Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method.
Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method.
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DOI：
10.1186/s13287-017-0738-4
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发表时间：
2017-12-29
影响因子：
7.5
通讯作者：
Skottman H
中科院分区：
文献类型：
--
作者：
Hongisto H;Ilmarinen T;Vattulainen M;Mikhailova A;Skottman H
人多能干细胞(HPSCs)为眼部细胞替代治疗提供了一种很有前途的细胞来源，但通常缺乏标准化和无异种的培养和分化方案。我们的目标是开发一种未分化的hPSCs的无异种和无饲养层细胞培养体系，以及获得眼部治疗靶细胞的有效方法：视网膜色素上皮(RPE)细胞和角膜缘上皮干细胞(LESCs)。多个遗传上不同的hPSC系适应于一个明确的、异种和无饲养层的培养系统，该系统由Essential 8™培养基和Laminin521基质组成。此后，利用无异种培养液和细胞外基质蛋白的组合，建立了向眼上皮细胞分化的两阶段方法。这两种分化方法共享相同的基本要素，在向所需细胞系的早期分化过程中只使用了少量的诱导修改。RPE细胞和LESCs经过几次独立的分化实验和无异种冷冻保存后的复苏进行了鉴定。这种明确的、无异种和无饲养层的培养系统提供了一种强大的方法来产生高质量的hPSCs，其染色体稳定性仅限于早期传代。在分化的第一周引入的诱导信号对谱系特征、细胞存活、甚至成熟的RPE特性都有很大的影响。衍生的RPE在插入物上成熟9周后，形成具有成熟紧密连接的功能性上皮单层，表达RPE基因和蛋白，并具有吞噬和关键生长因子分泌能力。高效的LESC分化导致细胞群体在第24天表达LESC标记，如p40/p63α。最后，我们建立了多能hPSCs、hPSC-RPE细胞和hPSC-LESCs的无异种冷冻库方案，并证明了解冻后的成功恢复。我们提出了高效、可扩展、定向分化高质量RPE细胞和LESC的方法。这两种临床相关的细胞类型是通过对相同的基本方法进行简单的诱导修改，然后进行贴壁培养、传代和冷冻库培养而产生的。本文的在线版本(doi：10.1186/s13287-0170738-4)包含补充材料，授权用户可以使用。
Human pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs). Multiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation. The defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing. We propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking. The online version of this article (doi:10.1186/s13287-017-0738-4) contains supplementary material, which is available to authorized users.
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影响因子：
3.7
作者：
Geng Z;Walsh PJ;Truong V;Hill C;Ebeling M;Kapphahn RJ;Montezuma SR;Yuan C;Roehrich H;Ferrington DA;Dutton JR
通讯作者：
Dutton JR
影响因子：
4.3
作者：
Haagdorens M;Van Acker SI;Van Gerwen V;Ní Dhubhghaill S;Koppen C;Tassignon MJ;Zakaria N
通讯作者：
Zakaria N
影响因子：
5.2
作者：
Metallo, Christian M.;Ji, Lin;Palecek, Sean P.
通讯作者：
Palecek, Sean P.
影响因子：
5.9
作者：
Avery, Stuart;Hirst, Adam J.;Baker, Duncan;Lim, Chin Yan;Alagaratnam, Sharmini;Skotheim, Rolf I.;Lothe, Ragnhild A.;Pera, Martin F.;Colman, Alan;Robson, Paul;Andrews, Peter W.;Knowles, Barbara B.
通讯作者：
Knowles, Barbara B.
影响因子：
4.6
作者：
Foster, James W.;Wahlin, Karl;Chakravarti, Shukti
通讯作者：
Chakravarti, Shukti