A Synthetic Peptide-Acrylate Surface for Production of Insulin-Producing Cells from Human Embryonic Stem Cells

Abstract

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable b-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serumfree conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigelcoated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.

Defined Culture of Human Embryonic Stem Cells and Xeno-Free Derivation of Retinal Pigmented Epithelial Cells on a Novel, Synthetic Substrate

“…3C). Markers of ectoderm (MAP2 [microtubule‐associated protein 2]) [34], mesoderm (Tbx6 [T‐box protein 6]) [35], and endoderm (AFP [α‐fetoprotein]) [52, 53] were detected. Taken together, these data demonstrate that Synthemax II‐SC is a suitable, ACF substrate for the maintenance of hESCs that express canonical pluripotency markers and can differentiate into cells expressing markers of all three germ layers.…”

“…ACF conditions do not use any animal-derived products, including human, and this distinguishes it from xeno-free conditions. Synthemax-R has been shown to support hESC and iPSC growth [31][32][33][34][35][36][37] and their differentiation into ocular cell types [32,37,38]. However, Synthemax-R is only available in limited styles of tissue culture plates, thus restricting its range of applications and its ability to scale up the manufacture of therapeutic cells.…”

Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity

“…Dishes coated with recombinant extracellular matrices (ECMs) such as recombinant vitronectin, laminin (laminin-511, laminin-521 and laminin-322), and fibronectin (CellStart) showed excellent performance for hPSC cultures in chemically defined and/or serum-containing media 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 . Dishes immobilized with oligopeptides derived from ECMs were also reported to maintain hPSC pluripotency in chemically defined medium 25 26 27 28 29 30 31 32 33 34 35 36 37 38 . Completely synthetic dishes, such as PMVE-alt-MA (poly(methyl vinyl ether-alt-maleic anhydride) 39 ), PMEDSAH (poly[2-(methacryloyloxy](ethyldimethyl-(3-sulfopropyl)ammoniumhydroxide 40 41 42 43 ), APMAAm (aminopropylmethacrylamide 44 ) and copoly(AEtMA-co-DEAEA) (copoly[2-(acryloyloxyethyl)] trimethylammonium-co-2-(diethylamino)ethyl acrylate] 45 ), have been also developed for hPSC culture in chemically defined medium, although there are no cell binding sites on the surfaces of the synthetic dishes.…”

A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)

“…Our analysis revealed several RGD binding integrins, such as integrin α 5 , were expressed at significantly higher levels in early hPSC-derived ectoderm cells than in proliferating hNPCs possibly explaining the need for both peptide sequences for the differentiation of hPSCs to early ectoderm cell types. Another broadly used peptide-based material, Corning® Synthemax®, has been used for the long-term culture of hPSCs (24) as well as their differentiation into retinal pigmented epithelial cells (21), mesenchymal stem cells (22), oligodendrocyte progenitor cells (23), cardiomyocytes (24), and insulin producing cells (25). In this study, we tested the same peptide sequence (peptide 12) that serves as the basis for Corning® Synthemax®.…”