Directed neuronal differentiation of human embryonic stem cells.
Directed neuronal differentiation of human embryonic stem cells.
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DOI：
10.1186/1471-2202-4-27
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发表时间：
2003-10-22
期刊：
影响因子：
2.4
通讯作者：
Condie BG
中科院分区：
文献类型：
--
作者：
Schulz TC;Palmarini GM;Noggle SA;Weiler DA;Mitalipova MM;Condie BG
我们开发了一种培养系统，用于人类胚胎干细胞（HESCs）向神经前体和神经元的有效和定向分化。人工传代维持HESC，并在胚状体分化之前分化为形态不同的OCT-4+/SSEA-4-单层细胞类型。胚状体在无血清条件下，在50%的人肝癌细胞系HepG2 （MedII）条件培养基中悬浮生长。HESC衍生的无血清胚样体在MedII条件培养基中培养约7-10天后，观察到神经前体群体。神经前体被组织成莲座，莲座由一个中心腔组成，周围环绕着一圈细胞，宽度为4至8个细胞。通过有丝分裂染色体的浓缩和磷酸化组蛋白H3免疫染色表明，莲座内的中心细胞正在增殖。在贴壁培养中，神经前体的莲座被大型交织的神经突网络所包围。免疫染色显示nestin在莲座和相关的非神经元细胞类型中表达，Map-2在莲座中呈径向表达。分化后的神经元表达Map-2和Neurofilament H标记物，一部分神经元表达酪氨酸羟化酶（多巴胺能神经元的标记物）。这种新颖的定向分化方法导致了HESCs中神经元培养物的高效衍生，包括表达酪氨酸羟化酶的神经元的分化。HESC形态分化为单层OCT-4+细胞，在无血清条件下直接获得胚状体。暴露于MedII条件培养基增强了神经前体的衍生，这是条件培养基对HESC影响的第一个例子。
We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons. HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII). A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.
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DOI：
10.1073/pnas.80.19.6126
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
STERNBERGER, LA;STERNBERGER, NH
通讯作者：
STERNBERGER, NH
影响因子：
46.9
作者：
Lee, SH;Lumelsky, N;McKay, RD
通讯作者：
McKay, RD
影响因子：
46.9
作者：
Reubinoff, BE;Itsykson, P;Ben-Hur, T
通讯作者：
Ben-Hur, T
影响因子：
9.2
作者：
Thomas, P;Beddington, R
通讯作者：
Beddington, R
影响因子：
56.9
作者：
Thomson, JA;Itskovitz-Eldor, J;Jones, JM
通讯作者：
Jones, JM