Moving toward Xeno-free Culture of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) were conventionally cultured on feeder cells that are isolated from mouse embryonic fibroblast (MEF). However, these culture components could contaminate the hPSCs and can limit the application of hPSCs for clinical use. On the other hand, we demonstrated that exogenous basic fibroblast growth factor (bFGF) could be omitted from the hPSC culture media if we used the suitable feeder cells. We also showed that although hPSCs can proliferate on the feeder-free culture system, however, genetic instability of hPSCs has been reported in such environment. Feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. We proposed the use of human cord blood-derived serum (hUCS) and showed a positive effect on culture of mesenchymal stem cells. The results showed that human foreskin fibroblasts (HFFs) cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, short population doubling times, high proliferation rates, and normal karyotypes after prolonged culture. These studies of hPSC xeno-free culture have been growing in both basic research and clinical trial. The data regarding the current clinical trials of using hPSCs convince the researchers not only about the possibility of application of hPSCs for cell-based therapy, but also the quality of established hPSC lines. Most of the hPSC lines that were published in the literature and registered in the National Institute of Health (NIH), hPSCreg of the European Union are not Good Manufacturing Practice (GMP) grade cell lines. Since one of the goals of using hPSCs is therapeutic purpose, GMP for derivation, cultivation, and handling the hPSCs are required. This chapter also reviews the state-of-the-art xeno-free culture system of hPSCs in the respect of future clinical applications

Human Embryonic Stem Cells: From Reproductive Medicine to Regenerative Medicine

Stem cells are the cells that have the capability to renew themselves and differentiate to the specific cell types. The successful of derivation of human embryonic stem cells (ESCs) in 1998, together with the discovery of reprogramming the human somatic cells into embryonic-like stage, so called “human induced pluripotent stemcells (iPSCs)” in 2007, dramatically increase the attention of the scientists and the public about the useof human ESCs or iPSCsforboth research and application. Although stem cells can be isolated and identified from several tissue of the human body such as limbal tissue, bone marrow or skin, the powerful of differentiation ability of human ESCs make these cell types a good candidates for cell replacement therapy. Recently, the successful of clinical trials using human ESCs for treating the patients who suffered from macular degeneration, underline the possibility of application of human ESCs for therapeutic purposes. In this review, the general biology of stem cells, source of human ESCs and the potential application of human ESCs were discussed

Comparative Analysis of Nuclear Transfer Embryo Derived Mouse Embryonic Stem Cells. Part I: Cellular characterization

Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. Karyotype analyses and cell growth studies did not reveal any significant variations. Despite some differences observed, the present study revealed that ntESC lines had similar differentiation competences compared to other ESCs. The results indicate that the observed differences may be related to the genotype rather than to the nuclear transfer technology

Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines

Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFβ1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system

The expression of the mRNA levels of IFN-α2, IFN-β1, IFN-γ, TNF-α, and IL-6 in the hNPCs following infection with and without H5N1 virus by quantitative RT-PCR.

<p>The mRNA expressions of IFN-α2, IFN-β1, IFN-γ, and IL-6 were not significantly difference, while the mRNA level expression of TNF-α was observed to be significantly up-regulated at 48 hpi, compared to that of the control groups. Data are expressed as mean fold changes with standard error compared to untreated controls after normalization with GAPDH mRNA expressions. Asterisks indicated statistically significant differences (<i>p</i>-value<0.05).</p

Immunostaining characterization and percentage of H5N1-positive cells of hNPCs following virus infection.

<p>Virus antigens were distributed within the cytoplasm and the nucleus of the infected cells (<b>A-C</b>), with peak percentage of virus-positive cells about 75% at 24 hpi, then declined to about 30% at 72 hpi (<b>D</b>). Scale bar in A-C ≈ 30 μm. Data represented are the mean ± standard error. Asterisks indicated statistically significant differences (<i>p</i>-value<0.05).</p