Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA 91343, USA.
Biochem Biophys Res Commun. 2012 Jan 13;417(2):897-902. doi: 10.1016/j.bbrc.2011.12.071. Epub 2011 Dec 22.
The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.
体外衍生肾系祖细胞对于肾细胞治疗和再生至关重要。尽管过去进行了广泛的研究，但从胚胎干细胞诱导肾系的方案仍未建立。在这项研究中，我们旨在通过遵循体内发育阶段，即中胚层诱导（阶段 I）、中胚层诱导（阶段 II）和肾系诱导（阶段 III），从小鼠胚胎干细胞（mESC）中诱导肾系。为了进行阶段 I 诱导，根据体内中胚层发育涉及的已知信号通路，即 Nodal、骨形态发生蛋白（BMPs）和 Wnt，我们发现，在第 0-2 天连续添加三种因子，即激活素-A（A），Nodal 信号的替代物，在第 2-4 天添加 A 加上 BMP-4（4），在第 4-6 天添加 A4 加上锂（L），Wnt 信号的替代物，是诱导中胚层标志物 Brachyury 最有效的方法。为了进行阶段 II 诱导，在第 6-8 天连续存在 A4L 的情况下添加视黄酸（R）最有效地诱导肾发生中胚层中间标志物，如 Pax2 和 Lim1。在这种条件下，超过 30%的细胞被染色为 Pax2 阳性，同时非中胚层标志物的表达减少。为了进行阶段 III 诱导，类似于肾脏发育过程中输尿管芽（UB）和后肾间充质（MM）之间的相互诱导，我们发现，暴露于 UB 和 MM 细胞衍生的条件培养基分别有效地诱导 MM 和 UB 标志物。我们还观察到从阶段 I 到阶段 III，细胞群体表达祖细胞标志物 CD24 的出现和逐渐增加。这些 CD24(+)细胞与阶段 I 时 Brachyury 的表达水平较高、阶段 II 时 Pax2 和 Lim1 的表达水平以及暴露于 UB 条件培养基后阶段 III 时 MM 标志物（如 WT1 和 Cadherin 11）相关。总之，我们的结果表明，通过追踪体内发育阶段的逐步诱导，从 mESC 中生成肾系祖细胞是有效的，CD24 可能是阶段 II 肾系细胞和阶段 III MM 细胞的有用表面标志物。
