Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development.

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.

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.

Perspectives on the cost of goods for hPSC banks for manufacture of cell therapies.

This report summarizes key issues contributing to the cost of preparing human pluripotent stem cell lines for use in cell therapy manufacturing based on discussion between stem cell banking experts from ten countries at a workshop session on ‘cost of goods’ for human pluripotent stem cell banking organized by the International Stem Cell Banking Initiative (ISCBI) held at the Korea National Institutes of Health in Korea (25 September 2019). In this report, we also build on the workshop discussion and highlight and discuss the full range of costs and unexpected challenges on resources for the delivery of stocks of hPSCs suitable for use as starting materials in the manufacture of stem cell-based medicines. The experiences of global leaders from different national resource centers highlight issues to consider in cost management and the possibilities for reducing costs while moving into the clinical application stage.

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.

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.

A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017.

This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed.

A Synthetic Hybrid Molecule for the Selective Removal of Human Pluripotent Stem Cells from Cell Mixtures.

A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probe 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs.

Efficient and scalable culture of single dissociated human pluripotent stem cells using recombinant E8 fragments of human laminin isoforms.

This unit describes a protocol for efficient expansion of human pluripotent stem cells (hPSCs). A key feature of this method is subculture of hPSCs by single-cell dissociation passaging on substrates coated with recombinant E8 fragments of human laminin isoforms (LM-E8s). LM-E8s, provide superior adhesion over intact laminin isoforms and Matrigel.

Selective elimination of human pluripotent stem cells by a marine natural product derivative.

One of the current obstacles to stem cell therapy is the tumorigenic potential of residual undifferentiated stem cells. The present study reports rediscovery of a synthetic derivative of okadaic acid, a marine polyether toxin, as a reagent that selectively induces the death of human pluripotent stem cells. Cell-based screening of 333 cytotoxic compounds identified methyl 27-deoxy-27-oxookadaate (molecule 1) as a substrate of two ATP-binding cassette (ABC) transporters, ABCB1 (MDR1) and ABCG2 (BCRP), whose expression is repressed in human embryonic stem cells and induced pluripotent stem cells.

Efficient Expansion of Dissociated Human Pluripotent Stem Cells Using a Synthetic Substrate.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human-induced pluripotent stem cells, are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes, large numbers of high-quality cells are essential. Recently, we showed that a biological substrate, recombinant E8 fragments of laminin isoforms, sustains long-term self-renewal of hPSCs in defined, xeno-free medium with dissociated single-cell passaging.

A chemical probe that labels human pluripotent stem cells.

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1.

Identification of small molecules that promote human embryonic stem cell self-renewal.

Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose, development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF).

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

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms.

Cardiomyocytes develop from anterior primitive streak cells induced by β-catenin activation and the blockage of BMP signaling in hESCs.

Cardiomyocytes arise from cells that migrate to the mid-to-anterior region of the primitive streak (PS) during embryogenesis. We previously showed that canonical Wnt/β-catenin pathway signaling leads to the development of nascent PS populations from human embryonic stem cells (hESCs) and that synergistic activation of the Wnt/β-catenin pathway and inhibition of bone morphogenetic protein (BMP) signaling by Noggin induced the formation of anterior PS cells. We herein demonstrate that anterior PS cells induced by the activation of β-catenin with Noggin differentiate into functional cardiomyocytes when cultured in suspension with BMP4 and fibroblast growth factor 2 (FGF2).

Role of SOX2 in maintaining pluripotency of human embryonic stem cells.

Human embryonic stem cell (ESC) pluripotency is thought to be regulated by several key transcription factors including OCT4, NANOG, and SOX2. Although the functions of OCT4 and NANOG in human ESCs are well defined, that of SOX2 has not been fully characterized. To investigate the role of SOX2, we modulated the level of SOX2 expression in human ESCs.

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.

In vitro hepatic maturation of human embryonic stem cells by using a mesenchymal cell line derived from murine fetal livers.

Hepatocytes derived from human embryonic stem cells (hESCs) are an attractive cell source for regenerative medicine. We previously reported the differentiation of hESCs into alpha-fetoprotein (AFP)-producing endodermal cells by using extracellular matrix and growth factors. We also reported the establishment of the MLSgt20 cell line, which was derived from mesenchymal cells residing in murine fetal livers and accelerated the hepatic maturation of both murine hepatic progenitor cells and murine ESCs.

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors.

Establishment of a cell line derived from a mouse fetal liver that has the characteristic to promote the hepatic maturation of mouse embryonic stem cells by a coculture method.

Stromal cells residing in murine fetal livers have the ability to promote the hepatic maturation of murine embryonic stem cells (ESCs) and hepatic progenitor cells (HPCs) 3848 in vitro. These stromal cells were isolated as the CD49f(+/-)CD45(-)Thy1(+)gp38(+) cell fraction. The present study established a murine fetal liver stromal cell line that induced hepatic maturation in mouse ESCs and HPCs.

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established.

Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells.

Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs.

Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells.

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs.