Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays.
Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays.
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DOI：
10.1038/srep07402
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发表时间：
2014-12-10
影响因子：
4.6
通讯作者：
Zhang N
中科院分区：
文献类型：
--
作者：
Pettinato G;Wen X;Zhang N
开发了一种简单、可扩展、可重复的技术，该技术允许从分离的人类诱导多能干细胞（hiPSCs）直接形成大量确定大小的同质和同步胚状体（EBs）。非细胞黏附水凝胶用于制造圆底微孔，以容纳解离的hiPSCs。不需要rho相关激酶抑制剂（ROCK-i）或离心，可以避免ROCK-i的副作用。除了非细胞黏附圆底微井外，成功形成EB的关键要求是每个微井的输入细胞密度。装载在微孔中的细胞太少或太多都会影响EB的形成过程。同时，我们已经测试了基于微孔的系统从分离的人类胚胎干细胞（hESCs）中形成均匀的hEB。在每个微孔的最佳输入细胞密度范围内，在没有ROCK-i和离心的情况下，成功地从分离的hESCs中生产出均匀的hEBs。hiPSC和hesc衍生的hEBs都表达了所有三个发育胚层的关键蛋白特征，证实了它们的EB特性。这种新的EB生产技术可能代表了一个从解离的人类多能干细胞（hPSCs）生产同质EB的通用平台。
A simple, scalable, and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i), or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel, we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers, confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs).
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