Minuth Will W, Denk Lucia, Meese Christine, Rachel Reinhard, Roessger Anne
Department of Molecular and Cellular Anatomy, University of Regensburg, University Street 31, D-93053 Regensburg, Germany.
Langmuir. 2009 Apr 21;25(8):4621-7. doi: 10.1021/la803858q.
In regenerative medicine, stem/progenitor cells are emerging as potential candidates for the treatment of renal failure. However, the mechanism of regeneration of renal tubules from stem/progenitor cells is not well-elucidated. In this study, a new method was developed for the generation of tubules replacing coating by extracellular matrix proteins. Renal stem/progenitor cells are mounted between layers of polyester fleece. This artificial interstitium supports spatial development of tubules within 13 days of perfusion culture in chemically defined Iscove's modified Dulbecco's medium (IMDM) containing aldosterone as the tubulogenic factor. Whole mount label by soybean agglutinin (SBA) showed that generated tubules exhibited a lumen and a continuously developed basal lamina. Immuno-labeling for cytokeratin Endo-A demonstrated the presence of isoprismatic epithelial cells, and laminin gamma1, occludin, and Na/K-ATPase alpha5 labeling revealed typical features of a polarized epithelium. To get first insight in the interface between tubules and polyester interstitium, transmission electron microscopy (TEM) was performed. The results showed that the generated tubules exhibited polar differentiation with a continuously developed basal lamina consisting of a lamina rara interna, lamina densa, and lamina rara externa. Collagen type III was found to be the linking molecule between the basal lamina and the surrounding polyester fibers by immuno labeling studies. Thus, the findings demonstrate that the spatial development involves the interface between the tubular basal lamina and the polyester interstitium of tubules and is not restricted to the epithelial portion.
在再生医学中，干细胞/祖细胞正成为治疗肾衰竭的潜在候选者。然而，干细胞/祖细胞再生肾小管的机制尚未得到充分阐明。在本研究中，开发了一种新方法来生成由细胞外基质蛋白替代包被的肾小管。肾干细胞/祖细胞被置于聚酯纤维层之间。这种人工间质在含有醛固酮作为肾小管生成因子的化学限定的伊斯科夫改良杜尔贝科培养基（IMDM）中灌注培养13天内支持肾小管的空间发育。用大豆凝集素（SBA）进行的整体标记显示，生成的肾小管呈现出管腔和持续发育的基底膜。细胞角蛋白Endo - A的免疫标记证明了等棱柱上皮细胞的存在，层粘连蛋白γ1、闭合蛋白和钠钾ATP酶α5标记揭示了极化上皮的典型特征。为了初步了解肾小管与聚酯间质之间的界面，进行了透射电子显微镜（TEM）检查。结果表明，生成的肾小管呈现出极性分化，具有由内疏松层、致密层和外疏松层组成的持续发育的基底膜。通过免疫标记研究发现，III型胶原蛋白是基底膜与周围聚酯纤维之间的连接分子。因此，这些发现表明空间发育涉及肾小管基底膜与肾小管聚酯间质之间的界面，且不限于上皮部分。
