Cell Therapy For Ischemic Disease Research Articles

An Injectable Decellularized Matrix That Improves Mesenchymal Stem Cell Engraftment for Therapeutic Angiogenesis.

Stem cell therapy has great potential for the treatment of ischemic diseases, but poor engraftment of implanted stem cells limits the therapeutic efficacy. Here, we developed an approximately 80 μm injectable decellularized matrix (IDM) to increase the angiogenic efficacy of mesenchymal stem cells by improving the engraftment of the stem cells implanted in to an ischemic tissue. Adhesion of human adipose tissue-derived stem cells (hADSCs) to the IDM enhanced the cell viability and upregulated angiogenic factors in vitro under either cell adhesion-suppressive conditions or hypoxic conditions, which simulated the microenvironment of ischemic tissues. In a murine ischemic-hindlimb model, hADSCs that were attached to the IDM and subsequently injected into an ischemic region showed better grafting and angiogenic factor expression. The hADSC-IDM implantation subsequently promoted the formation of microvessels, attenuated fibrosis, and increased blood perfusion in the ischemic region, as compared to implantation of hADSCs only. The IDM may be an effective off-the-shelf material that can enhance therapeutic efficacy of stem cell therapy for ischemic diseases.

Thromboxane Governs the Differentiation of Adipose-Derived Stromal Cells Toward Endothelial Cells In Vitro and In Vivo

Autologous adipose-derived stromal cells (ASCs) offer great promise as angiogenic cell therapy for ischemic diseases. Because of their limited self-renewal capacity and pluripotentiality, the therapeutic efficacy of ASCs is still relatively low. Thromboxane has been shown to play an important role in the maintenance of vascular homeostasis. However, little is known about the effects of thromboxane on ASC-mediated angiogenesis. To explore the role of the thromboxane-prostanoid receptor (TP) in mediating the angiogenic capacity of ASCs in vivo. ASCs were prepared from mouse epididymal fat pads and induced to differentiate into endothelial cells (ECs) by vascular endothelial growth factor. Cyclooxygenase-2 expression, thromboxane production, and TP expression were upregulated in ASCs on vascular endothelial growth factor treatment. Genetic deletion or pharmacological inhibition of TP in mouse or human ASCs accelerated EC differentiation and increased tube formation in vitro, enhanced angiogenesis in in vivo Matrigel plugs and ischemic mouse hindlimbs. TP deficiency resulted in a significant cellular accumulation of β-catenin by suppression of calpain-mediated degradation in ASCs. Knockdown of β-catenin completely abrogated the enhanced EC differentiation of TP-deficient ASCs, whereas inhibition of calpain reversed the suppressed angiogenic capacity of TP re-expressed ASCs. Moreover, TP was coupled with Gαq to induce calpain-mediated suppression of β-catenin signaling through calcium influx in ASCs. Thromboxane restrained EC differentiation of ASCs through TP-mediated repression of the calpain-dependent β-catenin signaling pathway. These results indicate that TP inhibition could be a promising strategy for therapy utilizing ASCs in the treatment of ischemic diseases.

Abstract P120: Improving the Angiogenic Abilities of Mobilized Peripheral Blood Stem Cells Achieved by Priming with Activated Platelet Supernatant for Regenerative Cell Therapy

Platelets play a critical role in hemostasis and also have ability to promote angiogenesis and tissue repair by secreting of numerous cytokine and making angiogenic condition. We investigated whether autologous ‘activated platelet supernatant (APS)’ has effect on enhancing pro-angiogenic potential of peripheral blood stem cells (PBSC) for stem cell-based therapy for ischemic diseases. Granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (mobPBSC) were isolated from healthy volunteers, while APS was collected from platelet rich plasma by thrombin activation. mobPBSCs were primed with APS (APS primed mobPBSCs) for 6 hours, and APS primed mobPBSCs characterized their angiogenic ability. For the safety analysis, we estimated the thrombogenicity of platelets in whole blood mixed with APS primed mobPBSCs by expression of glycoprotein IIb and IIIa on platelets. APS had a higher level of various cytokines, such as IL8, IL17, PDGF and VEGF than naïve platelet supernatants. And APS primed mobPBSCs had more expression of angiogenic factors, surface markers (i.e. CD34, CD31, and CXCR4) and integrins (integrin α5, β1 and β2) than Veh primed and Pre primed mobPBSC. Also APS primed mobPBSCs were polarized toward CD14++/CD16+ pro-angiogenic monocytes. And result in adhesion to endothelial cells and fibronectin which represents cell to cell and cell to extracellular matrix adhesion, respectively. The culture supernatant of APS-primed mobPBSCs contained high levels of IL8, IL10, IL17 and TNFα, and augmented proliferation and capillary network formation of HUVEC. In-vivo transplantation of APS-primed mobPBSC into athymic mice ischemic hindlimbs and Matrigel plugs elicited vessel differentiation and tissue repair. In thrombogenicity test, platelet activity increased after mixing whole blood with mobPBSC regardless of the priming agent. However, this was reduced by pretreatment of aspirin, which is an antiplatelet agent prescribed to patients with ischemic diseases. Our data demonstrate that mobPBSCs primed with APS improve angiogenic potential, and that can be adjunctive strategy to enhance the efficiency of stem cell therapy for ischemic diseases.

Expansion, Differentiation and Transplantation of Human Endothelial Progenitor Cells Derived from Umbilical Cord Blood CD34+ Cells

Transplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been developed as a cell therapy for ischemic diseases and hemophilia A, due to the capacities of these cells to repair vascular damage and produce factor VIII. The strategies and the mechanisms of this type of cell therapy have been extensively investigated in the past decades. However, given the problems associated with host immune responses to grafts in allotransplantation, the difficulties in obtaining adequate numbers of suitable cells for transplantation represent additional obstacles for a successful cell therapy. In the present study, human umbilical cord blood CD34+ mesenchymal cells (MCs), which generate low immune response, have been adopted as the source of endothelial progenitors. Our major goal is to establish a highly efficient approach to stimulate the expansion and subsequent differentiation of human endothelial progenitor cells (EPCs), in order to obtain sufficient quantities of healthy cells for therapeutic use. The CD34+ cells isolated from human umbilical cord blood were cultured for 6 days in a customized basal medium supplemented with a modified cytokine cocktail, including SCF, Flt-3L, TPO, IL-3, and GM-CSF. At the end of proliferation stage, cell number was calculated with Flow Cytometry, and cell morphology was characterized as well. The numbers of CD34+ cells and CD34+/CD133+/VEGFR-2+ early EPCs cells were found to be increased by approximately 108-fold and 41.9-fold, respectively, on day 6. In the differentiation stage, the attached cells were transferred to EGM-2 basal medium supplemented with FBS and additional nutrients, including VEGF, IGF, EGF, and FGF, for another 15 days. Following this two-step culture, the generated adherent cells were found to have proliferated more than 1200 folds over the initial EPCs, and they were identified as mature endothelial cells expressing CD31, vWF and FVIII. Furthermore, we developed a nonobese diabetic, severe combined immunodeficient (NOD/SCID) mouse model with portal sinusoidal endothelium injury. The NOD/SCID mice were treated with the produced EPCs/ECs through hepatic portal vein injection at the dose of 6x10^6 cells. Tissue examination demonstrated that GFP-labeled transplanted cells migrated and integrated into liver structure, where they expressed specific endothelial cell markers. Therefore, through our unique approach, we can provide an excellent source of healthy endothelial progenitors for cell therapy. DisclosuresQin:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

EACPT-0036 - The Beneficial Effect of Ultrasonic Microbubble Transfection on Aged Endothelial Progenitor Cells

Cell therapy for ischemic diseases by using autologous progenitor cells has been limited by the functional impairment of the cells, such as insufficient paracrine and angiogenic effects, owing to the presence of risk factors, including old age. Optimization of ultrasonic microbubble transfection (UMT) to increase the product of transferred gene in the progenitor cells is a potential strategy to improve the function of progenitor cells for clinical application. Therefore, we explored whether UMT has a beneficial effect on the angiogenic activity of aged endothelial progenitor cells (EPCs).

Activated platelet supernatant can augment the angiogenic potential of human peripheral blood stem cells mobilized from bone marrow by G-CSF

Platelets not only play a role in hemostasis, but they also promote angiogenesis and tissue recovery by releasing various cytokines and making an angiogenic milieu. Here, we examined autologous ‘activated platelet supernatant (APS)’ as a priming agent for stem cells; thereby enhance their pro-angiogenic potential and efficacy of stem cell-based therapy for ischemic diseases. The mobilized peripheral blood stem cells (mobPBSCs) were isolated from healthy volunteers after subcutaneous injection of granulocyte-colony stimulating factor. APS was collected separately from the platelet rich plasma after activation by thrombin. mobPBSCs were primed for 6h before analysis. Compared to naive platelet supernatants, APS had a higher level of various cytokines, such as IL8, IL17, PDGF and VEGF. APS-priming for 6h induced mobPBSCs to express key angiogenic factors, surface markers (i.e. CD34, CD31, and CXCR4) and integrins (integrins α5, β1 and β2). Also mobPBSCs were polarized toward CD14(++)/CD16(+) pro-angiogenic monocytes. The priming effect was reproduced by an in vitro reconstruction of APS. Through this phenotype, APS-priming increased cell–cell adhesion and cell–extracellular matrix adhesion. The culture supernatant of APS-primed mobPBSCs contained high levels of IL8, IL10, IL17 and TNFα, and augmented proliferation and capillary network formation of human umbilical vein endothelial cells. In vivo transplantation of APS-primed mobPBSCs into athymic mice ischemic hindlimbs and Matrigel plugs elicited vessel differentiation and tissue repair. In safety analysis, platelet activity increased after mixing with mobPBSCs regardless of priming, which was normalized by aspirin treatment. Collectively, our data identify that APS-priming can enhance the angiogenic potential of mobPBSCs, which can be used as an adjunctive strategy to improve the efficacy of cell therapy for ischemic diseases.

Genistein promotes endothelial colony-forming cell (ECFC) bioactivities and cardiac regeneration in myocardial infarction.

Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells in vivo because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model. Results: We found that genistein treatment enhanced ECFCs' migration and proliferation, which was accompanied by increases in the expression of ILK, α-parvin, F-actin, and phospholylation of ERK 1/2 signaling. Transplantation of genistein-stimulates ECFCs (GS-ECFCs) into myocardial ischemic sites in vivo induced cellular proliferation and secretion of angiogenic cytokines at the ischemic sites and thereby enhanced neovascularization and decreased myocardial fibrosis as well as improved cardiac function, as shown by echocardiography. Taken together, these data suggest that pretreatment of ECFCs with genistein prior to transplantation can improve the regenerative potential in ischemic tissues, providing a novel strategy in adult stem cell therapy for ischemic diseases.

PEP-1-CAT-Transduced Mesenchymal Stem Cells Acquire an Enhanced Viability and Promote Ischemia-Induced Angiogenesis

ObjectivePoor survival of mesenchymal stem cells (MSC) compromised the efficacy of stem cell therapy for ischemic diseases. The aim of this study is to investigate the role of PEP-1-CAT transduction in MSC survival and its effect on ischemia-induced angiogenesis.MethodsMSC apoptosis was evaluated by DAPI staining and quantified by Annexin V and PI double staining and Flow Cytometry. Malondialdehyde (MDA) content, lactate dehydrogenase (LDH) release, and Superoxide Dismutase (SOD) activities were simultaneously measured. MSC mitochondrial membrane potential was analyzed with JC-1 staining. MSC survival in rat muscles with gender-mismatched transplantation of the MSC after lower limb ischemia was assessed by detecting SRY expression. MSC apoptosis in ischemic area was determined by TUNEL assay. The effect of PEP-1-CAT-transduced MSC on angiogenesis in vivo was determined in the lower limb ischemia model.ResultsPEP-1-CAT transduction decreased MSC apoptosis rate while down-regulating MDA content and blocking LDH release as compared to the treatment with H2O2 or CAT. However, SOD activity was up-regulated in PEP-1-CAT-transduced cells. Consistent with its effect on MSC apoptosis, PEP-1-CAT restored H2O2-attenuated mitochondrial membrane potential. Mechanistically, PEP-1-CAT blocked H2O2-induced down-regulation of PI3K/Akt activity, an essential signaling pathway regulating MSC apoptosis. In vivo, the viability of MSC implanted into ischemic area in lower limb ischemia rat model was increased by four-fold when transduced with PEP-1-CAT. Importantly, PEP-1-CAT-transduced MSC significantly enhanced ischemia-induced angiogenesis by up-regulating VEGF expression.ConclusionsPEP-1-CAT-transduction was able to increase MSC viability by regulating PI3K/Akt activity, which stimulated ischemia-induced angiogenesis.

Highly angiogenic CXCR4+CD31+ monocyte subset derived from 3D culture of human peripheral blood

Ex vivo expansion of human circulating angiogenic cells is a major challenge in autologous cell therapy for ischemic diseases. Here, we demonstrate that hematosphere-derived CXCR4+CD31+ myeloid cells using peripheral blood possess robust proangiogenic capacity such as formation of vessel-like structures and tip cell-like morphology in Matrigel. We also found that CD31 positive myeloid cells are principal cellular component of hematospheres by magnetic cell sorting. Flow cytometry analysis showed that fresh peripheral blood contained 40.3 ± 15.2% of CXCR4+CD31+ myeloid cells, but at day 5 of hematosphere culture, most of myeloid cells were CXCR4+CD31+ by 86.9 ± 5.4%. Hematosphere culture significantly increased the production of angiogenic niche-supporting cytokines. Moreover, CD31-homophilic interaction and VEGF–VEGF receptor loop signaling were essential for sphere formation and acquisition of angiogenic capacity in hematospheres. Matrigel plug and ischemic hindlimb model provide in vivo evidence that hematosphere-derived myeloid cells have highly vasculogenic capacities, participate in new and mature vessel formation, and exert therapeutic effects on ischemic hindlimb. In conclusion, our strategy for ex vivo expansion of human CXCR4+CD31+ angiogenic cells using hematospheres provides an autologous therapeutic cell source for ischemic diseases and a new model for investigating the microenvironment of angiogenesis.

Three-Dimensional Cell Grafting Enhances the Angiogenic Efficacy of Human Umbilical Vein Endothelial Cells

Despite the great potential of cell therapy for ischemic disease, poor cell survival after engraftment in ischemic tissue limits its efficacy. Here we tested a hypothesis that three-dimensionally grafted human umbilical vein endothelial cell (HUVEC) spheroids would exhibit improved angiogenic efficacy following transplantation into mouse ischemic limbs compared with HUVECs prepared by conventional two-dimensional monolayer culture. One day after surgical induction of hindlimb ischemia in athymic mice, HUVECs cultured in monolayer or HUVEC spheroids were transplanted intramuscularly into ischemic limbs. Four weeks after the treatment, in the spheroid HUVEC transplantation group, we observed increased hypoxia-inducible factor-1α expression, decreased apoptosis, and increased HUVEC survival in the ischemic tissue compared with the monolayer HUVEC transplantation group. Transplantation of HUVEC spheroids also resulted in enhanced and prolonged secretion of paracrine factors as well as enhanced expression of factors involved in the recruitment of circulating angiogenic progenitor cells. In summary, transplantation of HUVECs as spheroids enhanced cell survival, increased paracrine factor secretion, and showed a potential as a therapeutic method to treat ischemic tissue damages by promoting angiogenesis.

Endothelial-like cells differentiated from bone marrow-derived mesenchymal stem cells preserve T-cell suppressive function (147.13)

Abstract Endothelial-like mesenchymal stem cells (EMSCs) differentiated from human mesenchymal stem cells (MSCs) have great potential in cell therapy for ischemic diseases and in tissue engineering for vascular grafts. Although they was proven to stimulate functional blood vessel formation in vivo, it still need to be investigated whether EMSCs harbor immunomodulatory capacity for applying immune-mediated vascular disorders. In this study, therefore, we compared in vivo angiogenic potential of EMSCs with MSCs and studied their immunosuppressive functions. In the phenotype analysis, differentiated EMSC showed several endothelial cell markers including vWF, TGF-beta and Flt-1. In in vitro angiogenesis analysis by using Matrigel and fibrin, they have augmented angiogeneic potential compared to MSCs. Moreover, EMSCs showed neovascularization potential superior to MSCs in subcutaneous transplantation of fibrin-islet-cell composite. In allogeneic mixed lymphocyte reaction, EMSCs showed preserved immunosuppressive function on T cell comparable to that of MSCs. Apoptosis induction and cell-cycle arrest mediated by cell free-culture supernatant were associated with EMSC-mediated T-cell immunosuppression, as evidenced by the increased P27kip and decreased cdk2, cyclin D2 and Bcl-2. These results suggest that ex-vivo expanded EMSC could be a useful source for postnatal neovascularization and for cell-based therapies of tissue damages involving immune reactions.

Cilostazol enhances integrin‐dependent homing of progenitor cells by activation of camp‐dependent protein kinase in synergy with Epac1

Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 μM) stimulated up-regulation of integrins β1, α1, and αv as well as 8-pCPT-2'-O-Me-cAMP (100 μM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 μM) and GGTI-298 (Rap1 inhibitor, 20 μM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N(6)-benzoyl-cAMP (100 μM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin β1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease.

Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells

Stem cells offer significant therapeutic promise for the treatment of ischemic disease. However, stem cells transplanted into ischemic tissue exhibit limited therapeutic efficacy due to poor engraftment in vivo. Several strategies for improving the survival and engraftment of stem cells in ischemic tissue have been developed including transplantation in combination with growth factor delivery, genetic modification of stem cells, and the use of cell-transplantation scaffolds. Here, we demonstrate that human adipose-derived stromal cells (hADSCs) cultured and grafted as spheroids exhibit improved therapeutic efficacy for ischemia treatment. hADSCs were cultured in monolayer or spheroids. Spheroid cultures were more effective in preconditioning hADSCs to a hypoxic environment, upregulating hypoxia-adaptive signals (i.e., stromal cell-derived factor-1α and hypoxia-inducible factor-1α), inhibiting apoptosis, and enhancing secretion of both angiogenic and anti-apoptotic factors (i.e., hepatocyte growth factor, vascular endothelial growth factor, and fibroblast growth factor 2) compared to monolayer cultures. Moreover, cell harvesting following spheroid cultures avoided damage to extracellular matrices due to harsh proteolytic enzyme treatment, thereby preventing anoikis (apoptosis induced by a lack of cell–matrix interaction). Following intramuscular transplantation to ischemic hindlimbs of athymic mice, hADSC spheroids showed improved cell survival, angiogenic factor secretion, neovascularization, and limb survival as compared to hADSCs grafted as dissociated cells. Taken together, spheroid cultures precondition hADSCs to a hypoxic environment, and grafting hADSCs as spheroids to ischemic limbs improves therapeutic efficacy for ischemia treatment due to enhanced cell survival and paracrine effects. Spheroid-based cell delivery could be a simple and effective strategy for improving stem cell therapy for ischemic diseases, eliminating the need for growth factor delivery, biomaterial scaffolds or genetic modification.

Combination of Injectable Multiple Growth Factor–Releasing Scaffolds and Cell Therapy as an Advanced Modality to Enhance Tissue Neovascularization

Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Priming With Angiopoietin-1 Augments the Vasculogenic Potential of the Peripheral Blood Stem Cells Mobilized With Granulocyte Colony-Stimulating Factor Through a Novel Tie2/Ets-1 Pathway

The low engraftment rate of stem/progenitor cells infused via the intracoronary route to the ischemic myocardium is one of the most important factors limiting the efficacy of cell therapy. We investigated the concept of priming peripheral blood stem cells enriched by granulocyte colony-stimulating factor mobilization and apheresis ((mob)PBSCs) with angiopoietin-1 (Ang1), to enhance the engraftment into the ischemic tissue and neovasculogenic potential. The expression of Tie2, the Ang1 receptor, was significantly higher in (mob)PBSCs than naïve peripheral blood mononuclear cells (19.2+/-3.0% versus 1.2+/-0.8% versus 1.2+/-0.2%; P<0.001 for (mob)PBSCs from acute myocardial infarction (AMI) patients with granulocyte colony-stimulating factor treatment for 3 days versus peripheral blood mononuclear cells from AMI patients versus peripheral blood mononuclear cells from stable angina patients). After 4 hours of cartilage oligomeric matrix protein (COMP)-Ang1 stimulation, (mob)PBSCs committed to the endothelial lineage with the induction of CD31 and VE-cadherin expression, mediated by Tie2/Ets-1 pathway. Priming of (mob)PBSCs with COMP-Ang1 induced the expression of alpha4beta1 and alpha5beta1 integrins, which are also Ets-1 downstream molecules, leading to enhanced adhesion to endothelial cells or fibronectin. In a rabbit ear ischemia/reperfusion model, priming of (mob)PBSCs with COMP-Ang1 improved first-pass engraftment to the distal vascular bed after intraarterial delivery. In a murine ischemic hind-limb model, intravascular delivery of primed (mob)PBSCs enhanced both engraftment and neovascularization. The short-term priming with COMP-Ang1 may be a feasible and promising option to activate (mob)PBSCs by enhancing differentiation and adhesiveness and to improve the efficacy of cell therapy for ischemic diseases.

Ex Vivo Nanofiber Expansion and Genetic Modification of Human Cord Blood-Derived Progenitor/Stem Cells Enhances Vasculogenesis

The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded approximately 225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF(164) and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

Adipose Tissue-Derived Stromal Cells as a Novel Option for Regenerative Cell Therapy

Adult stem cells hold great promise for use in tissue repair and regeneration, and the delivery of autologous progenitor cells into ischemic tissue is emerging as a novel therapeutic option. We and others have recently demonstrated the potential impact of adipose tissue-derived stromal cells (ADSC) on regenerative cell therapy for ischemic diseases. The main benefit of ADSC is that they can be easily harvested from patients by a simple, minimally invasive method and also easily cultured. Cultured ADSC can be induced to differentiate into not only adipocytes, but also bone, neurons or endothelial cells in certain conditions. Interestingly, they secrete a number of angiogenesis-related cytokines, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), which might be suitable for regenerative cell therapy for ischemic diseases. In the ischemic mouse hindlimb, the angiogenic score was improved in the ADSC-treated group. Moreover, recent reports demonstrated that these ADSC can also be induced to differentiate into cardiac myocytes. These adipose tissue-derived cells have potential in angiogenic cell therapy for ischemic disease, and might be applied for regenerative cell therapy instead of bone marrow cells in the near future.

Novel Autologous Cell Therapy in Ischemic Limb Disease Through Growth Factor Secretion by Cultured Adipose Tissue–Derived Stromal Cells

The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue-derived cells (ADSC) on angiogenic cell therapy. ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of ScaI and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and alpha-smooth muscle actin-positive cells in ischemic tissue. These adipose tissue-derived cells demonstrated potential as angiogenic cell therapy for ischemic disease, which appears to be mainly achieved by their ability to secrete angiogenic growth factors.