Green Howard, Easley Karen, Iuchi Shiro
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15625-30. doi: 10.1073/pnas.0307226100. Epub 2003 Dec 8.
Human embryonic stem cells injected into scid mice produce nodules containing differentiated somatic tissues. From the trypsinized cells of such a nodule, we have recovered keratinocytes that can be grown in cell culture. The method of recovery is sensitive enough to detect small numbers of keratinocytes formed in the nodule, but for purposes of analysis, it is preferable to study the development of the entire keratinocyte lineage in culture. The principle of our analysis is the successive appearance of markers, including transcription factors with considerable specificity for the keratinocyte (p63 and basonuclin) and differentiation markers characteristic of its final state (keratin 14 and involucrin). We have determined the order of marker succession during the time- and migration-dependent development of keratinocytes from single embryoid bodies in cell culture. Of the markers we have examined, p63 was the earliest to appear in the keratinocyte lineage. The successive accumulation of later markers provides increasing certainty of emergence of the definitive keratinocyte.
将人类胚胎干细胞注射到重症联合免疫缺陷小鼠体内会产生含有分化体细胞组织的结节。从这样一个结节的胰蛋白酶消化细胞中，我们分离出了能够在细胞培养中生长的角质形成细胞。这种分离方法足够灵敏，能够检测到结节中形成的少量角质形成细胞，但为了进行分析，在培养中研究整个角质形成细胞谱系的发育更为可取。我们分析的原理是标志物的相继出现，包括对角质形成细胞具有相当特异性的转录因子（p63和锌指蛋白）以及其终末状态特征性的分化标志物（角蛋白14和内披蛋白）。我们已经确定了在细胞培养中来自单个胚状体的角质形成细胞在时间和迁移依赖性发育过程中标志物相继出现的顺序。在我们检测的标志物中，p63是最早出现在角质形成细胞谱系中的。后期标志物的相继积累为确定的角质形成细胞的出现提供了越来越高的确定性。
