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Abstract The history of stem cell therapies is one of a limited number of clinical applications despite a vast therapeutic potential. Major breakthroughs in stem cell research have not yet enjoyed clinical success—all stem cell therapies bar hematopoietic stem cell transplantations remain experimental. With the increased risk of organ failure and neurodegenerative disease associated with our ability to push the boundaries of life expectancy comes an increased pressure to pioneer novel stem cell-based therapeutic approaches. We conclude that the failure of such therapies to achieve clinical translation stems from the polarising effect of the ethical debate around their use. The intractability of the ethical debate is double edged: legislators not only have placed tighter restrictions on certain stem cell therapies, but do so in favour of less controversial cells which will have worse outcomes for patients. It is by considering this relationship between the politics, ethics and science of stem cells that the reasons for the currently limited clinical significance of stem cell therapies be realised.

Long non-coding RNAs (lncRNAs) have been found to be involved in many biological processes, including the regulation of cell differentiation, but a complete characterization of lncRNA is still lacking. Additionally, there is evidence that lncRNAs interact with ribosomes, raising questions about their functions in cells. Here, we used a developmentally staged protocol to induce cardiogenic commitment of hESCs and then investigated the differential association of lncRNAs with polysomes. Our results identified lncRNAs in both the ribosome-free and polysome-bound fractions during cardiogenesis and showed a very well-defined temporal lncRNA association with polysomes. Clustering of lncRNAs was performed according to the gene expression patterns during the five timepoints analyzed. In addition, differential lncRNA recruitment to polysomes was observed when comparing the differentially expressed lncRNAs in the ribosome-free and polysome-bound fractions or when calculating the polysome-bound vs ribosome-free ratio. The association of lncRNAs with polysomes could represent an additional cytoplasmic role of lncRNAs, e.g., in translational regulation of mRNA expression. [ABSTRACT FROM AUTHOR]

Many of the earliest stem cell studies were conducted on cells isolated from tumors rather than from embryos. Of particular interest was research on embryonic carcinoma cells (EC), a type of stem cell derived from teratocarcinoma. The EC research laid the foundation for the later discovery of and subsequent work on embryonic stem cells (ESC). Both ESC isolated from the mouse (mESC) and then later from humans (hESC) shared not only pluripotency with their EC cousins, but also robust tumorigenicity as each readily form teratoma. Surprisingly, decades after the discovery of mESC, the question of what drives ESC to form tumors remains largely an open one. This gap in the field is particularly serious as stem cell tumorigenicity represents the key obstacle to the safe use of stem cell-based regenerative medicine therapies. Although some adult stem cell therapies appear to be safe, they have only a very narrow range of uses in human disease. Our understanding of the tumorigenicity of human induced pluripotent stem cells (IPSC), perhaps the most promising modality for future patient-specific regenerative medicine therapies, is rudimentary. However, IPSC are predicted to possess tumorigenic potential equal to or greater than that of ESC. Here, the links between pluripotency and tumorigenicity are explored. New methods for more accurately testing the tumorigenic potential of IPSC and of other stem cells applicable to regenerative medicine are proposed. Finally, the most promising emerging approaches for overcoming the challenges of stem cell tumorigenicity are highlighted.

Abstract Background Human embryonic stem cells (hESCs) potentially offer new routes to study, on the basis of the Developmental Origins of Health and Disease (DOHaD) concept, how the maternal environment during pregnancy influences the offspring’s health and can predispose to chronic disease in later life. Reactive oxygen species (ROS), antioxidant defences and cellular redox status play a key function in gene expression regulation and are involved in diabetes and metabolic syndromes as in ageing. Methods We have, therefore, designed an in vitro cell model of oxidative stress by exposing hESCs to hydrogen peroxide (H2O2) during 72 h, in order to resemble the period of preimplantation embryonic development. Results We have analysed the global gene expression profiles of hESCs (HUES3) exposed to non-cytotoxic H2O2 concentrations, using Illumina microarray HT-12 v4, and we found the differential expression of 569 upregulated and 485 downregulated genes. The most affected gene ontology categories were those related with RNA processing and splicing, oxidation reduction and sterol metabolic processes. We compared our findings with a published RNA-seq profiling dataset of human embryos developed in vitro, thereupon exposed to oxidative stress, and we observed that one of the common downregulated genes between this publication and our data, NEDD1, is involved in centrosome structure and function. Conclusions Therefore, we assessed the presence of supernumerary centrosomes and showed that the percentage of cells with more than two centrosomes increased acutely with H2O2 treatment in hESCs (HUES3 and 7) and in a control somatic cell line (Hs27), inducing a premature entry into senescence.

The human leukocyte antigen (HLA)-G gene seems to play a pivotal role in maternal tolerance to the fetus. Little is known about HLA-G expression and its molecular control during in vivo human embryogenesis. Human embryonic stem cells (hESC) provide an interesting in vitro model to study early human development. Different studies reported discrepant findings on whether HLA-G mRNA and protein are present or absent in hESC. Several lines of evidence indicate that promoter CpG methylation and 3′ untranslated region (3′UTR) polymorphisms may influence HLA-G expression.We investigated how HLA-G expression is linked to the patterns of promoter methylation and explored the role of the 3′UTR polymorphic sites and their binding microRNAs on the post-transcriptional regulation of HLA-G in eight hESC lines.We showed that, while the gross expression levels of HLA-G are controlled by promoter methylation, the genetic constitution of the HLA-G 3′UTR, more specifically the 14bp insertion in combination with the +3187A/A and +3142G/G SNP, plays a major role in HLA-G mRNA regulation in hESC.Our findings provide a solid first step towards future work using hESC as tools for the study of early human developmental processes in normal and pregnancy-related disorders such as preeclampsia.

We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency.

Cholangiocytes form a complex 3D network of bile ducts in the liver and contribute to liver function. The damage or destruction of cholangiocytes can lead to biliary diseases, and the shortage of cholangiocytes remains an obstacle for drug development targeting biliary diseases. Valproic acid (VPA) is a potent activator of Notch signaling pathway that is essential for cholangiocyte differentiation. Here, we report a VPA-based approach for cholangiocyte differentiation of human pluripotent stem cells. VPA activated Notch2 expression and upregulated HES-1, HEY-1, and Sox9 gene expression in hESC-derived hepatoblast. After 7 days treatment, VPA promoted successful differentiation of hepatoblast into cholangiocytes expressing cholangiocyte marker genes (AE2, AQP1, CFTR) and proteins (CK19 and CK7). In addition, the differentiated cholangiocytes formed bile duct-like structures after implantation into the spleen of NOD/SCID mice. Our results suggested that VPA can promote hESC differentiation to cholangiocyte-like cells. The induced cholangiocytes may serve as a potential cell source for both in vitro modeling and regenerative therapy of cholangiopathies. The findings can also support further development of small-molecule based differentiation protocols for cholangiocyte production., (© The Author(s) 2023. Published by Oxford University Press.)

The spatial localization of RNA within cells is closely related to its function and also involved in cell fate determination. However, the atlas of RNA distribution within cells and dynamic changes during the developmental process are largely unknown. In this study, five subcellular components, including cytoplasmic extract, membrane extract, soluble nuclear extract, chromatin-bound nuclear extract, and cytoskeletal extract, were isolated and the rules of subcellular RNA distribution in human embryonic stem cells (hESCs) and its change during hESC differentiation are summarized for the first time. The overall distribution patterns of coding and non-coding RNAs are revealed. Interestingly, some developmental genes are found to be transcribed but confined to the chromatin in undifferentiated hESC. Unexpectedly, alternative splicing and polyadenylation endow spatial heterogeneity among different isoforms of the same gene. Finally, the dynamic pattern of RNA distribution during hESC differentiation is characterized, which provides new clues for a comprehensive understanding hESC pluripotency and differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Formation of tissue models in 3 dimensions is more effective in recapitulating structure and function compared to their 2-dimensional (2D) counterparts. Formation of 3D engineered tissue to control shape and size can have important implications in biomedical research and in engineering applications such as biological soft robotics. While neural spheroids routinely are created during differentiation processes, further geometric control of in vitro neural models has not been demonstrated. Here, we present an approach to form functional in vitro neural tissue mimic (NTM) of different shapes using stem cells, a fibrin matrix, and 3D printed molds. We used murine-derived embryonic stem cells for optimizing cell-seeding protocols, characterization of the resulting internal structure of the construct, and remodeling of the extracellular matrix, as well as validation of electrophysiological activity. Then, we used these findings to biofabricate these constructs using neurons derived from human embryonic stem cells. This method can provide a large degree of design flexibility for development of in vitro functional neural tissue models of varying forms for therapeutic biomedical research, drug discovery, and disease modeling, and engineering applications. [ABSTRACT FROM AUTHOR]

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are used to study cardiogenesis and mechanisms of heart disease, and are being used in methods for toxiological screening of drugs. The phenotype of stem-cell-derived CMs should ideally resemble native CMs. Here, we compare embryonic/fetal CMs with hESC-derived CMs according to function and morphology. CM clusters were obtained from human embryonic/fetal hearts from elective terminated pregnancies before gestational week 12, and separated into atrial and ventricular tissues. Specific markers for embryonic CMs and primary cilia were visualized using immunofluorescence microscopy analysis. Contracting human embryonic cardiomyocyte (hECM) clusters morphologically and phenotypically resemble CMs in the embryonic/fetal heart. In addition, the contracting hECM clusters expressed primary cilia similar to that of cells in the embryonic/fetal heart. The electrophysiological characteristics of atrial and ventricular CMs were established by recording action potentials (APs) using sharp electrodes. In contrast to ventricular APs, atrial APs displayed a marked early repolarization followed by a plateau phase. hESC-CMs displayed a continuum of AP shapes. In all embryonic/fetal clusters, both atrial and ventricular, AP duration was prolonged by exposure to the KV11.1 channel inhibitor dofetilide (50 nM); however, the prolongation was not significant, possibly due to the relatively small number of experiments. This study provides novel information on APs and functional characteristics of atrial and ventricular CMs in first trimester hearts, and demonstrates that Kv11.1 channels play a functional role already at these early stages. These results provide information needed to validate methods being developed on the basis of in vitro-derived CMs from either hESC or iPSC, and although there was a good correlation between the morphology of the two types of CMs, differences in electrophysiological characteristics exist. [ABSTRACT FROM AUTHOR]

Abstract Cell replacement therapy is a promising approach for treatment of retinal degenerative diseases. Several protocols for the generation of photoreceptor precursors (PRP) from human embryonic stem cells (hESC) have been reported with variable efficiency. Herein, we show the advantages of use of size-controlled embryoid bodies in the ESC differentiation process using two differentiation protocols. We further explored cell-labeling methods for following the survival of PRP transplanted subretinally in rat eyes. Size-controlled embryoid bodies (EBs) generated using microwell dishes and non-size-controlled EBs generated using V-shaped 96-well plates were differentiated into PRP using two differentiation protocols. The differentiation protocols utilized two different combinations of growth factors. The first, Dkk1, Noggin, and IGF1, and the second protocol used IWR1e, SAG, and CHIR99021. Differentiation efficiency to PRP was analyzed by qPCR, immunocytochemistry, and fluorescence-assisted cell sorting (FACS). Size-controlled IWR1e yielded a significantly higher percent (86.4%) of PRP cells expressing CRX, compared with non-size-controlled IWR1e (51.4%, P = 0.026) or the size-controlled DKK1 protocol (70.5%, p = 0.007). In addition, the IWR1e differentiated cells exhibited a significantly higher fluorescence intensity of CRX immunostaining, compared with the DKK1 protocol, consistent with higher protein expression levels. The IWR1e cells exhibited higher maturation levels, as manifested by lower early neuronal marker PAX6 and pluripotency marker OCT4 levels compared with the DKK1 protocol. The expression of other late photoreceptor markers (NRL, recoverin) were similar among the differentiation groups. PRP cells were labeled by using hESC constitutively expressing EGFP or by AAV-GFP transduction. Finally, we transplanted the cells in the subretinal space of wild-type rats and monitored their survival over several weeks. The AAV2 serotype efficiently transduced the PRP cells, whereas other serotypes yielded low or no transduction. Following subretinal transplantation of GFP-labeled PRP, 63% of the cells were detected at 4 weeks post-transplantation. In conclusion, we show here that the IWR1e protocol using size-controlled EBs efficiently generated of PRP that could be labeled and followed in-vivo for weeks. The data from this study is an advance toward the goal of PRP transplantation therapy for retinal degenerative diseases. Graphical abstract Image 1 Highlights • Use of size-controlled EBs promotes the efficiency of PRP differentiation from hESC. • Generation of PRP with the IWR1e protocol is more efficient than a DKK1-based protocol. • AAV2 is superior to other AAV serotypes for transducing hESC-derived PRPs. • The generated PRP were transplanted and tracked for several weeks. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a chronic disease that affects hundreds of millions of people worldwide. Type 1 diabetes (T1D) is characterized by the lack of pancreatic β-cells that had been destroyed as a result of an autoimmune response. Therefore, in patients with T1D, the replacement therapy with functional β-cells derived from extrinsic sources could be a preferable option as compared to insulin treatment. Unfortunately, successful transplantation of whole pancreata or pancreatic islets into patients with diabetes is available only to a fraction of them due to the scarcity of donors. The rapid development of cell reprogramming methods made it possible to generate large numbers of human β-like cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). This review describes the basis of in vitro differentiaton protocols of β-like cells that mimic changes of the main signaling pathways during the key stages of human and murine pancreas development, which are described first. During the last 15 years it was found that there are no important differences between hESCs and hiPSCs in their differentiation capacities into β-like cells and the expression profiles of the key transcription factors. The in vitro produced β-like cells are immature as demonstrated by functional tests in rodents and single-cell transcriptomic and proteomic analyses. After the transplantation of the b cell progenitors into immunocompromised diabetic mice, a few weeks have to pass before the increased insulin levels in response to glucose load appear. There is a continuous progress in the development of open-type encapsulation devices which allow the vascularization of the transplanted cells and protect them against host's immune cells. The results of the first clinical trial of human partially differentiated endocrine progenitors of b cells transplanted into patients with T1D will be published in the year 2019. It is hoped that further improvements in the techniques of large-scale generation of the β-like cells derived from human pluripotent stem cells will bring us closer to their clinical application as a form of cause-directed therapy for people with diabetes. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) can differentiate into any cell lineage. Here, we report that ZEB1 and ZEB2 promote and inhibit mesodermal-to-myogenic specification of hESCs, respectively. Knockdown and/or overexpression experiments of ZEB1 , ZEB2 , or PAX7 in hESCs indicate that ZEB1 is required for hESC Nodal/Activin-mediated mesodermal specification and PAX7+ human myogenic progenitor (hMuP) generation, while ZEB2 inhibits these processes. ZEB1 downregulation induces neural markers, while ZEB2 downregulation induces mesodermal/myogenic markers. Mechanistically, ZEB1 binds to and transcriptionally activates the PAX7 promoter, while ZEB2 binds to and activates the promoter of the neural OTX2 marker. Transplanting ZEB1 or ZEB2 knocked down hMuPs into the muscles of a muscular dystrophy mouse model, showing that hMuP engraftment and generation of dystrophin-positive myofibers depend on ZEB1 and are inhibited by ZEB2. The mouse model results suggest that ZEB1 expression and/or downregulating ZEB2 in hESCs may also enhance hESC regenerative capacity for human muscular dystrophy therapy. [Display omitted] • ZEB1 is required for the generation of mesodermal and myogenic (PAX7+) progenitors • Downregulation of ZEB1 and ZEB2 induces neural and myogenic markers, respectively • ZEB1 and ZEB2 activate the promoters of myogenic PAX7 and neural OTX2 , respectively • Engraftment of myogenic precursors in mdx mice depends on ZEB1 and is inhibited by ZEB2 Ninfali et al. report that ZEB1 is required for the mesodermal-to-myogenic specification of human embryonic stem cells (hESCs) and their engraftment into mouse dystrophic muscles, while ZEB2 inhibits both processes. The study suggests that modulating ZEB1 and ZEB2 in hESCs could be an approach to enhance hESC regenerative capacity in muscular dystrophies. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells, namely human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) have a theoretical potential to differentiate into any type of human cells, they can be maintained in vitro for extended periods of time and can easily be genetically manipulated and expanded to clinically relevant cell numbers. As such, they can potentially serve as off-the-shelf sources of normal or genetically modified cells for tissue regenerative and other medical applications, including immune therapies. This presentation will provide an overview of the current state of pluripotent stem cells-based therapies, with the particular focus on pluripotent stem cell-derived immune cells. Our group and others have established that T cells with increased affinity for cancer epitopes can be derived from genetically modified pluripotent stem cells. Such T cells phenotypically and functionally fully resemble normal T cells and kill haplotype matched target cells in a dose dependent manner in vitro and in vivo. Practical considerations suggest that the pluripotent derived stem cell-derived T cells can therapeutically be superior to genetically modified T cells taken from patients' blood, as they can be expanded to significantly greater numbers sufficient for treatment of a large number of patients and properly analyzed for genotoxic effects of vector integration through DNA sequencing approaches. These types of cell therapies hold promise for the treatment of a broad array of conditions, including cancer, HIV infection, and immune insufficiency disorders. [ABSTRACT FROM AUTHOR]

The development of human erythroid cells has been mostly examined in models of adult hematopoiesis, while their early derivation during embryonic and fetal stages is largely unknown. We observed the development and maturation of erythroblasts derived from human pluripotent stem cells (hPSCs) by an efficient co-culture system. These hPSC-derived early erythroblasts initially showed definitive characteristics with a glycophorin A+ (GPA+) CD34lowCD36− phenotype and were distinct from adult CD34+ cell-derived ones. After losing CD34 expression, early GPA+CD36− erythroblasts matured into GPA+CD36low/+ stage as the latter expressed higher levels of β-globin along with a gradual loss of mesodermal and endothelial properties, and terminally suppressed CD36. We establish a unique in vitro model to trace the early development of hPSC-derived erythroblasts by serial expression of CD34, GPA, and CD36. Our findings may provide insight into the understanding of human early erythropoiesis and, ultimately, therapeutic potential.

The placenta is a fetal exchange organ connecting mother and baby that facilitates fetal growth in utero. DNA methylation is thought to impact placental development and function. Global DNA methylation studies using human placental lysates suggest that the placenta is uniquely hypomethylated compared to somatic tissue lysates, and this hypomethylation is thought to be important in conserving the unique placental gene expression patterns required for successful function. In the placental field, methylation has frequently been examined in tissue lysates, which contain mixed cell types that can confound results. To better understand how DNA methylation influences placentation, DNA from isolated first trimester trophoblast populations underwent reduced representation bisulfite sequencing and was compared to publicly available data of blastocyst-derived and somatic cell populations. First, this revealed that, unlike murine blastocysts, human trophectoderm and inner cell mass samples did not have significantly different levels of global methylation. Second, our work suggests that differences in global CpG methylation between trophoblasts and somatic cells are much smaller than previously reported. Rather, our findings suggest that different patterns of CpG methylation may be more important in epigenetically distinguishing the placenta from somatic cell populations, and these patterns of methylation may contribute to successful placental/trophoblast function.

Age-related macular degeneration (AMD) is a complex neurodegenerative visual disorder that causes profound physical and psychosocial effects. Visual impairment in AMD is caused by the loss of retinal pigmented epithelium (RPE) cells and the light-sensitive photoreceptor cells that they support. There is currently no effective treatment for the most common form of this disease (dry AMD). A new approach to treating AMD involves the transplantation of RPE cells derived from either human embryonic or induced pluripotent stem cells. Multiple clinical trials are being initiated using a variety of cell therapies. Although many animal models are available for AMD research, most do not recapitulate all aspects of the disease, hampering progress. However, the use of cultured RPE cells in AMD research is well established and, indeed, some of the more recently described RPE-based models show promise for investigating the molecular mechanisms of AMD and for screening drug candidates. Here, we discuss innovative cell-culture models of AMD and emerging stem-cell-based therapies for the treatment of this vision-robbing disease.

MicroRNAs (miRNAs) play crucial roles in pluripotency and differentiation of human Embryonic Stem Cells (hESCs). However, synergism among multiple miRNAs and their regulatory effects on stem cells are largely unknown. We investigated the synergistic regulations among miRNAs, which are differentially expressed in hESCs and fibroblasts (Fibs) to gain deeper insights into the regulatory mechanisms of miRNA-miRNA synergism in the differentiation of hESCs into Fibs. In this study, we identified miRNA targets incorporating differential expression profiles of miRNAs and genes in hESCs-Fibs with miRNA targeting features followed by enrichment analysis. We then built the active miRNA-miRNA synergistic network (MMSN) integrating miRNA-miRNA synergistic pairs, which identified 16 miRNA-miRNA closed communities. Further, topology assessment, community overlapping and functional studies revealed hsa-miR-873-5p as an important pluripotent miRNA which might be hindering hESCs differentiation into Fibs by targeting regulators of epithelial to mesenchymal transition (EMT). On the other hand, synergism among hsa-miR-98-5p, hsa-let-7i-5p, hsa-let-7g-5p, hsa-miR-30a-3p and hsa-miR-29b-2-5p is predicted to be highly essential for promoting differentiation of hESCs in to Fibs. This study deepens our understanding of miRNA synergism and its regulatory impacts on properties of stem cells. [ABSTRACT FROM AUTHOR]

The history of stem cell therapies is one of a limited number of clinical applications despite a vast therapeutic potential. Major breakthroughs in stem cell research have not yet enjoyed clinical success--all stem cell therapies bar hematopoietic stem cell transplantations remain experimental. With the increased risk of organ failure and neurodegenerative disease associated with our ability to push the boundaries of life expectancy comes an increased pressure to pioneer novel stem cell-based therapeutic approaches. We conclude that the failure of such therapies to achieve clinical translation stems from the polarising effect of the ethical debate around their use. The intractability of the ethical debate is double edged: legislators not only have placed tighter restrictions on certain stem cell therapies, but do so in favour of less controversial cells which will have worse outcomes for patients. It is by considering this relationship between the politics, ethics and science of stem cells that the reasons for the currently limited clinical significance of stem cell therapies be realised. [ABSTRACT FROM AUTHOR]

To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4. Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis. [ABSTRACT FROM AUTHOR]

As the field of science communication has matured over the past 50 years, there has been a significant move away from the conventional understanding that mass media's role in the public communication of science is limited to reporting new scientific discoveries. Media have been increasingly viewed as important for the legitimation of science and scholars have recognized their agenda-setting effects and ability to facilitate interaction between the public, scientific community, policymakers, interest groups, and other social actors. This article draws on analyses of news media coverage of stem cell research between 1998 and 2013 to demonstrate the active role of mass media in validating scientific claims about discoveries in the field and shaping the public understanding of key bioethical and policy issues. It further assesses whether media, in their attempts to construct the 'right' position, have instigated a rational-critical discourse on the controversy. I argue that media representations in different cultural contexts have largely failed to meet normative expectations about the democratization of public discussions on biomedical innovation, as set out in the public engagement with science and technology (PEST) model of science communication. Rather than deconstructing the major terms of science policy debates as framed by stem cell advocates and their opponents, media coverage has mostly replicated discussions in political and legislative arenas, presenting the controversy as a strict binary opposition. Media have rarely provided critical reflection on the hype surrounding breakthroughs in stem cell research, thus reinforcing the public's unrealistic expectations about the future of this biomedical innovation. [ABSTRACT FROM AUTHOR]

Although human embryonic stem cells (hESCs) were first derived almost 20 years ago, it was only recently acknowledged that they share closer molecular and functional identity to postimplantation lineage-primed murine epiblast stem cells than to naïve preimplantation inner cell mass-derived mouse ESCs (mESCs). A myriad of transcriptional, epigenetic, biochemical, and metabolic attributes have now been described that distinguish naïve and primed pluripotent states in both rodents and humans. Conventional hESCs and human induced pluripotent stem cells (hiPSCs) appear to lack many of the defining hallmarks of naïve mESCs. These include important features of the naïve ground state murine epiblast, such as an open epigenetic architecture, reduced lineage-primed gene expression, and chimera and germline competence following injection into a recipient blastocyst-stage embryo. Several transgenic and chemical methods were recently reported that appear to revert conventional human PSCs to mESC-like ground states. However, it remains unclear if subtle deviations in global transcription, cell signaling dependencies, and extent of epigenetic/metabolic shifts in these various human naïve-reverted pluripotent states represent true functional differences or alternatively the existence of distinct human pluripotent states along a spectrum. In this study, we review the current understanding and developmental features of various human pluripotency-associated phenotypes and discuss potential biological mechanisms that may support stable maintenance of an authentic epiblast-like ground state of human pluripotency. [ABSTRACT FROM AUTHOR]

Background & objectives: Diabetes is a global disease burden. Various stem cell types are being explored to serve as an alternative source of islets. This study was conducted to evaluate the ability of in-house developed human embryonic stem (hES) cells-derived pancreatic progenitors to ameliorate diabetic symptoms in mice. Methods: Pancreatic progenitors were packed in macro-capsules and transplanted into six male Swiss mice and four mice were taken as controls. Thirty days post-transplantation, diabetes was induced by streptozotocin treatment. Mice were then followed up for >100 days and body weight and blood glucose levels were regularly monitored. Results: Control mice lost weight, maintained high glucose levels and did not survive beyond 40 days, whereas transplanted group maintained body weight and four of the six mice had lowered blood glucose levels. About five-fold increase was observed in human C-peptide levels in the recipients of progenitor transplants as compared to diabetic control. Interpretation & conclusions: The beneficial effect of transplanted cells was not long-lasting. Further studies are required to critically evaluate and compare the potential of endogenous pluripotent stem cells and hES cells-derived progenitors before moving from bench to the bedside. [ABSTRACT FROM AUTHOR]

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models. [ABSTRACT FROM AUTHOR]

Background: Human embryonic stem cells (hESCs) potentially offer new routes to study, on the basis of the Developmental Origins of Health and Disease (DOHaD) concept, how the maternal environment during pregnancy influences the offspring's health and can predispose to chronic disease in later life. Reactive oxygen species (ROS), antioxidant defences and cellular redox status play a key function in gene expression regulation and are involved in diabetes and metabolic syndromes as in ageing. Methods: We have, therefore, designed an in vitro cell model of oxidative stress by exposing hESCs to hydrogen peroxide (H2O2) during 72 h, in order to resemble the period of preimplantation embryonic development. Results: We have analysed the global gene expression profiles of hESCs (HUES3) exposed to non-cytotoxic H2O2 concentrations, using Illumina microarray HT-12 v4, and we found the differential expression of 569 upregulated and 485 downregulated genes. The most affected gene ontology categories were those related with RNA processing and splicing, oxidation reduction and sterol metabolic processes. We compared our findings with a published RNA-seq profiling dataset of human embryos developed in vitro, thereupon exposed to oxidative stress, and we observed that one of the common downregulated genes between this publication and our data, NEDD1, is involved in centrosome structure and function. Conclusions: Therefore, we assessed the presence of supernumerary centrosomes and showed that the percentage of cells with more than two centrosomes increased acutely with H2O2 treatment in hESCs (HUES3 and 7) and in a control somatic cell line (Hs27), inducing a premature entry into senescence. [ABSTRACT FROM AUTHOR]

According to the judgement of the European Court of Justice in 2014, human parthenogenetic stem cells are excluded from the patenting prohibition of procedures based on hESC by the European Biopatent Directive, because human parthenotes are not human embryos. This article is based on the thesis that in light of the technological advances in the field of stem cell research, the attribution of the term 'human embryo' to certain entities on a descriptive level as well as the attribution of a normative protection status to certain entities based on the criterion of totipotency, are becoming increasingly unclear. The example of human parthenotes in particular demonstrates that totipotency is not at all a necessary condition for the attribution of the term 'human embryo'. Furthermore, the example of hiPSC and somatic cells particularly shows that totipotency is also not a sufficient condition for the attribution of a normative protection status to certain entities. Therefore, it is not a suitable criterion for distinguishing between human embryos worthy of protection and human non-embryos not worthy of protection. Consequently, this conclusion has repercussions for the patenting question. The strict delineation between an ethically problematic commercial use of human embryos and the concomitant patenting prohibition of hESC-based procedures and an ethically unproblematic commercial use of human non-embryos and the therefore either unrestrictedly permitted (cf. human parthenotes) or even unregulated (cf. hiPSC) patenting of procedures based on these alleged alternatives becomes increasingly blurred. [ABSTRACT FROM AUTHOR]

Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs. [ABSTRACT FROM AUTHOR]

Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene. To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation (MECP2T158M/T158M), hESC line expressing no MECP2 (MECP2-KO), congenic pair of wild-type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function. At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated from MECP2T158M/T158M, MECP2-KO, and V247fs-MT stem cell lines. Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the female Mecp2 heterozygous mice rescued several behavioral defects. Together, our study establishes a robust in vitro platform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT. [ABSTRACT FROM AUTHOR]

The human leukocyte antigen (HLA )- G gene seems to play a pivotal role in maternal tolerance to the fetus. Little is known about HLA-G expression and its molecular control during in vivo human embryogenesis. Human embryonic stem cells (hESC) provide an interesting in vitro model to study early human development. Different studies reported discrepant findings on whether HLA-G mRNA and protein are present or absent in hESC. Several lines of evidence indicate that promoter CpG methylation and 3′ untranslated region (3′UTR) polymorphisms may influence HLA-G expression. We investigated how HLA-G expression is linked to the patterns of promoter methylation and explored the role of the 3′UTR polymorphic sites and their binding microRNAs on the post-transcriptional regulation of HLA-G in eight hESC lines. We showed that, while the gross expression levels of HLA-G are controlled by promoter methylation, the genetic constitution of the HLA-G 3′UTR, more specifically the 14bp insertion in combination with the + 3187A/A and + 3142G/G SNP, plays a major role in HLA-G mRNA regulation in hESC. Our findings provide a solid first step towards future work using hESC as tools for the study of early human developmental processes in normal and pregnancy-related disorders such as preeclampsia. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorder is a neurodevelopmental disorder accompanied by weak immune system and neuroinflammation. Multiple factors contribute to etiology of autism spectrum disorder including genetic disorders, environmental substances/toxins, imbalanced immune system, encephalitis, and viral infections. Autism spectrum disorder is an incurable disease; however, it can be managed by educational and medical interventions. Human embryonic stem cell therapy has been shown to improve blood perfusion in the brain; thus, this therapy may be effective in improving motor skills, social skills, and cognition in patients with autism spectrum disorder. Method: Three pediatric patients with autism spectrum disorder were administered human embryonic stem cell therapy. Their treatment plan comprised 3 to 4 therapy sessions (T1, T2, T3, T4) that were 4 to 6 weeks in length, with 4- to 8-month gap phases separating each therapy session. Results: The patients showed improvements in eye coordination, writing, balancing, cognition, and speech and showed reduced hypersensitivity to noises and smells. Conclusion: The use of human embryonic stem cell therapy may be a safe and effective treatment for patients with autism spectrum disorder. Studies with larger sample sizes are needed to support the use of human embryonic stem cell therapy in this patient population. [ABSTRACT FROM AUTHOR]

The ability of human embryonic stem cells (hESCs) to proliferate indefinitely is attributed to its high telomerase activity and associated long telomere. However, factors regulating telomere length in hESCs remain largely uncharacterized. The aims of this study were, to identify factors which modulate telomere length of hESCs, and to determine if the telomere length of hESCs influences cellular senescence of its differentiated progeny cells. Telomerase reverse transcriptase (TERT) gene expression, telomerase activity and telomere length of hESCs cultured in different culture systems were compared. Genetically identical hESCs of different telomere lengths were differentiated into fibroblasts simultaneously, and the population doubling and cellular senescence levels were determined. We found that telomere lengths were significantly different in different culture systems and Fibroblast growth factor-2 (FGF-2) upregulated TERT expression, telomerase activity and telomere length via Wnt/β-catenin signaling pathway in hESCs in a significant manner. We also provide evidence that fibroblast differentiated from hESCs with longer telomere exhibited significant more population doublings and longer life span than those derived from hESCs with shorter telomeres. Thus, FGF-2 levels in hESCs culture systems can be manipulated to generate cells with longer telomere which would be advantageous in the applications of hESCs in regenerative medicine. [ABSTRACT FROM AUTHOR]

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here, we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes, respectively. Clusters of Wnt targets showed enrichment in specific biological functions, such as “gastrointestinal or skeletal development” in the Wnt-activated targets and “neural or immune system development” in the Wnt-repressed targets. Moreover, we adopted a high-throughput chromatin immunoprecipitation and deep sequencing (ChIP-seq) approach to identify the genomic regions through which Wnt-activated transcription factor TCF7L2 regulated transcription. We identified 83 Wnt direct target candidates, including the hindgut marker CDX2 and the genes relevant to morphogenesis ( MSX1 , MSX2 , LEF1 , T , PDGFRB etc. ) through combinatorial analysis of the RNA-seq and ChIP-seq data. Together, our study identified a series of direct and indirect Wnt targets in hindgut differentiation, and uncovered the diverse mechanisms of Wnt signaling in regulating multi-lineage differentiation. [ABSTRACT FROM AUTHOR]

Human embryonic stem (hES)-T3 (T3ES) cells were spontaneously differentiated into autogeneic fibroblast-like T3DF cells, as feeder cells with the capacity to support the growth of undifferentiated hES cells. The proteomes of undifferentiated T3ES cells and their differentiated T3DF fibroblasts were quantitatively compared. Several heterogeneous nuclear ribonucleoproteins and glycolytic enzymes, including l-lactate dehydrogenase A (M), were found to be abundantly and differentially expressed in T3ES cells and T3DF fibroblasts, respectively. Both miRNA and mRNA profiles from the undifferentiated T3ES cells and their differentiated T3DF fibroblasts had been previously determined. In this investigation, 206 genes were found to be targets of the four hES cell-specific miRNAs of miR-302d, miR-372, miR-200c, and/or miR-367 by using two-fold differential expression and inverse expression levels (highly negative correlations) of miRNAs to their target mRNAs. That YWHAZ (14-3-3 zeta) is a target of miR-302d and miR-372 was further confirmed by proteomic comparison between T3ES cells and their differentiated T3DF fibroblasts. According to GeneOntology analyses, almost 50% of these 206 target proteins are nuclear and are involved in gene transcription. Identifying the target mRNAs of hES cell-specific miRNAs will provide a better understanding of the complex regulatory networks in hES cells. Furthermore, these miRNA-targeted proteins play important roles in differentiation of hES cells and during embryo development.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Therefore, developing renewable sources of human liver tissue is attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and phenotypic instability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application and adoption is limited due to the reliance on variable biological components. Our study focus on developing 3D liver tissue under defined conditions. We demonstrate that 3D derived tissue can be generated at scale and implanted underneath the skin of mice. Excitingly, implanted human tissue provides support to mice with metabolic liver disease. This includes immunocompetent recipients. In addition to their clinical application, in vitro generated 3D tissues have important roles to perform in developing safe and efficacious medicines to treat human diseases. We demonstrate that stem cell derived 3D liver tissue exhibits liver cell phenotype for over one year in culture, providing an attractive resource for long-term disease modelling and screening studies. In conclusion, stem cell derived liver tissue has great potential for in vitro and in vivo endeavours. Our most recent advances will be presented at the meeting. [ABSTRACT FROM AUTHOR]

Human brain organoids provide unique platforms for modeling several aspects of human brain development and pathology. However, current brain organoid systems mostly lack the resolution to recapitulate the development of finer brain structures with subregional identity, including functionally distinct nuclei in the thalamus. Here, we report a method for converting human embryonic stem cells (hESCs) into ventral thalamic organoids (vThOs) with transcriptionally diverse nuclei identities. Notably, single-cell RNA sequencing revealed previously unachieved thalamic patterning with a thalamic reticular nucleus (TRN) signature, a GABAergic nucleus located in the ventral thalamus. Using vThOs, we explored the functions of TRN-specific, disease-associated genes patched domain containing 1 (PTCHD1) and receptor tyrosine-protein kinase (ERBB4) during human thalamic development. Perturbations in PTCHD1 or ERBB4 impaired neuronal functions in vThOs, albeit not affecting the overall thalamic lineage development. Together, vThOs present an experimental model for understanding nuclei-specific development and pathology in the thalamus of the human brain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Summary Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome-wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma is the sixth leading cause of cancer worldwide. The most common risk factors are carcinogens (tobacco, alcohol), and infection of the human papilloma virus. Surgery is still considered as the treatment of choice in case of head and neck cancer, followed by a reconstructive surgery to enhance the quality of life in the patients. However, the widespread use of artificial implants does not provide appropriate physiological activities and often cannot act as a long-term solution for the patients. Here we review the applicability of multiple stem cell types for tissue engineering of cartilage, trachea, vocal folds and nerves for head and neck injuries. The ability of the cells to self-renew and maintain their pluripotency state makes them an attractive tool in tissue engineering. [ABSTRACT FROM AUTHOR]