Transient naive reprogramming corrects hiPS cells functionally and epigenetically.

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.

The effects of microgravity on bone structure and function.

Humans are spending an increasing amount of time in space, where exposure to conditions of microgravity causes 1-2% bone loss per month in astronauts. Through data collected from astronauts, as well as animal and cellular experiments conducted in space, it is evident that microgravity induces skeletal deconditioning in weight-bearing bones. This review identifies contentions in current literature describing the effect of microgravity on non-weight-bearing bones, different bone compartments, as well as the skeletal recovery process in human and animal spaceflight data. Experiments in space are not readily available, and experimental designs are often limited due to logistical and technical reasons. This review introduces a plethora of on-ground research that elucidate the intricate process of bone loss, utilising technology that simulates microgravity. Observations from these studies are largely congruent to data obtained from spaceflight experiments, while offering more insights behind the molecular mechanisms leading to microgravity-induced bone loss. These insights are discussed herein, as well as how that knowledge has contributed to studies of current therapeutic agents. This review also points out discrepancies in existing data, highlighting knowledge gaps in our current understanding. Further dissection of the exact mechanisms of microgravity-induced bone loss will enable the development of more effective preventative and therapeutic measures to protect against bone loss, both in space and possibly on ground.

An integrated analysis of human myeloid cells identifies gaps in in vitro models of in vivo biology.

The Stemformatics myeloid atlas is an integrated transcriptome atlas of human macrophages and dendritic cells that systematically compares freshly isolated tissue-resident, cultured, and pluripotent stem cell-derived myeloid cells. Three classes of tissue-resident macrophage were identified: Kupffer cells and microglia; monocyte-associated; and tumor-associated macrophages. Culture had a major impact on all primary cell phenotypes. Pluripotent stem cell-derived macrophages were characterized by atypical expression of collagen and a highly efferocytotic phenotype. Myeloid subsets, and phenotypes associated with derivation, were reproducible across experimental series including data projected from single-cell studies, demonstrating that the atlas provides a robust reference for myeloid phenotypes. Implementation in Stemformatics.org allows users to visualize patterns of sample grouping or gene expression for user-selected conditions and supports temporary upload of your own microarray or RNA sequencing samples, including single-cell data, to benchmark against the atlas.

BRCA1 and BRCA2 associated breast cancer and the roles of current modelling systems in drug discovery.

For a drug candidate to be fully developed takes years and investment of hundreds of millions of dollars. There is no doubt that drug development is difficult and risky, but vital to protecting against devastating disease. This difficulty is clearly evident in BRCA1 and BRCA2 related breast cancer, with current treatment options largely confined to invasive surgical procedures, as well as chemotherapy and radiotherapy regimes which damage healthy tissue and can leave remnant disease. Consequently, patient survival and relapse rates are far from ideal, and new candidate treatments are needed. The preclinical stages of drug discovery are crucial to get right for translation to hospital beds. Disease models must take advantage of current technologies and be accurate for rapid and translatable treatments. Careful selection of cell lines must be coupled with high throughput techniques, with promising results trialled further in highly accurate humanised patient derived xenograft models. Traditional adherent drug screening should transition to 3D culture systems amenable to high throughput techniques if the gap between in vitro and in vivo studies is to be partially bridged. The possibility of organoid, induced pluripotent stem cell, and conditionally reprogrammed in vitro models is tantalising, however protocols are yet to be fully established. This review of BRCA1 and BRCA2 cancer biology and current modelling systems will hopefully guide the design of future drug discovery endeavours and highlight areas requiring improvement.

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

Hepatic Differentiation of Stem Cells in 2D and 3D Biomaterial Systems.

A critical shortage of donor livers for treating end-stage liver failure signifies the urgent need for alternative treatment options. Hepatocyte-like cells (HLC) derived from various stem cells represent a promising cell source for hepatocyte transplantation, liver tissue engineering, and development of a bioartificial liver assist device. At present, the protocols of hepatic differentiation of stem cells are optimized based on soluble chemical signals introduced in the culture medium and the HLC produced typically retain an immature phenotype. To promote further hepatic differentiation and maturation, biomaterials can be designed to recapitulate cell-extracellular matrix (ECM) interactions in both 2D and 3D configurations. In this review, we will summarize and compare various 2D and 3D biomaterial systems that have been applied to hepatic differentiation, and highlight their roles in presenting biochemical and physical cues to different stem cell sources.

Three-dimensional differentiation of human pluripotent stem cell-derived neural precursor cells using tailored porous polymer scaffolds.

This study investigates the utility of a tailored poly(ethylene glycol) diacrylate-crosslinked porous polymeric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demonstrating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed population of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregulation of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins ßIII-TUBULIN, NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, synchronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This tailored construct has potential application as an improved in vitro human neurogenesis model with utility in platform drug discovery programs. STATEMENT OF SIGNIFICANCE: The interconnected porosity of polyHIPE scaffolds exhibits the ability to support three-dimensional neural cell network formation due to limited resistance to cellular migration and re-organisation. The previously developed scaffold material displays mechanical properties similar to that of the mammalian brain. This research also employs the utility of pluripotent stem cell-derived neural cells which are of greater clinical relevance than primary neural cell lines. This scaffold material has future potential in better mimicking three-dimensional neural networks found in the human brain and may result in improved in vitro models for disease modelling and drug screening applications.

The application of cell surface markers to demarcate distinct human pluripotent states.

Recent advances in human pluripotent stem cell (hPSC) research have uncovered different subpopulations within stem cell cultures and have captured a range of pluripotent states that hold distinct molecular and functional properties. At the two ends of the pluripotency spectrum are naïve and primed hPSC, whereby naïve hPSC grown in stringent conditions recapitulate features of the preimplantation human embryo, and the conventionally grown primed hPSC align closer to the early postimplantation embryo. Investigating these cell types will help to define the mechanisms that control early development and should provide new insights into stem cell properties such as cell identity, differentiation and reprogramming. Monitoring cell surface marker expression provides a valuable approach to resolve complex cell populations, to directly compare between cell types, and to isolate viable cells for functional experiments. This review discusses the discovery and applications of cell surface markers to study human pluripotent cell types with a particular focus on the transitions between naïve and primed states. Highlighted areas for future study include the potential functions for the identified cell surface proteins in pluripotency, the production of new high-quality monoclonal antibodies to naïve-specific protein epitopes and the use of cell surface markers to characterise subpopulations within pluripotent states.

Designer macrophages: Pitfalls and opportunities for modelling macrophage phenotypes from pluripotent stem cells.

Macrophages are phagocytic immune cells resident in every tissue that are not only important for host defence, but are also involved in tissue homeostasis, injury, and disease. Despite increasingly sophisticated methods for in vitro macrophage isolation, expansion and activation over the past three decades, these have largely been restricted to modelling bone-marrow or blood-derived cells. The in vitro derivation of macrophages from human pluripotent stem cells provides new opportunities to study macrophage biology, including the factors that impact human myeloid development and those that induce macrophage activation. While sharing many of the functional characteristics of monocyte-derived macrophages, stem cell-derived macrophages may offer new opportunities to understand the role of development or tissue context in innate immune cell function. Immune responsiveness to pathogenic challenge is known to be impacted by a macrophage's history of prior exposure, as well as ontogeny and tissue context. Therefore, we explore the factors of in vitro derivation likely to influence macrophage phenotype and function.

Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media.

We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlex and TeSR-E8 media. Analysis of transcriptome of single cells revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Analysis of individual cells from four samples consisting of two different iPSC lines each grown in the two culture media revealed a shared subpopulation structure with three main subpopulations different in pluripotency states. By implementing a machine learning approach, we estimated that most cells within each subpopulation are very similar between all four samples. The single-cell RNA sequencing analysis of iPSC lines grown in both media reports the molecular signature in StemFlex medium and how it compares to that observed in the TeSR-E8 medium.

Physiological oxygen culture reveals retention of metabolic memory in human induced pluripotent stem cells.

Reprogramming somatic cells to a pluripotent cell state (induced Pluripotent Stem (iPS) cells) requires reprogramming of metabolism to support cell proliferation and pluripotency, most notably changes in carbohydrate turnover that reflect a shift from oxidative to glycolytic metabolism. Some aspects of iPS cell metabolism differ from embryonic stem (ES) cells, which may reflect a parental cell memory, or be a consequence of the reprogramming process. In this study, we compared the metabolism of 3 human iPS cell lines to assess the fidelity of metabolic reprogramming. When challenged with reduced oxygen concentration, ES cells have been shown to modulate carbohydrate use in a predictably way. In the same model, 2 of 3 iPS cell lines failed to regulate carbohydrate metabolism. Oxygen is a well-characterized regulator of cell function and embryo viability, and an inability of iPS cells to modulate metabolism in response to oxygen may indicate poor metabolic fidelity. As metabolism is linked to the regulation of the epigenome, assessment of metabolic responses of iPS cells to physiological stimuli during characterization is warranted to ensure complete cell reprogramming and as a measure of cell quality.

Long-Term Maintenance of Human Pluripotent Stem Cells on cRGDfK-Presenting Synthetic Surfaces.

Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.

Cell surface markers for the identification and study of human naive pluripotent stem cells.

Characterisation of mouse pluripotent stem cells has revealed two distinct pluripotent states, naive and primed, that maintain characteristics of the pre and post implanted epiblast respectively. Recent studies have developed several culture systems that seek to recapitulate the naive phenomenon in human pluripotent stem cells. Therefore, robust methods to isolate these cells will be fundamental to assess their potential in modelling human development and disease. Here we review current methods for human naive pluripotent culture and collate a list of cell surface antigens that have been identified as markers to differentiate naive from primed human pluripotent stem cells. While these culture systems do display marker variability, and not all antigens mentioned were assessed in all methods, this review provides a resource for researchers of the human naive pluripotent stem cell state. SSEA-4, SSEA-3, CD24, CD75, CD7, CD77, CD130/GP130, CD57, CD90 and NLGN4X were all found to have a +/- expression profile in at least 2 methods, while +/- expression of Tra-1-81, CDH3, CD172a, CD107b, CD229 was reported in one method. Often it was reported that naive and primed cells could be defined using a low/medium/high expression of the following antigens TRA-1-60, PCDH1, GPR64, MHC Class I, however these markers were more likely to display expression pattern differences between methods. Studies using mouse naive cells indicate that they may have benefits over primed cells in modelling development and disease, and while it is yet to be determined if the same can be said about a human naive state, tools to identify this population should greatly further the field.

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming.

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells.

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.

Gene expression variability as a unifying element of the pluripotency network.

Heterogeneity is a hallmark of stem cell populations, in part due to the molecular differences between cells undergoing self-renewal and those poised to differentiate. We examined phenotypic and molecular heterogeneity in pluripotent stem cell populations, using public gene expression data sets. A high degree of concordance was observed between global gene expression variability and the reported heterogeneity of different human pluripotent lines. Network analysis demonstrated that low-variability genes were the most highly connected, suggesting that these are the most stable elements of the gene regulatory network and are under the highest regulatory constraints. Known drivers of pluripotency were among these, with lowest expression variability of POU5F1 in cells with the highest capacity for self-renewal. Variability of gene expression provides a reliable measure of phenotypic and molecular heterogeneity and predicts those genes with the highest degree of regulatory constraint within the pluripotency network.

The hypoxia-inducible factor renders cancer cells more sensitive to vitamin C-induced toxicity.

Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells.

Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2.

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30(Hi)-GCTM-2(Hi) hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30(Neg)-GCTM-2(Neg)). In a further study, pluripotent stem cell-free samples of differentiated TG30(Neg)-GCTM-2(Neg) cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30(Hi)-GCTM-2(Hi) hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.