MNCs.

Dose-Dependent Contribution of CD34-Positive Cell Transplantation to Concurrent Vasculogenesis and Cardiomyogenesis for Functional Regenerative Recovery After Myocardial Infarction

Background— Multilineage developmental capacity of the CD34+ cells, especially into cardiomyocytes and smooth muscle cells (SMCs), is still controversial. In the present study we performed a series of experiments to prove our hypothesis that vasculogenesis and cardiomyogenesis after myocardial infarction (MI) may be dose-dependently enhanced after CD34+ cell transplantation. Methods and Results— Peripheral blood CD34+ cells were isolated from total mononuclear cells of patients with limb ischemia by apheresis after 5-day administration of granulocyte colony-stimulating factor. PBS and 1×103 (low), 1×105 (mid), or 5×105 (high) CD34+ cells were intramyocardially transplanted after ligation of the left anterior descending coronary artery of nude rats. Functional assessments with the use of echocardiography and a microtip conductance catheter at day 28 revealed dose-dependent preservation of left ventricular function by CD34+ cell transplantation. Necropsy examination disclosed dose-dependent augmentation of capillary density and dose-dependent inhibition of left ventricular fibrosis. Immunohistochemistry for human-specific brain natriuretic peptide demonstrated that human cardiomyocytes were dose-dependently observed in ischemic myocardium at day 28 (high, 2480±149; mid, 1860±141; low, 423±9; PBS, 0±0/mm2; P<0.05 for high versus mid and mid versus low). Immunostaining for smooth muscle actin and human leukocyte antigen or Ulex europaeus lectin type 1 also revealed dose-dependent vasculogenesis by endothelial cell and SMC development after CD34+ cell transplantation. Reverse transcriptase–polymerase chain reaction indicated that human-specific gene expression of cardiomyocyte (brain natriuretic peptide, cardiac troponin-I, myosin heavy chain, and Nkx 2.5), SMC (smooth muscle actin and sm22&agr;), and endothelial cell (CD31 and KDR) markers were dose-dependently augmented in MI tissue. Conclusions— Human CD34+ cell transplantation may have significant and dose-dependent potential for vasculogenesis and cardiomyogenesis with functional recovery from MI.

Fracture induced mobilization and incorporation of bone marrow-derived endothelial progenitor cells for bone healing†

We recently reported that systemic administration of peripheral blood (PB) CD34+ cells, an endothelial progenitor cell (EPC)‐enriched population, contributed to fracture healing via vasculogenesis/angiogenesis. However, pathophysiological role of EPCs in fracture healing process has not been fully clarified. Therefore, we investigated the hypothesis whether mobilization and incorporation of bone marrow (BM)‐derived EPCs may play a pivotal role in appropriate fracture healing. Serial examinations of Laser doppler perfusion imaging and histological capillary density revealed that neovascularization activity at the fracture site peaked at day 7 post‐fracture, the early phase of endochondral ossifification. Fluorescence‐activated cell sorting (FACS) analysis demonstrated that the frequency of BM cKit+Sca1+Lineage− (Lin−) cells and PB Sca1+Lin− cells, which are EPC‐enriched fractions, significantly increased post‐fracture. The Sca1+ EPC‐derived vasuculogenesis at the fracture site was confirmed by double immunohistochemistry for CD31 and Sca1. BM transplantation from transgenic donors expressing LacZ transcriptionally regulated by endothelial cell‐specific Tie‐2 promoter into wild type also provided direct evidence that EPCs contributing to enhanced neovascularization at the fracture site were specifically derived from BM. Animal model of systemic administration of PB Sca1+Lin− Green Fluorescent Protein (GFP)+ cells further confirmed incorporation of the mobilized EPCs into the fracture site for fracture healing. These findings indicate that fracture may induce mobilization of EPCs from BM to PB and recruitment of the mobilized EPCs into fracture sites, thereby augment neovascularization during the process of bone healing. EPCs may play an essential role in fracture healing by promoting a favorable environment through neovascularization in damaged skeletal tissue. J. Cell. Physiol. 215: 234–242, 2008.

Local Delivery of Granulocyte Colony Stimulating Factor-Mobilized CD34-Positive Progenitor Cells Using Bioscaffold for Modality of Unhealing Bone Fracture

We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor‐enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony‐stimulating factor‐mobilized peripheral blood (GM‐PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM‐PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM‐PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen‐ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 105 (Hi), 104 (Mid), or 103 (Lo) doses of GM‐PB CD34+ cells or phosphate‐buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM‐PB CD34+ cells, enhancement of the intrinsic angio‐osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM‐PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing.

Cardiomyocyte Formation by Skeletal Muscle-Derived Multi-Myogenic Stem Cells after Transplantation into Infarcted Myocardium

BACKGROUND Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported. METHODS AND RESULTS Skeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed. CONCLUSIONS AND SIGNIFICANCE Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum‐free quality and quantity control culture system for colony‐forming EPCs to enhance their regenerative potential. A culture with serum‐free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin‐6, and Flt‐3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony‐forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood‐CD133+ cells for 7 days produced a 52.9‐fold increase in total cell number and 3.28‐fold frequency in definitive EPC colony development, resulting in a 203.9‐fold increase in estimated total definitive EPC colony number in vitro. Pre‐ or post‐QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer‐tipped conductance catheter examinations 28 days post‐MI revealed significant preservation of left ventricular (LV) function in rats receiving pre‐ or post‐QQc cells compared with those receiving phosphate‐buffered saline. Assessments of global LV contractility indicated a dose‐dependent effect of pre‐ or post‐QQc cells and the superior potency of post‐QQc cells over pre‐QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post‐QQc cells compared with pre‐QQc cells. Our optimal serum‐free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

PlGF Repairs Myocardial Ischemia through Mechanisms of Angiogenesis, Cardioprotection and Recruitment of Myo-Angiogenic Competent Marrow Progenitors

Rationale Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected. Objective We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity. Methods and Results Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1+/Lin− (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage. Conclusions Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease.

Therapeutic Potential of Unrestricted Somatic Stem Cells Isolated from Placental Cord Blood for Cardiac Repair Post Myocardial Infarction

Objective—Unrestricted somatic stem cells (USSCs) were successfully identified from human cord blood. However, the efficacy of USSC transplantation for improving left ventricular (LV) function post myocardial infarction (MI) is still controversial. Methods and Results—PBS, 1×106 human fibroblasts (Fbr), 1×105 USSCs (LD), or 1×106 USSCs (HD) were transplanted intramyocardially 20 minutes after ligating the LAD of nude rats. Echocardiography and a microtip conductance catheter at day 28 revealed a dose-dependent improvement of LV function after USSC transplantation. Necropsy examination revealed dose-dependent augmentation of capillary density and inhibition of LV fibrosis. Dual-label immunohistochemistry for cardiac troponin-I and human nuclear antigen (HNA) demonstrated that human cardiomyocytes (CMCs) were dose-dependently generated in ischemic myocardium 28 days after USSC transplantation. Similarly, dual-label immunostaining for smooth muscle actin and class I human leukocyte antigen or that for von Willebrand factor and HNA also revealed a dose-dependent vasculogenesis after USSC transplantation. RT-PCR indicated that expression of human-specific genes of CMCs, smooth muscle cells, and endothelial cell markers in infarcted myocardium were significantly augmented in USSC-treated animals compared with control groups. Conclusions—USSC transplantation leads to functional improvement and recovery from MI and exhibits a significant and dose-dependent potential for concurrent cardiomyogenesis and vasculogenesis.

Synchrotron Radiation Coronary Microangiography for Morphometric and Physiological Evaluation of Myocardial Neovascularization Induced by Endothelial Progenitor Cell Transplantation

Background—Therapeutic effect of stem cell transplantation (SCTx) for myocardial neovascularization has been evaluated by histological capillary density in small animals. However, it has been technically difficult to obtain imaging evidence of collateral formation by conventional angiography. Methods and Results—Peripheral blood CD34+ and CD34− cells were isolated from patients with critical limb ischemia. PBS, CD34− cells, or CD34+ cells were intramyocardially transplanted after ligating LAD of nude rats. Coronary angiography of ex vivo beating hearts 5 and 28 days after the treatment was performed using the third generation synchrotron radiation microangiography (SRM), which has potential to visualize vessels as small as 20 &mgr;m in diameter. The SRM was performed pre and post sodium nitroprusside (SNP) to examine vascular physiology at each time point. Diameter of most collateral vessels was 20 to 120 &mgr;m, apparently invisible size in conventional angiography. Rentrop scores at day 28 pre and post SNP were significantly greater in CD34+ cell group than other groups (P<0.01). To quantify the extent of collateral formation, angiographic microvessel density (AMVD) in the occluded LAD area was analyzed. AMVD on day 28 post SNP, not pre SNP, was significantly augmented in CD34+ cell group than other groups (P<0.05). AMVD post SNP closely correlated with histological capillary density (R=0.82, P<0.0001). Conclusions—The SRM, capable of visualizing microvessels, may be useful for morphometric and physiological evaluation of coronary collateral formation by SCTx. The novel imaging system may be an essential tool in future preclinical/translational research of stem cell biology.

Lnk-dependent axis of SCF–cKit signal for osteogenesis in bone fracture healing

The therapeutic potential of hematopoietic stem cells/endothelial progenitor cells (HSCs/EPCs) for fracture healing has been demonstrated with evidence for enhanced vasculogenesis/angiogenesis and osteogenesis at the site of fracture. The adaptor protein Lnk has recently been identified as an essential inhibitor of stem cell factor (SCF)–cKit signaling during stem cell self-renewal, and Lnk-deficient mice demonstrate enhanced hematopoietic reconstitution. In this study, we investigated whether the loss of Lnk signaling enhances the regenerative response during fracture healing. Radiological and histological examination showed accelerated fracture healing and remodeling in Lnk-deficient mice compared with wild-type mice. Molecular, physiological, and morphological approaches showed that vasculogenesis/angiogenesis and osteogenesis were promoted in Lnk-deficient mice by the mobilization and recruitment of HSCs/EPCs via activation of the SCF–cKit signaling pathway in the perifracture zone, which established a favorable environment for bone healing and remodeling. In addition, osteoblasts (OBs) from Lnk-deficient mice had a greater potential for terminal differentiation in response to SCF–cKit signaling in vitro. These findings suggest that inhibition of Lnk may have therapeutic potential by promoting an environment conducive to vasculogenesis/angiogenesis and osteogenesis and by facilitating OB terminal differentiation, leading to enhanced fracture healing.