Department of Biosciences, Università degli Studi di Milano, Milano, Italy.
Circ Res. 2013 Aug 2;113(4):389-98. doi: 10.1161/CIRCRESAHA.113.301283. Epub 2013 Jun 10.
A cell-based biological pacemaker is based on the differentiation of stem cells and the selection of a population displaying the molecular and functional properties of native sinoatrial node (SAN) cardiomyocytes. So far, such selection has been hampered by the lack of proper markers. CD166 is specifically but transiently expressed in the mouse heart tube and sinus venosus, the prospective SAN.
We have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells.
We found that in embryonic day 10.5 mouse hearts, CD166 and HCN4, markers of the pacemaker tissue, are coexpressed. Sorting embryonic stem cells for CD166 expression at differentiation day 8 selects a population of pacemaker precursors. CD166+ cells express high levels of genes involved in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3) and low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5). In culture, CD166+ cells form an autorhythmic syncytium composed of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Isoproterenol increases (+57%) and acetylcholine decreases (-23%) the beating rate of CD166-selected cells, which express the β-adrenergic and muscarinic receptors. In cocultures, CD166-selected cells are able to pace neonatal ventricular myocytes at a rate faster than their own. Furthermore, CD166+ cells have lost pluripotency genes and do not form teratomas in vivo.
We demonstrated for the first time the isolation of a nonteratogenic population of cardiac precursors able to mature and form a fully functional SAN-like tissue.
基于干细胞的分化和选择具有天然窦房结（SAN）心肌细胞分子和功能特性的细胞群体，研制出了一种基于细胞的生物起搏器。到目前为止，这种选择一直受到缺乏合适标志物的阻碍。CD166 特异性但短暂地表达在小鼠心脏管和窦房静脉，即潜在的 SAN 中。
我们探索了使用 CD166 表达来分离分化胚胎干细胞中的 SAN 祖细胞的可能性。
我们发现，在胚胎第 10.5 天的小鼠心脏中，CD166 和 HCN4（起搏组织的标志物）共同表达。在分化第 8 天对胚胎干细胞进行 CD166 表达分选，可选择出一群起搏前体细胞。CD166+细胞表达高水平的 SAN 发育相关基因（Tbx18、Tbx3、Isl-1、Shox2）和功能相关基因（Cx30.2、HCN4、HCN1、CaV1.3），低水平表达心室基因（Cx43、Kv4.2、HCN2、Nkx2.5）。在培养中，CD166+细胞形成一个自动同步的合胞体，由形态上与鼠 SAN 心肌细胞相似且具有类似电生理特性的细胞组成。异丙肾上腺素增加（+57%）和乙酰胆碱减少（-23%）CD166 分选细胞的搏动率，这些细胞表达β肾上腺素能和毒蕈碱受体。在共培养中，CD166 分选细胞能够以比自身更快的速度起搏新生心室肌细胞。此外，CD166+细胞已失去多能性基因，且在体内不会形成畸胎瘤。
我们首次证明了可以分离出一种非致瘤性的心脏前体细胞群，其能够成熟并形成具有完全功能的类似 SAN 的组织。
