Lelkes P I, Galvan D L, Hayman G T, Goodwin T J, Chatman D Y, Cherian S, Garcia R M, Unsworth B R
Laboratory of Cell Biology, University of Wisconsin Medical School, Milwaukee Clinical Campus, 53201-0342, USA.
In Vitro Cell Dev Biol Anim. 1998 Apr;34(4):316-25. doi: 10.1007/s11626-998-0008-y.
We are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This "simulated microgravity" environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC12 cells, cultured under "simulated microgravity" conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells.
我们正在研究有助于神经内分泌器官组装和组织特异性分化的微环境线索。作为我们的体外模型，我们在一种新型细胞培养系统——美国国家航空航天局旋转壁式生物反应器（RWV）中培养大鼠肾上腺髓质嗜铬细胞瘤PC12细胞。RWV生物反应器中的这种“模拟微重力”环境，其特点是重力向量随机化且剪切应力最小化，已被证明有利于宏观组织组装并诱导组织特异性分化。我们假设RWV生物反应器中独特的培养条件可能会增强神经内分泌类器官的体外形成。为了验证我们的假设，我们评估了在慢转弯侧室（STLV）型RWV中培养长达20天的PC12细胞培养物中几种神经内分泌分化标志物的表达。通过形态学、免疫学、生物化学和分子技术评估PC12细胞的分化。在“模拟微重力”条件下培养的PC12细胞形成了直径达几毫米的宏观组织样类器官。同时，苯乙醇胺 - N - 甲基转移酶（PNMT）的表达增强，而其他儿茶酚胺合成酶的表达未增强。如在基因和蛋白质水平上所证实的，PNMT表达的增加伴随着该酶比活性的增加。此外，在STLV中培养20天后，我们观察到蛋白质酪氨酸磷酸化模式的改变以及AP - 1核转录因子复合物成员c - fos的激活延长。我们得出结论，RWV中的培养条件似乎选择性地激活了信号转导途径，导致PC12细胞的神经内分泌分化增强。
