Age-related BM-MNC dysfunction hampers neovascularization

Although ischemia-induced neovascularization is reportedly impaired with aging, the effect of aged-bone marrow mononuclear cells (BM-MNCs) on neovascularization has not been investigated. The neovascularization capacity of BM-MNCs obtained from 8-week-old mice (young) was compared to those obtained from 18-month-old mice (old), both in vivo and in vitro. Neovascularization in ischemic limbs was significantly impaired in old mice. Whereas transplantation of young BM-MNCs significantly improved blood perfusion, tissue capillary density, and vascular endothelial growth factor (VEGF) production in transplanted ischemic limbs, no such effects were observed with old BM-MNCs. Old BM-MNCs also showed a significant impairment of in vitro VEGF production and migratory capacity in response to VEGF. The number of Dil/lectin-positive cells was significantly lower in old mice, but there was no difference in the number of AC133(+)/CD34(+) and CD34(+)/VEGF-R2(+) positive cells between young and old BM-MNCs. Transplantation of young BM-MNCs improved neovascularization and VEGF production in the ischemic limbs of old recipients, with results that were similar to those obtained in young recipients. These results indicate that the neovascularization capacity of transplanted BM-MNCs is impaired with aging. However, aging does not hamper the revitalization of neovascularization in the murine host in response to transplantation of young BM-MNCs.     

CD133(+) human umbilical cord blood stem cells enhance angiogenesis in experimental chronic hepatic fibrosis

The in vivo angiogenic potential of transplanted human umbilical cord blood (UCB) CD133(+) stem cells in experimental chronic hepatic fibrosis induced by murine schistosomiasis was studied. Enriched cord blood-derived CD133(+) cells were cultured in primary medium for 3 weeks. Twenty-two weeks post-Schistosomiasis infection in mice, after reaching the chronic hepatic fibrotic stage, transplantation of stem cells was performed and mice were sacrificed 3 weeks later. Histopathology and electron microscopy showed an increase in newly formed blood vessels and a decrease in the fibrosis known for this stage of the disease. By immunohistochemical analysis the newly formed blood vessels showed positive expression of the human-specific angiogenic markers CD31, CD34 and von Willebrand factor. Few hepatocyte-like polygonal cells showed positive expression of human vascular endothelial growth factor and inducible nitric oxide synthase. The transplanted CD133(+) human stem cells primarily enhanced hepatic angiogenesis and neovascularization and contributed to repair in a paracrine manner by creating a permissive environment that enabled proliferation and survival of damaged cells rather than by direct differentiation to hepatocytes. A dual advantage of CD133(+) cell therapy in hepatic disease is suggested based on its capability of hematopoietic and endothelial differentiation.     

Endothelial differentiation potential of human monocyte-derived multipotential cells

We previously reported a unique CD14(+)CD45(+)CD34(+) type I collagen(+) cell fraction derived from human circulating CD14(+) monocytes, named monocyte-derived multipotential cells (MOMCs). This primitive cell population contains progenitors capable of differentiating along the mesenchymal and neuronal lineages. Here, we investigated whether MOMCs can also differentiate along the endothelial lineage. MOMCs treated with angiogenic growth factors for 7 days changed morphologically and adopted a caudate appearance with rod-shaped microtubulated structures resembling Weibel-Palade bodies. Almost every cell expressed CD31, CD144, vascular endothelial growth factor (VEGF) type 1 and 2 receptors, Tie-2, von Willebrand factor (vWF), endothelial nitric-oxide synthase, and CD146, but CD14/CD45 expression was markedly downregulated. Under these culture conditions, the MOMCs continued to proliferate for up to 7 days. Functional characteristics, including vWF release upon histamine stimulation and upregulated expression of VEGF and VEGF type 1 receptor in response to hypoxia, were indistinguishable between the MOMC-derived endothelial-like cells and cultured mature endothelial cells. The MOMCs responded to angiogenic stimuli and promoted the formation of mature endothelial cell tubules in Matrigel cultures. Finally, in xenogenic transplantation studies using a severe combined immunodeficient mouse model, syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMCs. Cotransplantation of the MOMCs promoted the formation of blood vessels, and more than 40% of the tumor vessel sections incorporated human endothelial cells derived from MOMCs. These findings indicate that human MOMCs can proliferate and differentiate along the endothelial lineage in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis.     

VEGFR1/CXCR4-positive progenitor cells modulate local inflammation and augment tissue perfusion by a SDF-1-dependent mechanism

Recruitment and retention of circulating progenitor cells at the site of injured or ischemic tissues facilitates adult neo-vascularization. We hypothesized that cell therapy could modulate local neo-vascularization through the vascular endothelial growth factor (VEGF)/stromal cell-derived factor-1 (SDF-1) axis and by paracrine effects on local endothelial cells. We isolated from rat bone marrow a subset of multipotent adult progenitor cell-derived progenitor cells (MDPC). In vitro, MDPCs secreted multiple cytokines related to inflammation and angiogenesis, including monocyte chemotactic protein-1, SDF-1, basic fibroblast growth factor, and VEGF, and expressed the chemokine receptors CXCR4 and VEGFR1. To investigate in vivo properties, we transplanted MDPCs into the ischemic hind limbs of rats. Elevated levels of the chemokine SDF-1 and colocalization of CD11b⁺ cells marked the initial phase of tissue remodeling after cell transplantation. Prolonged engraftment was observed in the adventitial–medial border region of arterioles of ischemic muscles. However, engrafted cells did not differentiate into endothelial or smooth muscle cells. Limb perfusion normalized 4 weeks after cell injection. Inhibition of SDF-1 reduced the engraftment of transplanted cells and decreased endothelial cell proliferation. These findings suggest a two-stage model whereby transplanted MDPCs modulate wound repair through recruitment of inflammatory cells to ischemic tissue. This is an important potential mechanism for cell transplantation, in addition to the direct modulation of local vascular cells through paracrine mechanisms.     

罗格列酮对CD34修饰脱细胞血管支架体内生长的影响

背景：早期研制的脱细胞血管基质支架上预载CD34+抗体会促进其再内皮化,但同时会加重支架内血管内膜增生。国内外研究证实过氧化物酶增殖体受体γ激动剂罗格列酮在体外可抑制平滑肌细胞增生及迁移,可减少血管损伤处内膜增生。 目的：进一步验证过氧化物酶增殖体受体γ激动剂罗格列酮对CD34抗体修饰脱细胞血管支架体内移植后平滑肌细胞生长及内膜增生的影响。 方法：获取新鲜兔颈动脉,应用光化学偶联法将CD34抗体固定到去细胞光氧化的血管支架上,构建抗体修饰的组织工程血管。将制备的血管分别移植于实验兔的颈动脉上,其中对照组予以移植单纯光氧化处理的脱细胞血管,CD34组予以CD34抗体预载的血管,罗格列酮组移植CD34抗体预载的血管并予喂养罗格列酮。 结果与结论：移植后10 d：对照组移植血管内皮样细胞数量稀少,CD34组和罗格列酮组可见较多的内皮样细胞覆盖;CD34组血管内膜较罗格列酮组厚,α-SMA染色显示CD34组血管平滑肌细胞数量较后者为多,其差异有显著性意义。移植后30 d：CD34组和罗格列酮组血管内皮样细胞基本覆盖管腔全层,对照组内皮样细胞数量仍较少;另外,CD34组血管内膜及管壁中可见大量的平滑肌样细胞及细胞外基质沉积,而罗格列酮组血管结构中平滑肌样细胞数量相对较少,内膜增生亦较轻。提示CD34修饰脱细胞血管支架可促进其内皮细胞的增生,罗格列酮可抑制血管支架中平滑肌细胞的增殖,减少内膜增生。     

不同时间移植人脐血CD34+细胞可影响心肌梗死大鼠心功能及血管内皮细胞生长因子的分泌

背景：干细胞移植可以改善心脏功能,改善预后。 目的：观察不同时间经静脉移植人脐血CD34+细胞对心肌梗死大鼠心功能及细胞因子分泌的影响。 方法：结扎冠状动脉左前降支制备Wistar大鼠心肌梗死模型,于梗死后1,5,10 ,30 d经尾静脉注入0.5 mL人脐血CD34+细胞(实验组)或磷酸盐缓冲溶液(对照组)。 结果与结论：与对照组相比,梗死后5,10 d实验组大鼠左室射血分数明显升高(P < 0.05),左室收缩末内径明显减小(P < 0.05),左室后壁增厚率更高(P < 0.05),毛细血管密度明显增加(P < 0.05),且以梗死后10 d移植大鼠心功能改善效果最明显(P < 0.05)。梗死后10 d实验组心肌局部血管内皮细胞生长因子最高(P < 0.05)。说明大鼠心肌梗死后5,10 d经静脉移植脐血CD34⁺细胞可明显改善心功能,梗死后10 d移植血管内皮细胞生长因子分泌更多,血管生成更多,对心功能的改善更明显;同时说明脐血单个核细胞移植可能是通过增加血管内皮细胞生长因子分泌,提高毛细血管密度来改善心功能的。     

Paraxial mesodermal progenitors derived from mouse embryonic stem cells contribute to muscle regeneration via differentiation into muscle satellite cells

Pluripotent embryonic stem (ES) cells hold great potential for cell-based therapies. Although several recent studies have reported the potential of ES cell-derived progenitors for skeletal muscle regeneration, how the cells contribute to reconstitution of the damaged myofibers has remained elusive. Here, we demonstrated the process of injured muscle regeneration by the engraftment of ES cell-derived mesodermal progenitors. Mesodermal progenitor cells were induced by a conventional differentiation system and isolated by flow cytometer of platelet-derived growth factor receptor-alpha (PDGFR-alpha), a marker of paraxial mesoderm, and vascular endothelial growth factor receptor-2 (VEGFR-2), a marker of lateral mesoderm. The PDGFR-alpha(+) population that represented the paraxial mesodermal character demonstrated significant engraftment when transplanted into the injured muscle of immunodeficient mouse. Moreover, the PDGFR-alpha(+) population could differentiate into the muscle satellite cells that were the stem cells of adult muscle and characterized by the expression of Pax7 and CD34. These ES cell-derived satellite cells could form functional mature myofibers in vitro and generate myofibers fused with the damaged host myofibers in vivo. On the other hand, the PDGFR-alpha(-)VEGFR-2(+) population that showed lateral mesodermal character exhibited restricted potential to differentiate into the satellite cells in injured muscle. Our results show the potential of ES cell-derived paraxial mesodermal progenitor cells to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation. This knowledge could be used to form the foundation of the development of stem cell therapies to repair diseased and damaged muscles.     

The impact of hyperglycemia and the presence of encapsulated islets on oxygenation within a bioartificial pancreas in the presence of mesenchymal stem cells in a diabetic Wistar rat model

This study investigates the potential of bone marrow (BM-MSCs) versus adipose mesenchymal stem cells (AMSCs) to potentiate the oxygenation of encapsulated islets in a subcutaneous bioartificial pancreas. Oxygen pressures (inside subcutaneous implants) were followed in vivo (by electronic paramagnetic resonance) in non-diabetic/diabetic rats transplanted with encapsulated porcine islets or empty implants up to 4 weeks post-transplantation. After graft explantation, neoangiogenesis surrounding the implants was assessed by histomorphometry. Angiogenic properties of BM-MSCs and AMSCs were first assessed in vitro by incubation of the cells in hypoxia chambers, under normoxic/hypoxic and hypo-/hyperglycemic conditions, followed by quantification of vascular endothelial growth factor (VEGF) release. Second, the in vivo aspect was studied by subcutaneous transplantation of encapsulated BM-MSCs and AMSCs in diabetic rats and assessment of the cells' angiogenic properties as described above. Diabetic state and islet encapsulation induced a significant decrease of oxygenation of the subcutaneous implant and an increased number of cells expressing VEGF. AMSCs demonstrated a significantly higher VEGF secretion than BM-MSCs in vitro. In vivo, AMSCs improved the implant's oxygenation and vascularization. Diabetes and islet encapsulation significantly reduced the oxygenation of a subcutaneous bioartificial pancreas. AMSCs can improve oxygenation by VEGF release in hypoxia and hyperglycemia states.     

Synergistic effect of adipose-derived stem cell therapy and bone marrow progenitor recruitment in ischemic heart

Human multipotent adipose-derived stem cells (hMADSCs) have recently been isolated featuring extensive expansion capacity ex vivo. We tested the hypothesis that hMADSC transplantation might contribute to cardiac functional recovery by its direct or indirect effect on myocardial infarction (MI). Nude rats were either transplanted with hMADSCs or PBS (control) in ischemic myocardium immediately following MI. Echocardiographical assessment of cardiac function after MI with hMADSCs showed significant improvement of each parameter compared to that with PBS. Histological analysis also showed significantly reduced infarct size and increased capillary density in peri-infarct myocardium by hMADSC treatment. However, remarkable transdifferentiation of hMADSCs into cardiac or vascular lineage cells was not observed. Despite the less transdifferentiation capacity, hMADSCs produced robust multiple pro-angiogenic growth factors and chemokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1α (SDF-1α). Specifically, hMADSC-derived SDF-1α had a crucial role for cooperative angiogenesis, with the paracrine effect of hMADSCs and Tie2-positive bone marrow (BM) progenitor recruitment in ischemic myocardium. hMADSCs exhibit a therapeutic effect on cardiac preservation following MI, with the production of VEGF, bFGF, and SDF-1α showing paracrine effects and endogenous BM stem/progenitor recruitment to ischemic myocardium rather than its direct contribution to tissue regeneration.     

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.     

Human squamous cell carcinomas of the head and neck chemoattract immune suppressive CD34(+) progenitor cells

CD34(+) progenitor cells have previously been shown to be mobilized in patients with squamous cell carcinoma of the head and neck (HNSCC). The present study showed that these CD34(+) cells inhibit the capacity of intratumoral lymphoid cells to become activated in response to stimulation through the TCR/CD3 complex. The mechanisms that could lead to the accumulation of CD34(+) cells within the tumor tissue were assessed. This was accomplished through in vitro studies that determined if HNSCC produce soluble factors that chemoattract CD34(+) cells. The migration of cord blood CD34(+) cells, which were used as a readily available source of progenitor cells, was stimulated by products derived from HNSCC explants and primary HNSCC cultures. This stimulated migration was due to chemotaxis because it was dependent on an increasing gradient of HNSCC-derived products. CD34(+) cells that were isolated from the peripheral blood of HNSCC patients were similarly chemoattracted to the HNSCC-derived products. The majority of the chemotactic activity produced by HNSCC could be attributed to vascular endothelial cell growth factor (VEGF). These studies indicate that HNSCC can chemoattract immune inhibitory CD34(+) progenitor cells through their production of VEGF.     

VEGF165转染大鼠血管内皮细胞对新生血管形成影响的实验研究

目的探讨血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）基因与增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）共表达载体转染大鼠血管内皮细胞，植入大鼠体内，观察诱导新生血管情况，为移植组织诱导新生血管形成奠定基础。方法对质粒pIRES2-EGFP／VEGF，岱进行扩增、纯化，以脂质体法转染大鼠血管内皮细胞并植入肾被膜下。采用荧光显微镜检测增强绿色荧光蛋白在内皮细胞中的表达，用流式细胞仪检测转染效率。用RT-PCR检测VEGF mRNA的表达。免疫组化检测VEGF在内皮细胞中的表达。切取移植肾脏组织标本,HE染色观察组织形态学变化。结果荧光显微镜观察到实验组内皮细胞有特异性的EGFP表达。流式细胞仪分析转染效率为13．06％。实验组血管内皮细胞胞核和胞浆中均有VEGF表达。RT-PCR显示实验组大鼠血管内皮细胞中有人源化VEGF165基因在mRNA水平表达。移植后14d，实验组大鼠肾被膜下可见成团的新生毛细血管网形成，而对照组及空白转染组尽管血管内皮细胞仍存活，但未形成明显血窦。结论转染VEGF是促进内皮细胞早期（14d内）形成新生血管的有效途径。     

Identification and characterization of hemoangiogenic progenitors during cynomolgus monkey embryonic stem cell differentiation

We identified intermediate-stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence-activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor-bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)-2(high) CD34(-) and VEGFR-2(high) CD34(+) cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR-2(high) cells, particularly that of VEGFR-2(high) CD34(+) cells, in a dose-dependent manner. Although either population of VEGFR-2(high) cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR-2(high) CD34(+) cells had greater hemoangiogenic potential. Both lineages developed from VEGFR-2(high) CD34(-)or VEGFR-2(high) CD34(+) precursor at the single-cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.     

Functional characterization of TPO-expanded CD34+ cord blood cells identifies CD34- CD61- cells as platelet-producing cells early after transplantation in NOD/SCID mice and rCD34+ cells as CAFC colony-forming cells

Transplantation of thrombopoietin (TPO)-expanded cord blood CD34(+) cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO-expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34(-) /CD61(-)/lineage(-) cells (Lin(-)), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin(-) dose dependence. The Lin(-) population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin(-) population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34(+) cells remaining after expansion (rCD34(+)) was similar to mice transplanted with nonexpanded CD34(+) cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area-forming cell colony-forming cells resided mostly in the rCD34(+) population. The pro-MK CD61(+) cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application.     

Derivation of endothelial cells from CD34- umbilical cord blood

CD34 is a transmembrane glycoprotein constitutively expressed on endothelial cells and hematopoietic stem cells. Use of CD34-recognizing antibodies has helped in the identification and isolation of CD34+ endothelial precursors from embryonic and adult tissues. However, CD34-null mice display no vascular abnormalities, demonstrating that CD34 antigen expression is not required for normal vascular development. Here we show that a CD34- cell population that includes endothelial cell precursors can be isolated from cord blood. In the presence of angiogenic factors, these cells mature to express the endothelial cell markers vascular endothelial-cadherin, vascular endothelial growth factor receptor-1 and -2, Tie-1 and -2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains), von Willebrand factor, and CD31 while maintaining their CD34- status, and can be expanded in vitro for over 20 passages. Moreover, in functional studies, these cells can undergo extracellular matrix-dependent morphogenic changes into capillary-like tubular structures. When transplanted into immunodeficient mice in conjunction with tumor cells or with the proangiogenic factor basic fibroblast growth factor, these cells can form functional microvessels arising along with host blood cells. These studies provide strong evidence for the existence of CD34- endothelial cell precursors in cord blood and suggest the use of ex vivo-expanded cord blood CD34- cells as a unique tool for the investigation of postnatal lineage diversification.     

Pluripotent stem cell-engineered cell sheets reassembled with defined cardiovascular populations ameliorate reduction in infarct heart function through cardiomyocyte-mediated neovascularization

Although stem cell therapy is a promising strategy for cardiac restoration, the heterogeneity of transplanted cells has been hampering the precise understanding of the cellular and molecular mechanisms. Previously, we established a cardiovascular cell differentiation system from mouse pluripotent stem cells, in which cardiomyocytes (CMs), endothelial cells (ECs), and mural cells (MCs) can be systematically induced and purified. Combining this with cell sheet technology, we generated cardiac tissue sheets reassembled with defined cardiovascular populations. Here, we show the potentials and mechanisms of cardiac tissue sheet transplantation in cardiac function after myocardial infarction (MI). Transplantation of the cardiac tissue sheet to a rat MI model showed significant and sustained improvement of systolic function accompanied by neovascularization. Reduction of the infarct wall thinning and fibrotic length indicated the attenuation of left ventricular remodeling. Cell tracing with species-specific fluorescent in situ hybridization after transplantation revealed a relatively early loss of transplanted cells and an increase in endogenous neovascularization in the proximity of the graft, suggesting an indirect angiogenic effect of cardiac tissue sheets rather than direct CM contributions. We prospectively dissected the functional mechanisms with cell type-controlled sheet analyses. Sheet CMs were the main source of vascular endothelial growth factor. Transplantation of sheets lacking CMs resulted in the disappearance of neovascularization and subsequent functional improvement, indicating that the beneficial effects of the sheet were achieved by sheet CMs. ECs and MCs enhanced the sheet functions and structural integration. Supplying CMs to ischemic regions with cellular interaction could be a strategic key in future cardiac cell therapy.