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Wei Zhu, Qiong Ding, Kristin Anfinson, Dina Ahram, Budd A Tucker, Markus H Kuehn; Induction of trabecular meshwork cells from human iPSCs by human primary trabecular meshwork cell conditioned media. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5707.
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© ARVO (1962-2015); The Authors (2016-present)
We recently developed experimental approaches to stimulate mouse induced pluripotent stem cells (iPSC) to assume a trabecular meshwork (TM) cell like phenotype. This approach is particularly valuable since it potentially provides an opportunity to create TM-like cells from human patients with glaucoma or other ocular pathologies. Here we demonstrate that human iPSCs, derived from patient fibroblasts, can also be induced to resemble TM cells.
Human primary TM cells derived from three human eye donors were cultured in Biopsy media (MEM-alpha, 10% FBS). After 24 hours the conditioned media was collected and pooled. The conditioned media was used to culture human iPSCs for 8 weeks. iPSCs-derived TM cells were collected every two weeks for RNA extraction. The gene expression profile was determined in iPSC, iPSC-derived TM cells, and primary TM cells.Temporal patterns associated with the transition to a TM-like phenotype were determined.
Primary TM cell conditioned media induces obvious morphological changes in human iPSC in as early as 2 weeks. Cells begin to migrate away from the iPSC colony, increase in size, and exhibit an elongated shape. 4-6 week after the start of the differentiation increasing numbers of cells can be observed that closely resemble primary TM cells.These morphological changes are accompanied by distinct changes in the gene expression pattern of the TM-like cells. Some TM markers exhibit a steady increase in expression levels (e.g. CHI3L1 or MYOC) during the process of differentiation. Others, e.g. TIMP3 (6.5 fold increase after 2 weeks) exhibit rapid induction without subsequent further increase. Some genes maintain relatively low levels of expression until differentiation is almost complete (e.g. LAMA4 11 fold increase only after 8 weeks).
Our findings demonstrate that iPSCs derived from adult human fibroblasts can be induced to closely resemble primary TM cells. These findings are similar to those obtained using mouse cells, although differentiation of human cells occurs more slowly. Furthermore, transition from iPSC to TM cells is transcriptionally complex, possibly mirroring distinct developmental stages. It is conceivable that transplantation of these cells could restore functionality to the TM in POAG patients and result in the maintenance of healthy IOP.
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