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首页> 美国卫生研究院文献>Cellular Reprogramming >Development Characterization and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized Hand-Guided Cloned and Parthenogenetic Embryos
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Development Characterization and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized Hand-Guided Cloned and Parthenogenetic Embryos

机译：体外受精手工克隆和孤雌生殖胚胎的水牛（Bubalus bubalis）胚胎干细胞系的发育鉴定和多能性分析

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We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium.
机译：我们介绍了从体外受精，体细胞核移植和单性胚泡的三个水牛胚胎干细胞（buESC）系的衍生，表征和多能性分析。开发了这些细胞系，用于以后分化为生殖系细胞和阐明涉及的信号通路。细胞系是从内部细胞团（ICM）中建立的，这些细胞团是从体外产生的胚泡中手动分离出来的。大多数ICM（45-55％）导致原代菌落的形成，这些原代菌落在8-10天后继代培养，随后导致形成三个buESC系，每种囊胚类型一个。所有细胞系均表达干细胞标记，例如碱性磷酸酶，OCT4，NANOG，SSEA1，SSEA4，TRA-1-60，TRA-1-81，SOX2，REX1，CD-90，STAT3和TELOMERASE。根据外胚层，中胚层和内胚层RNA和蛋白质标记物的确定，它们分化为所有三个胚层。所有细胞系均显示出多能性标记物的相等表达以及向所有三个胚层的等效分化潜能。静态悬浮培养衍生的胚状体（EB）与悬挂滴培养衍生的EB相比，在所有三个胚层标记中均表现出更高的表达。分析细菌层标记物表达时，与基于基因敲除血清替代品（KoSR）的分化相比，源自15％胎牛血清（FBS）的自发分化培养基的EB在所有三个细菌层中均表现出更大的分化介质。

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