Mina and Everard Goodman Faculty of Life Sciences, Israel.
Mina and Everard Goodman Faculty of Life Sciences, Israel; School of Optometry and Visual Science, Faculty of Life Science, Bar-Ilan University, Ramat-Gan, Israel; Institute for Nanotechnology and Advanced Materials (BINA) Bar-Ilan University, Ramat-Gan, Israel.
Exp Eye Res. 2019 Mar;180:29-38. doi: 10.1016/j.exer.2018.11.013. Epub 2018 Nov 14.
Cell replacement therapy is a promising approach for treatment of retinal degenerative diseases. Several protocols for the generation of photoreceptor precursors (PRP) from human embryonic stem cells (hESC) have been reported with variable efficiency. Herein, we show the advantages of use of size-controlled embryoid bodies in the ESC differentiation process using two differentiation protocols. We further explored cell-labeling methods for following the survival of PRP transplanted subretinally in rat eyes. Size-controlled embryoid bodies (EBs) generated using microwell dishes and non-size-controlled EBs generated using V-shaped 96-well plates were differentiated into PRP using two differentiation protocols. The differentiation protocols utilized two different combinations of growth factors. The first, Dkk1, Noggin, and IGF1, and the second protocol used IWR1e, SAG, and CHIR99021. Differentiation efficiency to PRP was analyzed by qPCR, immunocytochemistry, and fluorescence-assisted cell sorting (FACS). Size-controlled IWR1e yielded a significantly higher percent (86.4%) of PRP cells expressing CRX, compared with non-size-controlled IWR1e (51.4%, P = 0.026) or the size-controlled DKK1 protocol (70.5%, p = 0.007). In addition, the IWR1e differentiated cells exhibited a significantly higher fluorescence intensity of CRX immunostaining, compared with the DKK1 protocol, consistent with higher protein expression levels. The IWR1e cells exhibited higher maturation levels, as manifested by lower early neuronal marker PAX6 and pluripotency marker OCT4 levels compared with the DKK1 protocol. The expression of other late photoreceptor markers (NRL, recoverin) were similar among the differentiation groups. PRP cells were labeled by using hESC constitutively expressing EGFP or by AAV-GFP transduction. Finally, we transplanted the cells in the subretinal space of wild-type rats and monitored their survival over several weeks. The AAV2 serotype efficiently transduced the PRP cells, whereas other serotypes yielded low or no transduction. Following subretinal transplantation of GFP-labeled PRP, 63% of the cells were detected at 4 weeks post-transplantation. In conclusion, we show here that the IWR1e protocol using size-controlled EBs efficiently generated of PRP that could be labeled and followed in-vivo for weeks. The data from this study is an advance toward the goal of PRP transplantation therapy for retinal degenerative diseases.
细胞替代疗法是治疗视网膜退行性疾病的一种很有前途的方法。已经有几种从人胚胎干细胞（hESC）生成光感受器前体细胞（PRP）的方案被报道，但其效率各不相同。在此，我们展示了在 ESC 分化过程中使用尺寸可控的类胚体的优势，我们使用了两种分化方案。我们进一步探索了细胞标记方法，用于跟踪在大鼠眼睛的视网膜下移植的 PRP 的存活情况。使用微井板生成的尺寸可控的类胚体（EBs）和使用 V 形 96 孔板生成的非尺寸可控的 EBs ，使用两种分化方案将其分化为 PRP。这两种分化方案使用了两种不同的生长因子组合。第一种是 Dkk1、Noggin 和 IGF1，第二种方案则使用了 IWR1e、SAG 和 CHIR99021。通过 qPCR、免疫细胞化学和荧光辅助细胞分选（FACS）分析 PRP 的分化效率。与非尺寸可控的 IWR1e（51.4%，P=0.026）或尺寸可控的 DKK1 方案（70.5%，p=0.007）相比，尺寸可控的 IWR1e 产生了显著更高比例（86.4%）的表达 CRX 的 PRP 细胞。此外，与 DKK1 方案相比，IWR1e 分化的细胞的 CRX 免疫染色荧光强度显著更高，这与更高的蛋白表达水平一致。IWR1e 细胞表现出更高的成熟水平，表现为早期神经元标记物 PAX6 和多能性标记物 OCT4 的水平低于 DKK1 方案。其他晚期光感受器标记物（NRL、恢复蛋白）的表达在分化组之间相似。通过使用 hESC 组成型表达 EGFP 或通过 AAV-GFP 转导来标记 PRP 细胞。最后，我们将细胞移植到野生型大鼠的视网膜下腔，并在数周内监测其存活情况。AAV2 血清型能够有效地转导 PRP 细胞，而其他血清型的转导效率则较低或没有。在 GFP 标记的 PRP 细胞进行视网膜下移植后，在移植后 4 周时检测到 63%的细胞。总之，我们在此展示了使用尺寸可控的 EBs 的 IWR1e 方案能够有效地生成 PRP，这些 PRP 可以被标记并在体内跟踪数周。这项研究的数据是 PRP 移植治疗视网膜退行性疾病目标的一个进展。
