Iachininoto Maria Grazia, Capodimonti Sara, Podda Maria Vittoria, Valentini Caterina Giovanna, Bianchi Maria, Leone Antonio Maria, Teofili Luciana, Leone Giuseppe
Institute of Hematology, Catholic University of the Sacred Heart, Rome, Italy.
Institute of Human Physiology, Catholic University of the Sacred Heart, Rome, Italy.
Cytotherapy. 2015 Nov;17(11):1627-37. doi: 10.1016/j.jcyt.2015.07.012. Epub 2015 Aug 31.
Although bone marrow c-kit(+) progenitor cells support myocardial regeneration, the cardiomyocyte differentiation potential of umbilical cord blood (UCB) c-kit(+) cells is unknown.
UCB mononuclear cells (MNCs) and c-kit(+) cells purified by use of immunomagnetic beads were used. Cardiomyocyte differentiation was induced with (i) α-minimum essential medium (MEM) with cyclosporine A, (ii) α-MEM with bone morphogenic protein 4 (BMP-4) and transforming growth factor-β (TGF-β) or (iii) MEM with dexamethasone. The expression of cardiac markers (GATA4, GATA6, β-myosin heavy chain, α-sarcomeric actin and cardiac Troponin T) was investigated, and whole-cell current and voltage-clamp recordings were performed.
Although c-kit(+) cells revealed an immature gene profile, with high expression of CD34, CD133, aldehyde dehydrogenase-A1 and c-myc RNAs, purified c-kit(+) cells did not succeed in differentiating into cardiomyocyte-like cells in culture. In contrast, MNCs (either in α-MEM plus cyclosporine A or in α-MEM plus BMP-4 and TGF-β) produced large, adherent cells expressing several cardiac genes and exhibiting an excitable phenotype. Cardiomyocyte-like cell formation was prevented by removing the c-kit(+) cell fraction from MNCs. Furthermore, after co-culturing carboxyfluorescein diacetate succynimidyl ester (CFSE)-tracked c-kit(+) cells together with c-kit(-) cells, we found that cardiac Troponin T--expressing cells were also CFSE(+).
We show that UCB contains progenitors endowed with differentiation potential into cardiomyocytes: these cells reside in the c-kit(+) fraction and require the presence of abundant accessory cells to accomplish the differentiation. These preliminary observations provide the basis for consider the storage of autologous UCB in patients with prenatal diagnosis of congenital heart diseases potentially amenable by myocardial regenerative approaches.
尽管骨髓c-kit(+)祖细胞可支持心肌再生，但脐带血（UCB）c-kit(+)细胞向心肌细胞分化的潜能尚不清楚。
使用通过免疫磁珠纯化的UCB单核细胞（MNCs）和c-kit(+)细胞。采用以下方法诱导心肌细胞分化：（i）添加环孢素A的α-最低必需培养基（MEM）；（ii）添加骨形态发生蛋白4（BMP-4）和转化生长因子-β（TGF-β）的α-MEM；或（iii）添加地塞米松的MEM。研究心脏标志物（GATA4、GATA6、β-肌球蛋白重链、α-肌动蛋白和心肌肌钙蛋白T）的表达，并进行全细胞电流和电压钳记录。
尽管c-kit(+)细胞显示出不成熟的基因谱，CD34、CD133、醛脱氢酶-A1和c-myc RNA表达较高，但纯化的c-kit(+)细胞在培养中未能成功分化为心肌样细胞。相反，MNCs（在α-MEM加环孢素A或α-MEM加BMP-4和TGF-β中）产生了表达多种心脏基因并表现出可兴奋表型的大的贴壁细胞。通过从MNCs中去除c-kit(+)细胞部分可阻止心肌样细胞形成。此外，将羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）标记的c-kit(+)细胞与c-kit(-)细胞共培养后，我们发现表达心肌肌钙蛋白T的细胞也是CFSE(+)。
我们表明UCB含有具有向心肌细胞分化潜能的祖细胞：这些细胞存在于c-kit(+)部分中，并且需要大量辅助细胞的存在才能完成分化。这些初步观察结果为考虑在先天性心脏病产前诊断患者中储存自体UCB提供了依据，这些患者可能适合采用心肌再生方法治疗。
