Development of substrates for the culture of human pluripotent stem cells

Although human pluripotent stem cell (hPSC) lines were initially established in culture using feeder cells, the development of culture media and substrates is essential for safe, stable, high-quality, and efficient production of large numbers of cells. Many researchers are now culturing hPSCs in chemically defined media and on culture substrates without feeder cells. In this review, we first discuss the problems with Matrigel, which has long been used as a culture substrate. Then, we summarize the development of extracellular matrix proteins for hPSCs, which are now the mainstream alternative, and synthetic substrates that are expected to be the future mainstream alternative. We also highlight three-dimensional culture for suitable mass production of hPSCs

Overexpression of Nuclear Receptor 5A1 Induces and Maintains an Intermediate State of Conversion between Primed and Naive Pluripotency

Naive and primed human pluripotent stem cells (hPSCs) have provided useful insights into the regulation of pluripotency. However, the molecular mechanisms regulating naive conversion remain elusive. Here, we report intermediate naive conversion induced by overexpressing nuclear receptor 5A1 (NR5A1) in hPSCs. The cells displayed some naive features, such as clonogenicity, glycogen synthase kinase 3β, and mitogen-activated protein kinase (MAPK) independence, expression of naive-associated genes, and two activated X chromosomes, but lacked others, such as KLF17 expression, transforming growth factor β independence, and imprinted gene demethylation. Notably, NR5A1 negated MAPK activation by fibroblast growth factor 2, leading to cell-autonomous self-renewal independent of MAPK inhibition. These phenotypes may be associated with naive conversion, and were regulated by a DPPA2/4-dependent pathway that activates the selective expression of naive-associated genes. This study increases our understanding of the mechanisms regulating the conversion from primed to naive pluripotency

Efficient derivation and banking of clinical-grade human embryonic stem cell lines in accordance with Japanese regulations

[Introduction] We recently established clinical-grade human embryonic stem cell (hESC) line KthES11 in accordance with current good manufacturing practice standards in Japan. Despite this success, the establishment efficiency was very low at 7.1% in the first period. [Methods] To establish clinical-grade hESC lines, we used xeno-free chemically defined medium StemFit AK03N with the LM-E8 fragments instead of feeder cells. The protocol was then optimized, especially in the early culture phase. [Results] We established five hESC lines (KthES12, KthES13, KthES14, KthES15, and KthES16) with 45.5% efficiency. All five hESC lines showed typical hESC-like morphology, a normal karyotype, pluripotent state, and differentiation potential for all three germ layers. Furthermore, we developed efficient procedures to prepare master cell stocks for clinical-grade hESC lines and an efficient strategy for quality control testing. [Conclusions] Our master cell stocks of hESC lines may contribute to therapeutic applications using human pluripotent stem cells in Japan and other countries

Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells

Miyazaki, T. et al.. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat. Commun. 3:1236 doi: 10.1038/ncomms2231 (2012)

Efficient integration of transgenes into a defined locus in human embryonic stem cells

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency

Generation of clinical-grade human embryonic stem cell line KthES11 according to Japanese regulations

The human embryonic stem cell line, KthES11, is derived from a normal healthy blastocyst donated for clinical research. The inner cell mass (ICM) was isolated using mechanical dissection and plated on laminin fragments. Cell line derivation, its propagation and storage were performed without feeders in an animal product-free environment according to current Good Manufacturing Practice (cGMP) standards. KthES11 shows a normal karyotype, pluripotent state and differentiation to the three germ layers. The cell line was further validated for sterility, mycoplasma-free, antibiotic residues and specific human pathogens