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的心血管再生有望成为治疗严重心力衰竭的有效新方法。然而，有各种情况下，作为心衰的原因，如心肌缺血，心肌病，或其他。因此，需要根据每个患者的病情，通过自体骨髓干细胞的植入来开发一种量身定制的心力衰竭治疗方法。首先，我们尝试通过植入体外TGF-β-预编程CD117 （c-kit）阳性（CD117^+）骨髓干细胞来再生梗死心肌。从gfp转基因小鼠和正常C57/BL6小鼠骨髓单个核细胞中分离CD117^+细胞。用TGF-β培养CD117^+细胞实现成肌分化。我们发现tgf - β增加了肌球蛋白、肌钙蛋白、连接蛋白43、GATA-4和NKx-2.5的细胞表达，这表明它诱导了CD117^+细胞的成肌分化。在急性心肌梗死模型中，我们还尝试通过在心肌内植入未经处理（新分离）或预先编程（培养24小时）的5 ng/mL TGF-β_1) CD117^+细胞来再生梗死心肌。组织学分析显示，TGF-β-预编程细胞植入后左室前壁有新生心肌，未处理细胞未见。TGF-β-预编程细胞移植后左心室百分比分数缩短明显高于未处理的CD117^+细胞。上述结果表明，体外TGF-β-预编程增加了CD117^+干细胞的成肌分化，有助于提高心肌再生能力。其次，我们尝试在植入前通过体外缺氧刺激增强骨髓细胞的血管生成潜能。收集骨髓细胞，在低氧（2% O_2）和常氧条件下培养。培养24 h后，将细胞注射到缺血大鼠后肢。缺氧培养使骨髓细胞中VEGF、VE-cadherin和Flk-1 mRNA的表达增加2.5 ~ 5倍。缺氧培养细胞移植2周后，缺血后肢微血管密度和血流速率明显高于正常缺氧培养细胞。上述结果表明，体外缺氧预刺激提高了VEGF mRNA的表达和骨髓细胞内皮分化，从而提高了诱导治疗性血管生成的效力。骨髓干细胞经TGF-β体外预处理和缺氧预处理后，对损伤心肌和血管的再生效果均有明显改善。骨髓干细胞在植入前进行不同的体外预处理，似乎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