Fate conversion of developing kidney cells

该项目旨在利用基因工程和发育生物学知识控制发育中肾脏的细胞命运。后肾间充质可分化为肾小球、近端和远端肾小管等多种组织的上皮细胞，因此含有多能祖细胞。祖细胞在Wnt刺激后上皮化，随后Notch2在近端命运的建立中起重要作用，所述近端命运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细胞命运决定，并使所需的细胞谱系，这将是有益的，为未来的细胞治疗的肾脏。为了达到这一目的，我们产生了两种小鼠品系：一种仅在肾间充质祖细胞中表达Cre重组酶，另一种含有两侧为loxP位点（Cre靶）的终止序列，随后是Notch2的细胞内结构域，Notch2是组成性活性形式。我们通过使用同源重组到胚胎干细胞的ROSA 26基因座中来产生后者。当这两个品系的小鼠杂交时，终止序列被切除，Notch2在肾脏的祖细胞中被激活。事实上，所有的双杂合子出生后不久死亡，肾脏大小显着减少。我们有初步的数据表明，祖细胞过早耗尽的激活Notch2，并需要确认与免疫染色和其他方法。通过使用小鼠，我们计划最终开发一种方法来诱导祖细胞向肾小球和其他谱系分化，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.