Andrei Gennadyevich Fadeev

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

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.

Synthetic Surfaces for Human Embryonic Stem Cell Culture

Human embryonic stem cells (hESCs) have two properties that distinguish them from other cell types: self-renewal, the ability to propagate indefinitely in culture, and pluripotency, the ability to differentiate into any type of specialized cells found in the human body. These properties provide the foundation for the development of hESC-derived cell-based therapeutics, where specific cell types derived by differentiation of hESCs become a therapeutic agent that cures the disease or restores the function of damaged organs or tissue. To make this a reality, several technologies must be developed to provide an unlimited and consistent supply of hESC-derived cells for clinical use. These include robust and scalable methods for production of undifferentiated hESCs, differentiation of the hESCs into desirable cell types, recovery, purification, storage and transportation of the derived cells to the location of use, and methods and techniques for delivery of the therapeutic cells to a human body to provide health benefits. Since the derivation of the first hESC lines by Thomson, J. et al. (Thomson, 1998) and Reubinoff, B. et al. (Reubinoff et al., 2000), hundreds of new lines have been established and propagated under various cell culture conditions. Historically, hESCs were maintained in complex culture systems under poorly defined conditions comprising mouse or human feeder cell layers and medium containing fetal bovine serum (FBS) or serum replacement to provide an extracellular matrix (ECM)-rich environment for cell adhesion, as well as soluble growth factors for self-renewal. It is highly desirable that the cell culture systems utilized for therapeutic cells, including cell culture surfaces and the media, are well defined (all components are known and characterized and their abundance is controlled) and of nonanimal origin or xeno-free (do not contain biological materials of a non-human nature). Establishment of the first human embryonic stem cell line (Thomson, 1998) was accomplished by extending to hESCs a cell culture system developed for culturing mouse embryonic stem cells that is based on inactivated mouse embryonic fibroblasts (MEF) as a feeder layer. Soon after the first reports on isolation of human pluripotent cells came realization that feeder-free cell culture is essential for production of cells for transplantation (Donovan & Gearhart, 2001; Pera et al., 2000); (Pedersen, 2002). Back in 2000 this looked like a challenge that would require a very long time to overcome, as 19 prior years of using MEFs to support stem cell culture of non-human cells did not result in significant understanding of what exactly MEFs provide for stem cells. To make matters worse, there was experimental evidence showing that neither MEF conditioned medium nor ECM