Structure and Barrier Properties of Human Embryonic Stem Cell–Derived Retinal Pigment Epithelial Cells Are Affected by Extracellular Matrix Protein Coating

Abstract

Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstartÔ and MatrigelÔ. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

Biomimetic collagen I and IV double layer Langmuir–Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells

“…For control samples, substrates were dip-coated with collagen protein solutions as described previously [11,27]. Briefly, substrates were rinsed with DPBS, and immersed in collagen protein solution at a concentration of 5 μg cm -2 for 3 hours at +37 o C. The following substrates were used for dip-coating: freshly cleaved mica for AFM measurements, SPR sensor substrates for SPR, borosilicate glass coverslips for immunofluorescence stainings and commercial PET cell culture inserts with 5 µm pore size for cell culture studies.…”

“…Undifferentiated hESCs were differentiated into RPE cells as described previously [11]. culture studies as we have previously used Col IV for enrichment and maturation of hPSC-RPE cells [11,27].…”

“…After 42 Differences in relative expression level of RPE65 gene were studied further with quantitative real-time PCR (qPCR) as described previously [27]. Samples were collected from two individual experiments.…”

“…The RPE65 protein expression was studied further after 42 days of culture on double layer LS films with Western blotting as previously described [27]. Mouse anti-RPE65 (1:5000, Millipore) and mouse anti-β-actin (1:5000, Santa Cruz) primary antibodies were used.…”

“…In addition, gelatin, which is collagen that has been processed to remove its higher organized structure, has been widely studied with RPE [24][25][26]. To the best of our knowledge, collagen type IV has been assessed with RPE cells and hESC-RPE cells merely in the form of protein coating [27][28][29]. Despite the success with collagen in biomedical applications, mimicking the natural fiber orientation and structure remains a major challenge [30][31][32].…”