The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.

Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions

A simple, robust, chemically defined method for generating cardiomyocytes from human pluripotent stem cells is described. It should enable the identification of conditions for maturation of these cells.

/pdf/chemically-defined-generation-of-human-cardiomyocytes-100ug2pq9h.pdf

Chemically defined generation of human cardiomyocytes

The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine.

/pdf/materials-as-stem-cell-regulators-e55d1lh2ny.pdf

Materials as stem cell regulators

Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells to Cardiomyocytes: A Methods Overview

The cell microenvironment has emerged as a key determinant of cell behavior and function in development, physiology, and pathophysiology. The extracellular matrix (ECM) within the cell microenvironment serves not only as a structural foundation for cells but also as a source of three-dimensional (3D) biochemical and biophysical cues that trigger and regulate cell behaviors. Increasing evidence suggests that the 3D character of the microenvironment is required for development of many critical cell responses observed in vivo, fueling a surge in the development of functional and biomimetic materials for engineering the 3D cell microenvironment. Progress in the design of such materials has improved control of cell behaviors in 3D and advanced the fields of tissue regeneration, in vitro tissue models, large-scale cell differentiation, immunotherapy, and gene therapy. However, the field is still in its infancy, and discoveries about the nature of cell–microenvironment interactions continue to overturn much earl...

Functional and Biomimetic Materials for Engineering of the Three-Dimensional Cell Microenvironment

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

Synthetic surfaces capable of supporting culture of eukaryotic cells including stem cells and undifferentiated human embryonic stem cells in a chemically defined medium include a swellable (meth)acrylate layer and a polypeptide conjugated to the swellable (meth)acrylate layer. The swellable (meth)acrylate layer may be formed by polymerizing monomers in a composition that includes a carboxyl group-containing (meth)acrylate monomer, a cross-linking (di- or higher-functional) (meth)acrylate monomer, and a hydrophilic monomer capable of polymerizing with the carboxyl group-containing (meth)acrylate monomer and the cross-linking (meth)acrylate monomer. The swellable (meth)acrylate layer has an equilibrium water content in water of between about 5% and about 70%. The conjugated peptide may include an RGD amino acid sequence.

Synthetic surfaces for culturing cells in chemically defined media

Plasma treated cyclic polyolefin copolymer surfaces having enhanced binding density for binding biologically active agents and cells are provided. These plasma treated cyclic polyolefin copolymer surfaces may be further enhanced for binding biologically active agents or cells by the application of conjugates. Methods of making and characterizing treated polymer surfaces are also provided.

Reactive surface on a polymeric substrate

Synthetic surfaces capable of supporting culture of undifferentiated human embryonic stem cells in a chemically defined medium include a swellable (meth)acrylate layer and a peptide conjugated to the swellable (meth)acrylate layer. The swellable (meth)acrylate layer may be formed by polymerizing monomers in a composition that includes hydroxyethyl methacrylate, 2-carboxyehylacrylate, and tetra(ethylene glycol) dimethacrylate. The conjugated peptide may include an amino acid sequence of XaanProGlnValThrArgGlyAspValPheThrMetPro, where n is an integer from 0 to 3 and where Xaa is any amino acid. Further, disclosed herein is a swellable (meth)acrylate synthetic surface which can be sterilized by gamma irradiation.

Swellable (meth)acrylate surfaces for culturing cells in chemically defined media

A method for producing a cell culture article having a synthetic polymer layer for incubating with cells includes diluting one or more (meth)acrylate monomers in a solvent and dispersing the diluted monomers on a surface of the cell culture article. Some or substantially all of the solvent is removed and the monomers are then polymerized on the surface of the article to form the synthetic polymer layer attached to the surface of the article.

Cell culture article and screening

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