Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation
Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation
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DOI：
10.1073/pnas.0803078105
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发表时间：
2008-08-19
影响因子：
11.1
通讯作者：
Sasai, Yoshiki
中科院分区：
文献类型：
--
作者：
Wataya, Takafumi;Ando, Satoshi;Sasai, Yoshiki
胚胎干细胞在无血清条件下以漂浮集合体的形式培养时，可分化为神经外胚祖细胞。在这里，我们证明了在分化的早期阶段严格去除外源性模式因子可以在小鼠ES细胞来源的神经外胚层细胞中高效地产生下丘脑嘴端样祖细胞(Rax(+)/Six3(+)/Vax1(+))。使用无生长因子的化学定义的培养液是至关重要的，即使是细胞培养中常用的外源性胰岛素的存在，也会强烈地抑制通过Akt依赖的途径进行的分化。ES细胞来源的RAX(+)前体细胞产生OTP(+)/Brn2(+)神经元前体(下丘脑头背侧神经元的特征)，随后产生大细胞加压素能神经元，在刺激下有效地释放激素。下丘脑吻腹侧前体细胞和神经元的分化标记物是由ES细胞来源的RAX(+)前体细胞经Shh处理后诱导的。因此，在没有外源性生长因子的情况下，ES细胞来源的神经外胚层细胞会自发分化为吻侧(特别是吻侧-背侧)的下丘脑样前体细胞，如在体内观察到的那样，这些前体细胞以循序渐进的方式产生特有的下丘脑神经内分泌神经元。这些发现表明，外源性模式信号的最小化，而不是诱导信号的添加，在下丘脑嘴端多能细胞来源的神经前体的指定中起着关键作用。
Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax(+)/Six3(+)/Vax1(+)) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax(+) progenitors generate Otp(+)/Brn2(+) neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax(+) progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.