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

通过植入自体骨髓干细胞来再生心血管有望成为治疗严重心力衰竭的有效新疗法。然而，作为心力衰竭的原因，也有各种情况，如心肌缺血、心肌病或其他。首先，我们尝试通过移植转化生长因子-β预编程序的CD117(c-Kit)阳性(CD117^+)的骨髓干细胞来再生梗死心肌。从GFP转基因小鼠和正常C57/BL6小鼠的骨髓单个核细胞中分离出CD117+细胞。转化生长因子-β诱导CD117+细胞向肌样细胞分化。我们发现，转化生长因子-β可上调细胞肌球蛋白、肌钙蛋白、连接蛋白-43、GATA-4和NKx-2.5的表达，提示其可诱导CD117^+细胞向肌源性分化。使用急性心肌梗死…为了建立更多的急性心肌梗死模型，我们还尝试了将未经处理的(新分离的)或预编程的(含5 ng/mL转化生长因子-β_1的培养24小时)CD117^+细胞植入心肌内以再生梗死心肌。组织学分析显示，在移植转化生长因子-β预编程细胞后，左室前壁有新生的心肌，但没有未经处理的细胞。此外，移植转化生长因子-β-预编程细胞后的左室短轴缩短率显著高于未处理的CD117^+细胞。这些结果表明，体外转化生长因子-β-预编程增加了CD117^+干细胞的肌源性分化，有助于提高心肌再生的潜能。第二，我们试图在植入前通过体外低氧刺激增强骨髓细胞的血管生成能力。收集骨髓细胞，在低氧(2%O2)或常氧条件下培养。培养24 h后，将细胞注入大鼠缺血后肢。低氧培养使骨髓细胞中血管内皮生长因子、VE-钙粘附素和Flk-1的mRNA表达从2.5倍增加到5倍。缺氧培养细胞移植后2周，缺血后肢微血管密度和血流速度也明显高于常氧培养细胞。这些结果表明，体外低氧预刺激增加了骨髓细胞血管内皮生长因子mRNA的表达和内皮细胞的分化，有助于提高骨髓干细胞诱导治疗性血管生成的能力。转化生长因子-β和低氧预处理骨髓干细胞分别能够提高其对损伤心肌或血管再生的效果。似乎可以通过植入前不同的骨髓干细胞体外预处理来开发针对心力衰竭的量身定制的治疗方法。较少

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