Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins.

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation.

Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin.

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.