iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications.

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

Sodium valproate rescues expression of TRANK1 in iPSC-derived neural cells that carry a genetic variant associated with serious mental illness.

Biological characterization of genetic variants identified in genome-wide association studies (GWAS) remains a substantial challenge. Here we used human-induced pluripotent stem cells (iPSC) and their neural derivatives to characterize common variants on chromosome 3p22 that have been associated by GWAS with major mental illnesses. IPSC-derived neural progenitor cells carrying the risk allele of the single nucleotide polymorphism (SNP), rs9834970, displayed lower baseline TRANK1 expression that was rescued by chronic treatment with therapeutic dosages of valproic acid (VPA). VPA had the greatest effects on TRANK1 expression in iPSC, NPC, and astrocytes. Although rs9834970 has no known function, we demonstrated that a nearby SNP, rs906482, strongly affects binding by the transcription factor, CTCF, and that the high-affinity allele usually occurs on haplotypes carrying the rs9834970 risk allele. Decreased expression of TRANK1 perturbed expression of many genes involved in neural development and differentiation. These findings have important implications for the pathophysiology of major mental illnesses and the development of novel therapeutics.

High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair.

Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2'-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs. Stem Cells 2018;36:1501-1513.

Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

Metabolic Quadrivalency in RSeT Human Embryonic Stem Cells.

One of the most important properties of human embryonic stem cells (hESCs) is related to their pluripotent states. In our recent study, we identified a previously unrecognized pluripotent state induced by RSeT medium. This state makes primed hESCs resistant to conversion to naïve pluripotent state. In this study, we have further characterized the metabolic features in these RSeT hESCs, including metabolic gene expression, metabolomic analysis, and various functional assays. The commonly reported metabolic modes include glycolysis or both glycolysis and oxidative phosphorylation (i.e., metabolic bivalency) in pluripotent stem cells. However, besides the presence of metabolic bivalency, RSeT hESCs exhibited a unique metabolome with additional fatty acid oxidation and imbalanced nucleotide metabolism. This metabolic quadrivalency is linked to hESC growth independent of oxygen tension and restricted capacity for naïve reprogramming in these cells. Thus, this study provides new insights into pluripotent state transitions and metabolic stress-associated hPSC growth in vitro.

Resistance to Naïve and Formative Pluripotency Conversion in RSeT Human Embryonic Stem Cells.

One of the most important properties of human embryonic stem cells (hESCs) is related to their primed and naïve pluripotent states. Our previous meta-analysis indicates the existence of heterogeneous pluripotent states derived from diverse naïve protocols. In this study, we have characterized a commercial medium (RSeT)-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent hypoxic growth conditions as required by naïve hESCs, in which some RSeT cells (e.g., H1 cells) exhibit much lower single cell plating efficiency, having altered or much retarded cell growth under both normoxia and hypoxia. Evidently, hPSCs lack many transcriptomic hallmarks of naïve and formative pluripotency (a phase between naive and primed states). Integrative transcriptome analysis suggests our primed and RSeT hESCs are close to the early stage of post-implantation embryos, similar to the previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs did not express naïve surface markers such as CD75, SUSD2, and CD130 at a significant level. Biochemically, RSeT hESCs exhibit a differential dependency of FGF2 and co-independency of both Janus kinase (JAK) and TGFß signaling in a cell-line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of formative pluripotency. Our data suggest that human naïve pluripotent potentials may be restricted in RSeT medium. Hence, this study provides new insights into pluripotent state transitions in vitro.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

Multivariate meta-analysis reveals global transcriptomic signatures underlying distinct human naive-like pluripotent states.

The ground or naive pluripotent state of human pluripotent stem cells (hPSCs), which was initially established in mouse embryonic stem cells (mESCs), is an emerging and tentative concept. To verify this vital concept in hPSCs, we performed a multivariate meta-analysis of major hPSC datasets via the combined analytic powers of percentile normalization, principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and SC3 consensus clustering. This robust bioinformatics approach has significantly improved the predictive values of our meta-analysis. Accordingly, we revealed various similarities or dissimilarities between some naive-like hPSCs (NLPs) generated from different laboratories. Our analysis confirms some previous studies and provides new evidence concerning the existence of three distinct naive-like pluripotent states. Moreover, our study offers global transcriptomic markers that define diverse pluripotent states under various hPSC growth protocols.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Human embryoid bodies as a 3D tissue model of the extracellular matrix and α-dystroglycanopathies.

The basal lamina is a specialized sheet of dense extracellular matrix (ECM) linked to the plasma membrane of specific cell types in their tissue context, which serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies and specific types of brain malformation. The latter three pathologies occur in the α-dystroglycanopathies, which are caused by dysfunction of the ECM receptor α-dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted owing to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells that model the basal lamina. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In α-dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy reveal ultrastructural basal lamina defects and reduced ECM accumulation. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in the α-dystroglycanopathies. Finally, we apply this system to evaluate an experimental ribitol supplement therapy on genetically diverse α-dystroglycanopathy patient samples.This article has an associated First Person interview with the first author of the paper.

Generation of human induced pluripotent stem cell line (NIDCRi001-A) from a Muenke syndrome patient with an FGFR3 p.Pro250Arg mutation.

Muenke syndrome is the leading genetic cause of craniosynostosis and results in a variety of disabling clinical phenotypes. To model the disease and study the pathogenic mechanisms, a human induced pluripotent stem cell (hiPSC) line was generated from a patient diagnosed with Muenke syndrome. Successful reprogramming was validated by morphological features, karyotyping, loss of reprogramming factors, expression of pluripotency markers, mutation analysis and teratoma formation.

Pluripotent Stem Cell Platforms for Drug Discovery.

Use of human pluripotent stem cells (hPSCs) and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery (hPDD). Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. In this review, we dissect representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR, the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis.

CYP3A5 Mediates Effects of Cocaine on Human Neocorticogenesis: Studies using an In Vitro 3D Self-Organized hPSC Model with a Single Cortex-Like Unit.

Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.

Toward xeno-free culture of human embryonic stem cells.

The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.

Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development.

Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage. Transgene expression is consistent with the developmental expression of PLP, with cells at all stages of oligodendrocyte differentiation clearly visualized. These animals were analyzed to establish the time course of oligodendrocyte progenitor migration, proliferation, and differentiation in neonatal cortex. In these animals, two populations of NG2 proteoglycan-positive oligodendrocyte progenitor cells were identified that exist in postnatal subventricular zone and cortex. These two populations are distinguished by the presence or absence of PLP gene expression. Thus, PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ. EGFP(+)/NG2(+) cells are present in the cortex from postnatal day 1, and they remain in the cortex as undifferentiated oligodendrocyte progenitors for up to 3 weeks before myelination begins. These data could be explained by the presence of an important inhibitor of oligodendrocyte differentiation in the cortex during this period, which is downregulated in a region-specific manner to allow myelination. On the other hand, it is possible that oligodendrocyte progenitor cells remain undifferentiated in cortex until an essential signal is produced in situ to induce differentiation.

Functional consequences of 17q21.31/WNT3-WNT9B amplification in hPSCs with respect to neural differentiation.

Human pluripotent stem cell (hPSC) lines exhibit repeated patterns of genetic variation, which can alter in vitro properties as well as suitability for clinical use. We examined associations between copy-number variations (CNVs) on chromosome 17 and hPSC mesodiencephalic dopaminergic (mDA) differentiation. Among 24 hPSC lines, two karyotypically normal lines, BG03 and CT3, and BG01V2, with trisomy 17, exhibited amplification of the WNT3/WNT9B region and rapid mDA differentiation. In hPSC lines with amplified WNT3/WNT9B, basic fibroblast growth factor (bFGF) signaling through mitogen-activated protein kinase (MAPK)/ERK amplifies canonical WNT signaling by phosphorylating LRP6, resulting in enhanced undifferentiated proliferation. When bFGF is absent, noncanonical WNT signaling becomes dominant due to upregulation of SIAH2, enhancing JNK signaling and promoting loss of pluripotency. When bFGF is present during mDA differentiation, stabilization of canonical WNT signaling causes upregulation of LMX1A and mDA induction. Therefore, CNVs in 17q21.31, a "hot spot" for genetic variation, have multiple and complex effects on hPSC cellular phenotype.

Alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.

Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, 3D environmental cues, and modes of multicellular association. In this Protocol Review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications.