Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, BS2 8HW, UK.
Stem Cell Rev Rep. 2013 Jun;9(3):350-9. doi: 10.1007/s12015-011-9316-9.
Transplantation of antigenic-separated stem cells for human cardiovascular diseases such as myocardial infarction needs to be supported by experimental studies that allow refinement of the procedure. In this study we investigated optimising a protocol for the expansion and subsequent differentiation of human umbilical cord blood (HUCB) derived CD133(+) stem cells into a cardiomyocyte-like lineage. CD133(+) cells from HUCB were selected first by immunomagnetic separation and their purity was confirmed by flow cytometry analysis. For expansion and differentiation we developed a novel culture medium recipe that involves sequential signalling factors. Briefly, CD133(+) cells were expanded for 6 days under optimal serum-free conditions in combination with fibronectin and assessed by microscopy and AlamarBlue proliferation assay. Expanded CD133(+) cells were then plated in a cardiac differentiation promoting medium and cultured up to 4 weeks. With this protocol HUCB-CD133(+) cells can be regularly expanded in serum-free medium to obtain recovery and growth in vitro up to 6 folds. The addition of recombinant human thrombopoietin to the remaining factors of the expanding medium was associated with larger cell expansion. Expanded UCB CD133(+) cells showed a cardiomyocyte-like phenotype following differentiation in vitro through expressing intracellular cardiac specific markers including cardiac-specific α-actin, myosin heavy chain and troponin I. This change in phenotype was associated with the expression of cardiac-specific transcription factors Gata-4 and MEF2C. In addition, the change in phenotype was associated with an upregulation of nuclear receptor transcription factors including PPAR α, PPARγ, RXR α and RXRβ. We believe our protocol represents a significant advancement and overcome the technical hurdle of deriving cardiomyogenic-like cells from HUCB CD133(+) stem cells. In addition, it has the required attributes of simplicity and consistency. This will permit more robust manipulation of these cells towards better engraftment and repair in patients with myocardial infarction.
用于人类心血管疾病（如心肌梗死）的抗原分离干细胞移植需要得到实验研究的支持，以完善该程序。在这项研究中，我们研究了优化人脐血（HUCB）来源 CD133(+)干细胞向心肌样谱系分化的方案。首先通过免疫磁珠分离法分离 HUCB 中的 CD133(+)细胞，并通过流式细胞术分析确认其纯度。为了扩增和分化，我们开发了一种新的培养配方，涉及顺序信号因子。简而言之，将 CD133(+)细胞在最佳无血清条件下与纤连蛋白一起扩增 6 天，并通过显微镜和 AlamarBlue 增殖测定进行评估。然后，将扩增的 CD133(+)细胞接种在促进心脏分化的培养基中，并培养长达 4 周。通过该方案，HUCB-CD133(+)细胞可以在无血清培养基中定期扩增，以获得体外恢复和生长至 6 倍。在扩展培养基中添加重组人血小板生成素（rhTPO）与剩余因子一起可使细胞大量扩增。扩增的 UCB CD133(+)细胞在体外分化后表现出心肌样表型，通过表达细胞内心脏特异性标志物，包括心脏特异性α-肌动蛋白、肌球蛋白重链和肌钙蛋白 I。这种表型的改变与心脏特异性转录因子 Gata-4 和 MEF2C 的表达有关。此外，表型的改变与核受体转录因子的上调有关，包括 PPARα、PPARγ、RXRα和 RXRβ。我们相信我们的方案是一个重大的进步，克服了从 HUCB CD133(+)干细胞中获得心肌样细胞的技术障碍。此外，它具有简单和一致的必要属性。这将允许对这些细胞进行更有力的操作，以更好地在心肌梗死患者中植入和修复。
