Multipotent Stem Cells from Trabecular Meshwork Become Phagocytic TM Cells

Abstract

PURPOSE.To isolate and characterize stem cells from human trabecular meshwork (TM) and to investigate the potential of these stem cells to differentiate into TM cells. METHODS. Human trabecular meshwork stem cells (TMSCs) were isolated as side population cells by fluorescence-activated cell sorting or isolated by clonal cultures. Passaged TMSCs were compared with primary TM cells by immunostaining and quantitative RT-PCR. TMSC purity was assessed by flow cytometry and TMSC multipotency was examined by induction of neural cells, adipocytes, keratocytes, or TM cells. Differential gene expression was detected by quantitative RT-PCR, immunostaining, and immunoblotting. TM cell function was evaluated by phagocytic assay using inactivated Staphylococcus aureus bioparticles. RESULTS. Side population and clonal isolated cells expressed stem cell markers ABCG2, Notch1, OCT-3/4, AnkG, and MUC1 but not TM markers AQP1, MGP, CHI3L1, or TIMP3. Passaged TMSCs are a homogeneous population with Ͼ95% cells positive to CD73, CD90, CD166, or Bmi1. TMSCs exhibited multipotent ability of differentiation into a variety of cell types with expression of neural markers neurofilament, ␤-tubulin III, GFAP; or keratocyte-specific markers keratan sulfate and keratocan; or adipocyte markers ap2 and leptin. TMSC readily differentiated into TM cells with phagocytic function and expression of TM markers AQP1, CHI3L1, and TIMP3. CONCLUSIONS. TMSCs, isolated as side population or as clones, express specific stem cell markers, are homogeneous and multipotent, with the ability to differentiate into phagocytic TM cells. These cells offer a potential for development of a novel stem cell-based therapy for glaucoma. (Invest Ophthalmol Vis Sci. 2012;53:1566 -1575

Cyp1b1 Mediates Periostin Regulation of Trabecular Meshwork Development by Suppression of Oxidative Stress

“…To further confirm the identity of these cells as TM cells, we detected the expression of TM cell markers, including alphasmooth muscle actin (47,48), ADAMTS10 (49), CHI3L1 (50-52), and AQP1 (50,53,54). Our FACS, Western blot, and quantitative PCR (qPCR) analyses confirmed positive expression of all these markers in Cyp1b1 ϩ/ϩ and Cyp1b1 Ϫ/Ϫ TM cells (Fig.…”

Transplantation of iPSC-derived TM cells rescues glaucoma phenotypes in vivo

“…Loss of TM cells has been suspected to contribute to aqueous humor outflow deficiencies and, conversely, it has been hypothesized that replacing lost cells can restore function to the eye (39). Previous studies by this and other laboratories have indicated that either multipotent cells isolated from the TM (40,41) or iPSC may be useful for this purpose (23,42). Indeed, recently published data have indicated that iPSC can restore some functional aspects in an organ culture system.…”

Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure

“…Using SP cell sorting to purify stem cells was discovered in 1996 38 and has been used to isolate many kinds of adult stem cells. Since there are not enough cells from human TM tissue for direct SP cell sorting, we 39,40 first cultured and passaged cells from human TM in stem cell growth medium (SCGM) containing multipurpose reduced-serum media (Opti-MEM) supplemented with 5% fetal bovine serum (FBS), 10 ng/mL epidermal growth factor (EGF), 100 mg/mL bovine pituitary extract, 20 mg/mL ascorbic acid, 200 mg/mL calcium chloride, 0.08% chondroitin sulfate, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 50 mg/mL gentamicin. After 2 to 3 passages, 5 · 10 5 -2 · 10 6 cells were incubated at 1 · 10 6 cells/mL in prewarmed DMEM with 2% FBS and either 5 mg/mL Hoechst 33342 41 or 10 mM DyeCycle Violet (DCV) 42 for 100 min at 37°C.…”