Development of tailor-made regenerative therapy for heart failure by using autologous bone marrow stem cells

自体骨髓干细胞移植再生心血管有望成为治疗严重心力衰竭的有效新方法。然而，存在各种情况作为心力衰竭的原因，诸如心肌缺血、心肌病等。因此，需要根据患者的具体情况，通过移植自体骨髓干细胞来治疗心力衰竭。首先，我们尝试通过体外移植TGF-β-preprogrammed CD 117（c-kit）-positive（CD 117 ^+）骨髓干细胞来再生梗死心肌。从GFP转基因或正常C57/BL 6小鼠的骨髓单个核细胞中分离出CD 117 ^+细胞。用TGF-β诱导CD 117 ^+细胞向肌细胞分化。我们发现TGF-β可增加肌球蛋白、肌钙蛋白、连接蛋白-43、加塔-4和NKx-2.5的表达，这表明TGF-β可诱导CD 117 ^+细胞的肌源性分化。使用急性心肌 关于我们 在急性心肌梗死模型中，我们还尝试通过心肌内植入未处理的（新分离的）或预编程的（用5 ng/mL TGF-β_1培养24小时）CD 117 ^+细胞来再生梗死心肌。组织学分析显示，植入TGF-β-预编程细胞后，左心室前壁新生心肌，而非未处理细胞。此外，植入TGF-β-预编程细胞后左心室缩短百分比显著高于植入未经处理的CD 117 ^+细胞后。这些结果表明，体外TGF-β预编程可提高CD 117 ^+干细胞的成肌分化能力，从而提高其心肌再生能力。其次，我们尝试在植入前通过体外低氧刺激来增强骨髓细胞的血管生成能力。收集骨髓细胞，在低氧（2%O_2）或常氧条件下培养。培养24小时后，将细胞注射到大鼠缺血后肢中。低氧培养增加了骨髓细胞中VEGF、VE-钙粘蛋白和Flk-1的mRNA表达，从2.5倍增加到5倍。缺氧培养的细胞移植2周后，缺血后肢的微血管密度和血流速度也明显高于常氧培养的细胞。上述结果表明，低氧预刺激可提高骨髓干细胞VEGF mRNA的表达和内皮细胞的分化，从而提高其诱导治疗性血管生成的能力; TGF-β或低氧预处理骨髓干细胞可分别提高其促进损伤心肌或血管再生的作用。通过对骨髓干细胞移植前进行不同的体外预处理，可以为心力衰竭患者提供量身定制的治疗方案。少

Cardiovascular regeneration by the implantation of autologous bone marrow stem cells is expected as an effective new therapy for serious heart failure. However, there are various cases as a cause of heart failure, such as myocardial ischemia, cardiomyopathy, or others. So, it is requested to develop a tailor-made therapy for heart failure by the implantation of autologous bone marrow stem cells, according to each patient's condition.At first, we tried to regenerate infarcted myocardium by implanting ex vivo TGF-β-preprogrammed CD117 (c-kit)-positive (CD117^+) bone marrow stem cells. CD117^+ cells were isolated from the bone marrow mononuclear cells of GFP-transgenic or normal C57/BL6 mice. The myogenic differentiation of CD117^+ cells was achieved by cultivation with TGF-β. We found that TGF-beta increased the cellular expression of myosin, troponins, connexin-43, GATA-4, and NKx-2.5, which suggested that it induced the myogenic differentiation of CD117^+ cells. Using an acute myocardi … More al infarction model, we also tried to regenerate infarcted myocardium by implanting untreated (newly isolated) or preprogrammed (24 hours of cultivation with 5 ng/mL TGF-β_1) CD117^+ cells intramyocardially. Histological analysis revealed newly regenerated myocardium in the left ventricular anterior wall after the implantation of TGF-β-preprogrammed cells but not untreated cells. Furthermore, the left ventricular percent fraction shortening was significantly higher after the implantation of TGF-β-preprogrammed cells than after the implantation of untreated CD117^+ cells. These results indicated that ex vivo TGF-β-preprogramming increased the myogenic differentiation of CD117^+ stem cells, which contributed to improved potency for the myocardial regeneration.Secondly, we tried to enhance the angiogenic potential of bone marrow cells by ex vivo hypoxia stimulation before implantation. Bone marrow cells were collected and cultured under hypoxia (2% O_2) or normoxia conditions. Cells were also injected into the ischemic hindlimb of rats after 24 h of culture. Hypoxia culture increased the mRNA expression of VEGF, VE-cadherin, and Flk-1 from 2.5- to five-fold in bone marrow cells. The microvessel density and blood flow rate in the ischemic hindlimbs were also significantly higher 2 weeks after implantation with hypoxia-cultured cells than with normoxia-cultured cells. These results indicated that ex vivo hypoxia pre-stimulation increased the VEGF mRNA expression and endothelial differentiation of bone marrow cells, which contributed to improved potency for the induction of therapeutic angiogenesis.Ex vivo pre-treatment of the marrow stem cell by TGF-β or hypoxia was able to improve their effect for regenerating injured myocardium or vessels, respectively. It seemed that the tailor-made therapy for heart failure could be developed by different ex vivo pre-treatments of bone marrow stem cells before implantation. Less