Fate conversion of developing kidney cells

本项目旨在利用基因工程和发育生物学的知识来控制发育中的肾脏的细胞命运。后肾间质产生多种组织的上皮，包括肾小球、肾小管近端和远端，因此含有多能祖细胞。祖细胞在Wnt刺激下上皮化，随后Notch2在近端命运的建立中起重要作用，其中包括肾小球足细胞和近端小管。如果我们能够操纵祖细胞的命运决定，使细胞谱系如我们所愿，这将有利于未来的肾脏细胞治疗。为了达到这一目的，我们培养了两种小鼠：一种仅在肾间充质祖细胞中表达Cre重组酶，另一种含有loxP位点（Cre靶点）的停止序列，随后是Notch2的细胞内结构域，这是一种组成活性形式。我们通过对胚胎干细胞的ROSA26位点进行同源重组产生了后者。当这两种小鼠杂交时，停止序列被切除，Notch2在肾脏祖细胞中被激活。事实上，所有的双杂合子在出生后不久就死亡，肾脏大小明显缩小。我们有初步的数据表明，由于Notch2的激活，祖细胞过早耗尽，需要用免疫染色和其他方法来证实。通过使用小鼠，我们计划最终开发一种方法，将祖细胞诱导到肾小球和其他谱系，这将有助于肾脏再生。

This project aims at controlling cell fates of the developing kidney, by utilizing genetic engineering and the knowledge of developmental biology. The metanephric mesenchyme gives rise to epithelia of multiple tissues including glomeruli, proximal and distal renal tubules, thus contains multipotent progenitors. The progenitors epithelialize upon Wnt stimulation and subsequently Notch2 plays an important role for establishment of the proximal fate, which includes glomerular podocytes and proximal tubules. If we can manipulate the cells fate decision of the progenitors and make the desired cell lineages as we wish, it would be beneficial for future cell therapy of the kidney. To approach to this end, we generated two lines of mice: one expressing a Cre recombinase only in the kidney mesenchymal progenitors and the other harboring stop sequences flanked by loxP sites (Cre targets) followed by the intracellular domain of Notch2, the constitutively active form. We generated the latter by using homologous recombination into the ROSA26 locus of the embryonic stem cells. When these two lines of mice were crossed, the stop sequences were excised out and Notch2 was activated in the progenitors of the kidney. Indeed all the double heterozygotes died shortly after birth and the kidney size was reduced remarkably. We have preliminary data indicating that the progenitors were prematurely depleted by the activation of Notch2, and need to confirm it with immunostaining and other methods. By using the mice, we plan to develop eventually a method to induce the progenitors toward the glomeruli and other lineages, which would help regenerate the kidney.