Affiliation:
1. Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021, China
2. Institute of Regenerative Medicine and Laboratory Technology Innovation, Qingdao University, Qingdao 266021, China
3. Qingdao Haier Biotech Co., Ltd., Qingdao 266109, China
4. Advanced Innovation Center for Big Data-Based Precision Medicine, Beijing University of Aeronautics and Astronautics-Capital Medical University, Beijing 100083, China
Abstract
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.
Funder
National Key Research and Development Program
National Marine Pilot Laboratory of the Ministry of Science and Technology
National Natural Science Foundation of China
Taishan Scholar Youth Expert Program
Qingdao Key Technology and Industrialization Project
Natural Science Foundation of Shandong Province
Shandong Key Research and Development Program
Qingdao Science and Technology Huimin Project
Project of Qingdao Haier Biotech Co., Ltd.
Project of Shandong Land Urban–Rural Integration Development Group Co., Ltd.