Isolation and Expansion of Multipotent Progenitors from Human Trabecular Meshwork

Abstract

To expand multi-potent progenitors from human trabecular meshwork (TM), we have created a new optimized method on two-dimensional (2D) followed by three–dimensional (3D) Matrigel in modified embryonic stem cell medium supplemented with 5% fetal bovine serum (MESCM + 5% FBS). The expanded TM cells were small cuboidal cells expressing TM markers such as AQP1, MGP, CHI3L1, and AnkG, embryonic stem cell (ESC) markers such as Oct4, Sox2, Nanog, and ABCG2, and neural crest (NC) markers such as p75NTR, FOXD3, Sox9, Sox10, and MSX1. Although expanded cells lost expression of these markers after passage, the cells regained the markers when Passage 2 cells were seeded on 3D Matrigel through activation of canonical BMP signaling. Such restored progenitors could differentiate into corneal endothelial cells, adipocytes, and chondrocytes but not keratocytes or osteocytes. Therefore, we have concluded that human TM harbors multipotent progenitors that can be effectively isolated and expanded using 2D Matrigel in MESCM + 5% FBS. This unique in vitro model system can be used to understand how TM is altered in glaucoma and whether such TM progenitor cells might one day be used for treating glaucoma or corneal endothelial dysfunction.

Human Trabecular Meshwork Progenitors

“…In contrast, TM cells cultured in MESCM+5% FBS could be expanded to P8 with a total cell doubling of 16. Based on our experiment results, culture of TM cells in MESCM+5% FBS, not MESCM alone on 2D Matrigel is the best condition for their expansion TM cells can be isolated by collagenase and expanded on coated Matrigel in MESCM+5%FBS up to 7 passages with 16 cell doublings [1]. Immunostaining showed that these cells at the time of isolation (D0) expressed TM markers such as AQP1, CHI3L1, MGP and AnkGs [1], similar to what have been previously reported [22,23].…”

“…Immunostaining showed that these cells at the time of isolation (D0) expressed TM markers such as AQP1, CHI3L1, MGP and AnkGs [1], similar to what have been previously reported [22,23]. Compared with that of the D0 cluster immediately isolated by collagenase, qRT-PCR revealed a significant decline in expression of these markers as well as ESC and NC markers by P2 cells [1], suggesting that 2D matrigel cannot retain their progenitor status. This conclusion was confirmed by qTR-PCR of TM cell markers, ESC and NC markers.…”

“…Based on our experiment results, culture of TM cells in MESCM+5% FBS, not MESCM alone on 2D Matrigel is the best condition for their expansion TM cells can be isolated by collagenase and expanded on coated Matrigel in MESCM+5%FBS up to 7 passages with 16 cell doublings [1]. Immunostaining showed that these cells at the time of isolation (D0) expressed TM markers such as AQP1, CHI3L1, MGP and AnkGs [1], similar to what have been previously reported [22,23]. Compared with that of the D0 cluster immediately isolated by collagenase, qRT-PCR revealed a significant decline in expression of these markers as well as ESC and NC markers by P2 cells [1], suggesting that 2D matrigel cannot retain their progenitor status.…”

“…When reseeded on 3D Matrigel at P3 for 2 days following the method from Li [24,25], these cells formed spheres, but not from P2 cells on coated Matrigel. Addition of Noggin abolished nuclear translocation of Nanog in cells seeded on 3D Matrigel [1]. In addition, the transcript level of TM cell markers CHI3L1, MGP, AnkG, ESC and NC markers Klf4, Nanog, Oct4, Sox2, SSEA4, Foxd3, Msx10, Sox9, Sox10 and PDGFRβ was significantly increased compared that from P2 cells.…”

Trabecular meshwork cells are a valuable resource for cellular therapy of glaucoma

“…In fact, we found that TM cells did not express detectable keratocan transcripts by RT-real-time PCR and cannot be induced into keratocyte-like cells even after the cells were cultured in 3-D Matrigel. 53 All Du's findings were that TM cells could be induced into adipose like cells. 62 Therefore, it is important to thoroughly characterize TM progenitors using TM cell markers, embryo stem cell markers, and neural crest progenitor markers.…”