Machine Learning Models of Phase Contrast Images Predict Efficiency of Human Pluripotent Stem Cell Differentiation to Cardiomyocytes

Background The number of pharmaceutical companies developing cell and gene therapy (CGT) products, categorized into cell therapy products (CTPs) and gene therapy products (GTPs), has increased, expanding patient access to approved treatments. Compared to conventional modalities, such as chemical compounds and biopharmaceuticals, both CTPs and GTPs pose significant challenges in their development. This study analyzed the characteristics of pharmaceutical companies associated with entry into CGT product development. Methods Focusing on 309 pharmaceutical companies worldwide with marketed products, we analyzed entry into development and the numbers of CTPs and GTPs. We explored firm characteristics associated with entry into CTP and GTP development, as well as their respective proportions within company pipelines, using multiple regression models. Results Approximately 30% of firms included CTPs or GTPs in their product baskets. Larger firms were more inclined to pursue CGT product development. Compared with companies headquartered elsewhere, Japanese companies were more likely to develop CTPs, maintaining a higher proportion of these agents in their baskets. The tendency towards home market entry was more pronounced for CTPs and GTPs than for chemical moieties and proteins. Firms with experience in biopharmaceuticals were more likely to develop GTPs, demonstrating higher proportions of these agents than firms without such experience. While larger sales tended to enhance entry into both CTP and GTP development, research and development expenditure played a greater role in driving entry into CTP development than into GTP development. Conclusions These findings underscore the distinct characteristics of CTPs and GTPs and offer valuable insights into pharmaceutical innovation research.

Toxicology has long relied on animal models in a tedious approach to understanding risk of exposure to an uncharacterized molecule. Stem cell-derived tissues can be made in high purity, quality, and quantity to enable a new approach to this problem. Currently, stem cell-derived tissues are primarily "generic" genetic backgrounds; the future will see the integration of various genetic backgrounds and complex three-dimensional models to create truly unique in vitro organoids. This minireview focuses on the state of the art of a number of stem cell-derived tissues and details their application in toxicology.

Reverse transcription, quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression by transcript abundance. Expression of target genes is normalized to expression of stable reference genes to account for sample preparation variability. Thus, the identification and validation of stably expressed reference genes is crucial for making accurate, quantitative, statistical conclusions in gene expression studies. Traditional housekeeping genes identified decades ago based on high and relatively stable expression are often used, although many have shown these to not be valid, particularly in highly dynamic systems such as stem cell differentiation. In this study we outline a rational approach to identify stable reference genes valid throughout human pluripotent stem cell (hPSC) differentiation to hPSC-derived cardiomyocytes (hPSC-CMs). Several publicly available transcriptomic data sets were analyzed to identify genes with low variability in expression throughout differentiation. These putative novel reference genes were subsequently validated in RT-qPCR analyses to assess their stability under various perturbations, including maturation during extended culture, lactate purification, and various differentiation efficiencies. Expression in hPSC-CMs was also compared with whole human heart tissue. A core set of three novel reference genes (EDF1, DDB1, and ZNF384) exhibited robust stability across the conditions tested, whereas expression of the traditional housekeeping genes tested (ACTB, B2M, GAPDH, and RPL13A) varied significantly under these conditions. Impact statement This article presents an unbiased method for the selection and validation of novel reference genes for real-time quantitative polymerase chain reaction normalization using data from RNA sequencing datasets. This method identified more robust and stable reference genes for gene expression studies during human pluripotent stem cell differentiation to cardiomyocytes than commonly used reference genes. This study also provides a roadmap for identifying reference genes for assessing gene expression during other dynamic cellular processes, including stem cell differentiation to other cell types.

Introduction: Innovations in human pluripotent stem cell research and their application in therapeutics have seen a giant leap in the past decade. Patent applications related to human pluripotent stem cell generation, culture and differentiation show an ever-increasing trend worldwide with hundreds of patents being applied for every year. With the turn of the second decade in stem cell patenting, a review of the latest patents issued will be significant.Areas covered: The growing need in healthcare sector has revolutionized stem cell application in clinical therapeutics by extending in unprecedented dimensions. With the potential of being able to differentiate into any desired adult cell lineage, human pluripotent stem cells find a wide range of applicability in clinical as well as cosmetic therapy. Moreover, the recent innovation of isolating a disease-specific pluripotent stem cell has opened new horizons to stem cell application in cell therapy. This review gives an overview of significant international patents granted on innovations in human pluripotent stem cell differentiation methodologies between 2009 and 2014.Expert opinion: The discovery of human pluripotent stem cells and their immense potential in clinical therapeutics has increasingly channeled scientific research in their orientation. Although being widely used to fathom human physiology, the trend in stem cell application is slowly shifting toward disease-modeling, drug safety evaluation and toxicity-testing. And in order to probe those unexplored realms of stem cell applications, a unified approach from the scientific community is imperative.

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

896. Local Direct Injection of BMP2-Modified Fibroblasts Induces Ectopic Bone Formation in Immunocompetent Mice

A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor β+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.

JAM-A as a potential surface marker of human pluripotent stem cells.

WNT3 Is a Biomarker Capable of Predicting the Definitive Endoderm Differentiation Potential of hESCs

Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.

Foreign Particulate Characterization Methodologies for Cell and Gene Therapy Products

Heme oxygenase-1 induction promotes human pluripotent stem cell differentiation into endothelial cells and vascular repair

Modulation of integrin and E-cadherin-mediated adhesions to spatially control heterogeneity in human pluripotent stem cell differentiation

Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media

To the Editor: The molecular mechanisms and the control of smooth muscle cell (SMC) differentiation have been extensively investigated because of its therapeutic potential.1 To date, different cell types have been used to study SMC differentiation, including a variety of mouse embryonic stem cells,2 adult stem cells,3,4 and others.5 Because several fundamental differences exist between mouse and human embryonic development,6 lack of a good model system to study human SMC differentiation has hampered the progress of translating SMC knowledge to novel clinical therapies. Human embryonic stem (hES) cells provide a valuable source of cells for studying human cell differentiation and developing therapeutic potentials in regenerative medicine. Since the initial report describing the derivation of hES cells,7 a variety of studies have established in vitro differentiation strategies to several lineages. Recently, it has been demonstrated that vascular progenitors derived from hES cells could be differentiated into endothelial cells and SMCs by endothelial …