Feng Pengchao, Wang Wenyan, Xu Wenhua, Cao Qilong, Zhu Wei
Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021, China.
Institute of Regenerative Medicine and Laboratory Technology Innovation, Qingdao University, Qingdao 266021, China.
Bioengineering (Basel). 2023 Mar 25;10(4):410. doi: 10.3390/bioengineering10040410.
The emergence of induced pluripotent stem cell (iPSC) technology has provided a new approach to regenerating decellularized trabecular meshwork (TM) in glaucoma. We have previously generated iPSC-derived TM (iPSC-TM) using a medium conditioned by TM cells and verified its function in tissue regeneration. Because of the heterogeneity of iPSCs and the isolated TM cells, iPSC-TM cells appear to be heterogeneous, which impedes our understanding of how the decellularized TM may be regenerated. Herein, we developed a protocol based on a magnetic-activated cell sorting (MACS) system or an immunopanning (IP) method for sorting integrin subunit alpha 6 (ITGA6)-positive iPSC-TM, an example of the iPSC-TM subpopulation. We first analyzed the purification efficiency of these two approaches by flow cytometry. In addition, we also determined cell viability by analyzing the morphologies of the purified cells. To conclude, the MACS-based purification could yield a higher ratio of ITGA6-positive iPSC-TM and maintain a relatively higher cell viability than the IP-based method, allowing for the preparation of any iPSC-TM subpopulation of interest and facilitating a better understanding of the regenerative mechanism of iPSC-based therapy.
诱导多能干细胞（iPSC）技术的出现为青光眼患者再生去细胞小梁网（TM）提供了一种新方法。我们之前使用经TM细胞条件培养的培养基生成了iPSC来源的TM（iPSC-TM），并验证了其在组织再生中的功能。由于iPSC和分离的TM细胞的异质性，iPSC-TM细胞似乎也是异质性的，这阻碍了我们对去细胞TM如何再生的理解。在此，我们开发了一种基于磁激活细胞分选（MACS）系统或免疫淘选（IP）方法的方案，用于分选整合素亚基α6（ITGA6）阳性的iPSC-TM，这是iPSC-TM亚群的一个例子。我们首先通过流式细胞术分析了这两种方法的纯化效率。此外，我们还通过分析纯化细胞的形态来确定细胞活力。总之，基于MACS的纯化方法能够产生更高比例的ITGA6阳性iPSC-TM，并且比基于IP的方法保持相对更高的细胞活力，从而能够制备任何感兴趣的iPSC-TM亚群，并有助于更好地理解基于iPSC的治疗的再生机制。
