Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells

Abstract

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™ 1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™ -coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR™ 1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.

In situ tissue engineering with synthetic self-assembling peptide nanofiber scaffolds, PuraMatrix, for mucosal regeneration in the rat middle-ear

“…Biological ECM, eg, Matrigel (BD Biosciences, San Jose, CA, USA), which consists of type IV collagen, laminin, and heparan sulfate proteoglycan, seems to help create a suitable microenvironment. 12,13 Moreover, due to their innate structural and compositional similarities to ECM, hydrogels have been used as the material of choice in many applications in regenerative medicine and have been used as drug and cell carriers in the field of tissue engineering. 14,15 Hydrogels are three-dimensional networks formed from hydrophilic homopolymers, copolymers, or macromers.…”

“…[14][15][16] However, with regard to their clinical application, biological ECM and biological hydrogels carry the risk of contamination. 8,13 On the other hand, synthetic hydrogels, consisting of neutrally charged synthetic monomers, such as poly(2-hydroxyethyl methacrylate) (PHEMA), poly(vinyl acetate), and poly(ethylene glycol) (PEG) hydrogels do not have the drawback of contamination with infectious pathogens and might be ideal for tissue engineering. Much of the success with synthetic hydrogels in tissue engineering is due to the use of PHEMA hydrogels.…”

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

“…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.…”

Potential for pharmacological manipulation of human embryonic stem cells

“…Commonly used media and growth substrates are generally not well defined and may be contaminated by pathogens or xenogens (Martin et al, 2005). For this reason, many laboratories have attempted to develop fully defined conditions for hESC growth and in doing so have identified many cytokines and growth factors, such as WNT proteins, fibroblast growth factor (FGF), heparin, TGF-b, insulin-like growth factor II (IGF-II), activin A, platelet derived growth factor (PDGF) and neurotrophins (Dravid et al, 2005;Pebay et al, 2005;Vallier et al, 2005;Pyle et al, 2006;Xiao et al, 2006;Bendall et al, 2007;Furue et al, 2008;Montes et al, 2009) and growth surfaces (Klim et al, 2010;Mei et al, 2010;Melkoumian et al, 2010;Rodin et al, 2010;Villa-Diaz et al, 2010;Irwin et al, 2011;Lee et al, 2011;Nandivada et al, 2011;Saha et al, 2011), which allow for clonal feeder-free growth and subsequent differentiation. One such commercial success is the mTeSR® defined media from StemCell Technologies, which allow for both hESC and human-induced pluripotent stem cell (hiPSC) growth on Matrigel extracellular matrix with no additional growth factors (Thomson et al, 1998;Ludwig et al, 2006;Takahashi et al, 2007).…”