Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.
Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.
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DOI：
10.1083/jcb.143.7.2045
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发表时间：
1998-12-28
期刊：
影响因子：
--
通讯作者：
Hescheler J
中科院分区：
文献类型：
--
作者：
Kolossov E;Fleischmann BK;Liu Q;Bloch W;Viatchenko-Karpinski S;Manzke O;Ji GJ;Bohlen H;Addicks K;Hescheler J
与终末分化的心肌细胞相反，对哺乳动物心肌细胞（前体细胞）在自发收缩开始前的特征知之甚少。迄今为止，对这些细胞的功能研究是不可能的，因为相应阶段的小鼠胚胎非常小，并且由于缺乏横纹和自发收缩，无法鉴定心脏前体细胞。在本研究中，我们使用小鼠胚胎干（ES，D3细胞系）细胞系统在体外分化的心肌细胞。为了鉴定心脏前体细胞，我们用含有心脏α-肌动蛋白启动子控制下的绿色荧光蛋白（GFP）基因的载体稳定转染ES细胞。首先，在收缩开始前2天检测ES细胞来源的细胞聚集体（胚状体[EBs]）中的荧光区域。由于Ca 2+稳态在心脏功能中起着关键作用，我们研究了GFP标记的心脏前体细胞和早期心肌细胞中Ca 2+通道和Ca 2+释放位点的建立。膜片钳和Ca ~（2+）成像实验证实了EB发育第7天开始的L型Ca ~（2+）电流（伊卡）的功能表达。在第7天，使用10 mM Ca 2+作为电荷载体，伊卡以非常低的密度4 pA/pF.伊卡的生物物理和药理学性质证明类似于终末分化的心肌细胞。在心脏前体细胞中，发现伊卡已经在cAMP依赖性磷酸化的控制下，因为蛋白激酶A的催化亚基的细胞内输注导致1.7倍的刺激。腺苷酸环化酶激活剂毛喉素没有影响。在GFP阳性和GFP阴性细胞的所有分化阶段都存在IP 3敏感的细胞内Ca 2+储存和Ca 2 +-ATP酶。功能ryanodine敏感的Ca 2+商店，检测咖啡因诱导的Ca 2+释放，出现在大多数GFP阳性细胞伊卡后1-2天。共表达的伊卡和ryanodine敏感的Ca 2+商店在第10天的发展正好与大多数EB自发收缩的开始。因此，电压依赖性L型Ca 2+通道（VDCC）的功能性表达是早期心肌形成的标志，而IP 3受体和肌浆Ca 2 +-ATP酶在心肌形成开始之前表达。有趣的是，与VDCC相比，兰尼碱受体/敏感商店的功能表达延迟。
In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1–2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.
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影响因子：
20.1
作者：
CHIN, TK;FRIEDMAN, WF;KLITZNER, TS
通讯作者：
KLITZNER, TS
影响因子：
20.1
作者：
GANIM, JR;LUO, WS;KRANIAS, EG
通讯作者：
KRANIAS, EG
影响因子：
5.5
作者：
HIROTA, A;KAMINO, K;YADA, T
通讯作者：
YADA, T
影响因子：
3.6
作者：
KLITZNER, TS;FRIEDMAN, WF
通讯作者：
FRIEDMAN, WF
影响因子：
--
作者：
MASUDA, H;SPERELAKIS, N
通讯作者：
SPERELAKIS, N