The effects of the physical properties of culture substrates on the growth and differentiation of human embryonic stem cells
ABSTRACT The physical factors of cell-culture environment have received little attention despite their anticipated significant role in human embryonic stem cell (hESC) culture optimization. Here we show that hESC culture conditions can be optimized by utilizing polyethylene terephthalate (PET) membranes whose defined pore densities (PDs) determine membrane surface hardness. The PET membranes with 1-4 × 10(6) pores/cm(2) (0.291-0.345 GPa) supported the adherence and survival of hESCs without matrix coating. Furthermore, PET membrane with 4 × 10(6) pores/cm(2) (0.345 GPa) supported optimal hESC self renewal as well as by the increase in cell proliferation. The expression level and activity of Rho-associated kinase (ROCK) were specifically down-regulated in hESCs cultured on the optimal PET membrane. We suggest that PET membranes of a defined PD/hardness provide an excellent culture substrate for the maintenance of uniform and undifferentiated hESCs.
- SourceAvailable from: Irina V. Voronkina[Show abstract] [Hide abstract]
ABSTRACT: We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.Cell and Tissue Biology 01/2013; 7(1). DOI:10.1134/S1990519X13010094
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ABSTRACT: The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products which can be cost-prohibitive for large scale production and effective lineage specific differentiation protocols also still remain relatively undefined and are inefficient in producing large amounts of cells for therapeutic use. Further, the mechanisms and signalling pathways which mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents which can boost hESC pluripotency/self-renewal and survival and has allowed great increases in the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.British Journal of Pharmacology 04/2012; 169(2). DOI:10.1111/j.1476-5381.2012.01978.x · 4.99 Impact Factor
Conference Paper: The effect of introducing TeO2 on 2.7 µm emission in ZBLAN glass[Show abstract] [Hide abstract]
ABSTRACT: A novel erbium ions doped fluoro-tellurite glasses were prepared, and intense 2.7 μm emission was observed. The present Er3+-doped fluoro-tellurite glasses with large 2.7 μm emission cross-sections (6.32×10-21 cm2) indicates that it has a very promising application for solid state lasers around 3 μm.Advanced Infocomm Technology (ICAIT), 2013 6th International Conference on; 01/2013