Department of Embryology and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium.
Hum Reprod. 2009 Nov;24(11):2709-17. doi: 10.1093/humrep/dep262. Epub 2009 Jul 24.
Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres.
Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs).
Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype.
We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.
最近，我们证明了 4 细胞期人类胚胎的单个卵裂球能够发育成具有内细胞团和滋养外胚层的囊胚。为了进一步研究 4 细胞期的潜能，我们旨在从单个卵裂球中获得多能性人类胚胎干细胞（hESC）。
将 4 个 4 细胞期胚胎在植入前发育的第 2 天分裂，将 16 个卵裂球分别在连续培养基中培养。在第 3 或第 4 天，将卵裂球衍生的胚胎种植在灭活的小鼠胚胎成纤维细胞（MEF）上。
16 个卵裂球中有 10 个衍生的桑椹胚附着在 MEF 上，其中 2 个产生了一个外胚层。它们被机械传代到新鲜的 MEF 上，如囊胚 ICM 衍生的 hESC 所述，并通过免疫细胞化学和/或 RT-PCR 显示出典型的干细胞标志物表达。体内多能性通过注射免疫缺陷小鼠后获得的畸胎瘤中存在所有三个胚层得到证实。第一个 hESC 系显示出一种镶嵌型正常/异常 46,XX,dup(7)(q33qter),del(18)(q23qter) 核型。第二个 hESC 系显示出正常的 46,XY 核型。
我们报告了从四个分裂的 4 细胞期人类胚胎的单个卵裂球成功地衍生和鉴定了两个 hESC 系。这两个 hESC 系来自不同的胚胎，证明至少一个 4 细胞期的卵裂球是多能性的。
