Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins

“…We therefore evaluated surfaces displaying a cyclic RGD-containing peptide (cRGD) known to interact with high affinity with the α V β 3 and α V β 5 integrins (21). Additionally, we assessed RGD-containing peptides derived from bone sialoprotein [BSP(RGD)] (26) and vitronectin [VTN(RGD)] (22). We included a laminin-derived ligand for integrin α V β 3 and α 5 β 1 (C-16) (27) and a cyclic peptide integrin α 5 β 1 ligand (CRRETAWAC) (28).…”

“…Such defined substrata have been developed for the long-term culture of undifferentiated hPS cells in defined media. A number of surfaces have been described including recombinant proteins (14)(15)(16), fully synthetic polymers (17)(18)(19), and peptide-modified surfaces (20)(21)(22)(23). Surfaces that present bioactive peptides have the advantage that they can be programmed to interact with specific cell-surface macromolecules such as the glycosaminoglycans (GAGs) and integrins.…”

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions

“…Here, we develop a technology that notably provides expansion rates greater than the gold standard of feeder substrates; works with enzymatic passaging; and is compatible with current manipulations involving hESCs/hiPSCs, including gene modification and reprogramming. Importantly, in contrast to the previous reports that used polymers and/or peptides to coat the dish (10)(11)(12)14), our UV/ozone (UV) treatment of common research materials is an attractive alternative because it is inexpensive and mass production of appropriately treated culture dishes for numerous applications should be straightforward.…”

“…A variety of substrates are used in these methods: synthetic polymers (11,14), peptide-modified surfaces (10,12), embryonic ECM laminin isoforms (7), fibronectin from ECM with a small molecule mixture (9), and various vitronectin proteins (4,6). Some of these culture methods use animal products, which makes potential transplantation applications problematic (21).…”

“…induced pluripotent stem cells (hiPSCs) have the potential to grow indefinitely in culture and to make any cell in the adult human body, they represent important resources for regenerative medicine (1) and for disease modeling efforts that seek to recreate a patient's disease pathogenesis in the laboratory (2,3). hESCs and hiPSCs are typically grown on a "feeder" cell layer of mitotically inactivated mouse embryonic fibroblasts (mEFs) or on "feeder-free" culture systems composed of a variety of ECM/ serum proteins coated onto tissue culture dishes (4)(5)(6)(7)(8)(9) or synthetic materials (10)(11)(12)(13)(14). Nearly all these systems have been reported to promote hESC/hiPSC self-renewal when the cells are passaged in multicellular clumps and seeded at a suitably high cell density.…”