Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone.

Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.

Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation.

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D-pellet culture led to significant increases in the expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific -10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N-acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co-IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B-SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC-chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators.

Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by endometrial epithelial cells during human embryo implantation.

STUDY QUESTION: How does the human embryo breach the endometrial epithelium at implantation? SUMMARY ANSWER: Embryo attachment to the endometrial epithelium promotes the formation of multinuclear syncytiotrophoblast from trophectoderm, which goes on to breach the epithelial layer. WHAT IS KNOWN ALREADY: A significant proportion of natural conceptions and assisted reproduction treatments fail due to unsuccessful implantation. The trophectoderm lineage of the embryo attaches to the endometrial epithelium before breaching this barrier to implant into the endometrium. Trophectoderm-derived syncytiotrophoblast has been observed in recent in vitro cultures of peri-implantation embryos, and historical histology has shown invasive syncytiotrophoblast in embryos that have invaded beyond the epithelium, but the cell type mediating invasion of the epithelial layer at implantation is unknown. STUDY DESIGN, SIZE, DURATION: Fresh and frozen human blastocyst-stage embryos (n = 46) or human trophoblast stem cell (TSC) spheroids were co-cultured with confluent monolayers of the Ishikawa endometrial epithelial cell line to model the epithelial phase of implantation in vitro. Systems biology approaches with published transcriptomic datasets were used to model the epithelial phase of implantation in silico. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were consented for research. Day 6 blastocysts were co-cultured with Ishikawa cell layers until Day 8, and human TSC spheroids modelling blastocyst trophectoderm were co-cultured with Ishikawa cell layers for 48 h. Embryo and TSC morphology was assessed by immunofluorescence microscopy, and TSC differentiation by real-time quantitative PCR (RT-qPCR) and ELISA. Single-cell human blastocyst transcriptomes, and bulk transcriptomes of TSC and primary human endometrial epithelium were used to model the trophectoderm-epithelium interaction in silico. Hypernetworks, pathway analysis, random forest machine learning and RNA velocity were employed to identify gene networks associated with implantation. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos co-cultured with Ishikawa cell layers from Day 6 to 8 breached the epithelial layer (37/46), and syncytiotrophoblast was seen in all of these. Syncytiotrophoblast was observed at the embryo-epithelium interface before breaching, and syncytiotrophoblast mediated all pioneering breaching events observed (7/7 events). Multiple independent syncytiotrophoblast regions were seen in 26/46 embryos, suggesting derivation from different regions of trophectoderm. Human TSC spheroids co-cultured with Ishikawa layers also exhibited syncytiotrophoblast formation upon invasion into the epithelium. RT-qPCR comparison of TSC spheroids in isolated culture and co-culture demonstrated epithelium-induced upregulation of syncytiotrophoblast genes CGB (P = 0.03) and SDC1 (P = 0.008), and ELISA revealed the induction of hCGß secretion (P = 0.03). Secretory-phase primary endometrial epithelium surface transcriptomes were used to identify trophectoderm surface binding partners to model the embryo-epithelium interface. Hypernetwork analysis established a group of 25 epithelium-interacting trophectoderm genes that were highly connected to the rest of the trophectoderm transcriptome, and epithelium-coupled gene networks in cells of the polar region of the trophectoderm exhibited greater connectivity (P < 0.001) and more organized connections (P < 0.0001) than those in the mural region. Pathway analysis revealed a striking similarity with syncytiotrophoblast differentiation, as 4/6 most highly activated pathways upon TSC-syncytiotrophoblast differentiation (false discovery rate (FDR < 0.026)) were represented in the most enriched pathways of epithelium-coupled gene networks in both polar and mural trophectoderm (FDR < 0.001). Random forest machine learning also showed that 80% of the endometrial epithelium-interacting trophectoderm genes identified in the hypernetwork could be quantified as classifiers of TSC-syncytiotrophoblast differentiation. This multi-model approach suggests that invasive syncytiotrophoblast formation from both polar and mural trophectoderm is promoted by attachment to the endometrial epithelium to enable embryonic invasion. LARGE SCALE DATA: No omics datasets were generated in this study, and those used from previously published studies are cited. LIMITATIONS, REASONS FOR CAUTION: In vitro and in silico models may not recapitulate the dynamic embryo-endometrial interactions that occur in vivo. The influence of other cellular compartments in the endometrium, including decidual stromal cells and leukocytes, was not represented in these models. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the mechanism of human embryo breaching of the epithelium and the gene networks involved is crucial to improve implantation success rates after assisted reproduction. Moreover, early trophoblast lineages arising at the epithelial phase of implantation form the blueprint for the placenta and thus underpin foetal growth trajectories, pregnancy health and offspring health. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Wellbeing of Women, Diabetes UK, the NIHR Local Comprehensive Research Network and Manchester Clinical Research Facility, and the Department of Health Scientist Practitioner Training Scheme. None of the authors has any conflict of interest to declare.

Pluripotent stem cells for skeletal tissue engineering.

Here, we review the use of human pluripotent stem cells for skeletal tissue engineering. A number of approaches have been used for generating cartilage and bone from both human embryonic stem cells and induced pluripotent stem cells. These range from protocols relying on intrinsic cell interactions and signals from co-cultured cells to those attempting to recapitulate the series of steps occurring during mammalian skeletal development. The importance of generating authentic tissues rather than just differentiated cells is emphasized and enabling technologies for doing this are reported. We also review the different methods for characterization of skeletal cells and constructs at the tissue and single-cell level, and indicate newer resources not yet fully utilized in this field. There have been many challenges in this research area but the technologies to overcome these are beginning to appear, often adopted from related fields. This makes it more likely that cost-effective and efficacious human pluripotent stem cell-engineered constructs may become available for skeletal repair in the near future.

Emulating Human Tissues and Organs: A Bioprinting Perspective Toward Personalized Medicine.

The lack of in vitro tissue and organ models capable of mimicking human physiology severely hinders the development and clinical translation of therapies and drugs with higher in vivo efficacy. Bioprinting allow us to fill this gap and generate 3D tissue analogues with complex functional and structural organization through the precise spatial positioning of multiple materials and cells. In this review, we report the latest developments in terms of bioprinting technologies for the manufacturing of cellular constructs with particular emphasis on material extrusion, jetting, and vat photopolymerization. We then describe the different base polymers employed in the formulation of bioinks for bioprinting and examine the strategies used to tailor their properties according to both processability and tissue maturation requirements. By relating function to organization in human development, we examine the potential of pluripotent stem cells in the context of bioprinting toward a new generation of tissue models for personalized medicine. We also highlight the most relevant attempts to engineer artificial models for the study of human organogenesis, disease, and drug screening. Finally, we discuss the most pressing challenges, opportunities, and future prospects in the field of bioprinting for tissue engineering (TE) and regenerative medicine (RM).

The expression and activity of Toll-like receptors in the preimplantation human embryo suggest a new role for innate immunity.

STUDY QUESTION: Is the innate immunity system active in early human embryo development? SUMMARY ANSWER: The pattern recognition receptors and innate immunity Toll-like receptor (TLR) genes are widely expressed in preimplantation human embryos and the pathway appears to be active in response to TLR ligands. WHAT IS KNOWN ALREADY: Early human embryos are highly sensitive to their local environment, however relatively little is known about how embryos detect and respond to specific environmental cues. While the maternal immune response is known to be key to the establishment of pregnancy at implantation, the ability of human embryos to detect and signal the presence of pathogens is unknown. STUDY DESIGN, SIZE, DURATION: Expression of TLR family and related genes in human embryos was assessed by analysis of published transcriptome data (n = 40). Day 5 (D-5) human embryos (n = 25) were cultured in the presence of known TLR ligands and gene expression and cytokine production measured compared to controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to Day 6 (D-6) in the presence of the TLR3 and TLR5 ligands Poly (I: C) and flagellin, with gene expression measured by quantitative PCR and cytokine release into medium measured using cytometric bead arrays. MAIN RESULTS AND THE ROLE OF CHANCE: TLR and related genes, including downstream signalling molecules, were expressed variably at all human embryo developmental stages. Results showed the strongest expression in the blastocyst for TLRs 9 and 5, and throughout development for TLRs 9, 5, 2, 6 and 7. Stimulation of Day 5 blastocysts with TLR3 and TLR5 ligands Poly (I: C) and flagellin produced changes in mRNA expression levels of TLR genes, including the hyaluronan-mediated motility receptor (HMMR), TLR5, TLR7, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and monocyte chemoattractant Protein-1 (MCP-1) (P < 0.05, P < 0.001 compared to unstimulated controls), and release into culture medium of cytokines and chemokines, notably IL8 (P = 0.00005 and 0.01277 for flagellin and Poly (I: C), respectively). LIMITATIONS, REASONS FOR CAUTION: This was a descriptive and experimental study which suggests that the TLR system is active in human embryos and capable of function, but does not confirm any particular role. Although we identified embryonic transcripts for a range of TLR genes, the expression patterns were not always consistent across published studies and expression levels of some genes were low, leaving open the possibility that these were expressed from the maternal rather than embryonic genome. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report of the expression and activity of a number of components of the innate immunity TLR system in human embryos. Understanding the role of TLRs during preimplantation human development may be important to reveal immunological mechanisms and potential clinical markers of embryo quality and pregnancy initiation during natural conception and in ART. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Ministry of Higher Education, The State of Libya, the UK Medical Research Council, and the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility and the European Union's Horizon 2020 Research and Innovation Programmes under the Marie Sklodowska-Curie Grant Agreement No. 812660 (DohART-NET). In accordance with H2020 rules, no new human embryos were sacrificed for research activities performed from the EU funding, which concerned only in silico analyses of recorded time-lapse and transcriptomics datasets. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: n/a.

A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

PURPOSE: To investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). METHODS: Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. RESULTS: The consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. CONCLUSION: KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

Correction: A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The role of Trp53 in the mouse embryonic response to DNA damage.

Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation.

In vitro culture during assisted reproduction technologies (ART) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2h in medium with osmolarity raised by 400mOsm induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation, and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1ß, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1ß challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1-/- presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

MiR-145 suppresses embryo-epithelial juxtacrine communication at implantation by modulating maternal IGF1R.

Successful implantation requires the synchronization of viable embryonic development with endometrial receptivity. The mechanisms allowing for the initiation of crosstalk between the embryo and the endometrium remain elusive; however, recent studies have revealed that there are alterations in endometrial microRNAs (miRs) in women suffering repeated implantation failure and that one of the altered miRs is miR-145. We assessed the role of miR-145 and its target IGF1R, in early implantation. miR-145 overexpression and IGF1R knockdown were achieved in Ishikawa endometrial cells. Quantitative PCR, western blotting and 3'UTR luciferase reporter assays confirmed that IGF1R is a direct target of miR-145 in the endometrium. Attachment of mouse embryos or IGF1-coated beads to endometrial epithelial cells was used to study the effects of altered miR-145 and/or IGF1R expression on early implantation events. miR-145 overexpression or specific reduction of IGF1R impaired attachment in both cases. An IGF1R target protector prevented the miR-145-mediated reduction in IGF1R and reversed the effect of miR-145 overexpression on attachment. The data demonstrate that miR-145 influences embryo attachment by reducing the level of IGF1R in endometrium.

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

STUDY QUESTION: How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model? SUMMARY ANSWER: Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium. WHAT IS KNOWN ALREADY: In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models. STUDY DESIGN, SIZE, DURATION: This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001. MAIN RESULTS AND THE ROLE OF CHANCE: Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared.

Maternal nutrition modifies trophoblast giant cell phenotype and fetal growth in mice.

Mammalian placentation is dependent upon the action of trophoblast cells at the time of implantation. Appropriate fetal growth, regulated by maternal nutrition and nutrient transport across the placenta, is a critical factor for adult offspring long-term health. We have demonstrated that a mouse maternal low-protein diet (LPD) fed exclusively during preimplantation development (Emb-LPD) increases offspring growth but programmes adult cardiovascular and metabolic disease. In this study, we investigate the impact of maternal nutrition on post-implantation trophoblast phenotype and fetal growth. Ectoplacental cone explants were isolated at day 8 of gestation from female mice fed either normal protein diet (NPD: 18% casein), LPD (9% casein) or Emb-LPD and cultured in vitro. We observed enhanced spreading and cell division within proliferative and secondary trophoblast giant cells (TGCs) emerging from explants isolated from LPD-fed females when compared with NPD and Emb-LPD explants after 24 and 48 h. Moreover, both LPD and Emb-LPD explants showed substantial expansion of TGC area during 24-48 h, not observed in NPD. No difference in invasive capacity was observed between treatments using Matrigel transwell migration assays. At day 17 of gestation, LPD- and Emb-LPD-fed conceptuses displayed smaller placentas and larger fetuses respectively, resulting in increased fetal:placental ratios in both groups compared with NPD conceptuses. Analysis of placental and yolk sac nutrient signalling within the mammalian target of rapamycin complex 1 pathway revealed similar levels of total and phosphorylated downstream targets across groups. These data demonstrate that early post-implantation embryos modify trophoblast phenotype to regulate fetal growth under conditions of poor maternal nutrition.

Comparative proteomic analysis of supportive and unsupportive extracellular matrix substrates for human embryonic stem cell maintenance.

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.

A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFß deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

Graphene oxide activates canonical TGFß signalling in a human chondrocyte cell line via increased plasma membrane tension.

Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is critical for developing articular cartilage regeneration therapies but remains poorly understood. The objectives of this work were to elucidate the effects of GO on the key chondrogenic signalling pathway - TGFß and identify the mechanism through which signal activation is achieved in human chondrocytes. Activation of canonical signalling was validated through GO-induced SMAD-2 phosphorylation and upregulation of known TGFß response genes, while the use of a TGFß signalling reporter assay allowed us to identify the onset of GO-induced signal activation which has not been previously reported. Importantly, we investigate the cell-material interactions and molecular mechanisms behind these effects, establishing a novel link between GO, the plasma membrane and intracellular signalling. By leveraging fluorescent lifetime imaging (FLIM) and a membrane tension probe, we reveal GO-mediated increases in plasma membrane tension, in real-time for the first time. Furthermore, we report the activation of mechanosensory pathways which are known to be regulated by changes in plasma membrane tension and reveal the activation of endogenous latent TGFß in the presence of GO, providing a mechanism for signal activation. The data presented here are critical to understanding the chondroinductive properties of GO and are important for the implementation of GO in regenerative medicine.

Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.

Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but is still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development (referred to as the RAPID-E protocol). Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II, but additionally expressed Collagen X, indicative of hypertrophy. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations.

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.