Abstract
Purpose:
Tissue/organ development in human is a highly orchestrated complex process, regulated in part by the surrounding extracellular matrix (ECM) proteins and cell surface integrins. Every complex tissue in our body including retina has its own ECM configuration that plays critical role in cellular differentiation, adhesion, migration, and maturation. In this study, we applied a 3 D in vitro suspension culture system to direct differentiation of human induced pluripotent stem (iPS) cells into primitive optic vesicles and studied the ECM remodeling during early events of retinogenesis.<br />
Methods:
Defined sizes of 9000 cells embryoid bodies (EBs) were formed from dissociated iPS cells and directed differentiation towards neuroretina development. Expression of different markers of eye field and retinal progenitors were confirmed at different time points. RT Profiler PCR Array from Qiagen was applied to study the expression pattern of ECM & adhesion molecules in developing EBs and further confirmed by immunofluroescence assay.<br />
Results:
EBs undergoing 3D in vitro differentiation started expressing different neural and eye field markers, including Otx2, Sox2, Rx, LHX2, Six6, and PAX6 at different time points of differentiation. Retinal progenitor marker, CHX10 was observed on day 16 onwards. Several cell adhesion family members like E-cadherin, ICAM1, CD11A, CD11B, VLA-6 were downregulated while neural and retina specific cell adhesion molecules NCAM1, NPRAP, VLA-1 and CD49D were upregulated. Several glycoproteins that were previously shown to play key role during retinogenesis namely Tenascin-c, CD44, and fibronectin I were upregulated. Developing EBs were rich in laminin, collagen, and vitronectin.<br />
Conclusions:
Our finding shows that developing retina in EBs undergoing 3D suspension culture differentiation expressed several ECMs and adhesion molecules known to play critical role during retinogenesis in vivo. Stem cell derived in vitro model can help for further functional characterization of ECM remodeling during development and various retinal pathologies. <br />