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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.

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 alpha 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.

Background: Numerous studies have documented the in vitro differentiation of human pluripotent stem cells (hPSCs) into kidney cells. Fewer studies have followed the fates of such kidney precursor cells (KPCs) inside animals, a more life-like setting. Here, we tested the hypothesis that implanting hPSC-derived KPCs into an in vivo milieu surgically engineered to be highly vascular would enhance their maturation into kidney tissues. Methods: 3D printed chambers containing KPCs were implanted into the thighs of adult immunodeficient mice. In some chambers, an arterial and venous flow-through (AVFT) was surgically fashioned. After 3 weeks and 3 months, implants were studied by histology, using qualitative and quantitative methods. Results: After 3 weeks, chambers containing AVFTs were richer in small vessels than contralateral chambers without AVFTs. Glomeruli with capillary loops and diverse types of tubules were detected in all chambers. At 3 months, chambers contained only rudimentary tubules and glomeruli that appeared avascular. In chambers with AVFTs, prominent areas of muscle-like cells were also detected near tubules and the abnormal tissues immunostained for transforming growth factor β1. These features have similarities to renal dysplasia, a typical histological signature of human congenital kidney malformations. Conclusions: This study urges a note of caution regarding the in vivo fates of hPSC-derived kidney precursors, with pathological differentiation appearing to follow a period of increased vascularity.

Human‐induced pluripotent stem cells (hiPSCs) are reprogrammed somatic cells and are an excellent cell source for tissue engineering applications, disease modeling, and for understanding human development. HiPSC lines have now been generated from a diverse range of somatic cell types and have been reported to retain an epigenetic memory of their somatic origin. To date, the reprogramming of a true ligament has not been reported. The aim of this study is to generate iPSCs from human anterior cruciate ligament (ACL) cells. ACL cells from three above‐knee amputation donors, with donor matched dermal fibroblasts (DFs) were tested for reprogramming using an existing DF reprogramming protocol. ACL cells were, however, more sensitive than donor matched DF to transforming growth factor‐β (TGF‐β); displaying marked contraction, increased proliferation and increased TNC and COMP expression in vitro, which hindered reprogramming to iPSCs. Modification of the protocol by scoring the cell monolayer or by removal of TGF‐β during ACL reprogramming resulted in emerging colonies being easier to identify and extract, increasing reprogramming efficiency. Following 30 passages in culture, the generated ACL derived iPSCs displayed pluripotency markers, normal karyotype and can successfully differentiate to cells of the three embryonic germ layers. This study illustrates it is possible to generate hiPSCs from ligament and identifies optimized ligament reprogramming conditions. ACL derived iPSCs may provide a promising cell source for ligament and related tissue engineering applications. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:92–104, 2020

Kabuki syndrome (KS) is a rare multisystem disorder, characterized by intellectual disability, growth delay, and distinctive craniofacial features. It is mostly caused by de novo mutations of KMT2D, which is responsible for histone H3lysine 4 mono-methylation (H3K4me1) that marks active and poised enhancers. We assessed the impact of KMT2D mutations on chromatin and transcriptional regulation in a cohort of multiple KS1 tissues, including primary patient samples and disease-relevant lineages, namely cortical neurons (iN), neural crest stem cells (NCSC), and mesenchymal cells (MC). In parallel, we generated an isogenic line derived from human embryonic stem cells (hESC) for the stepwise characterization of neural precursors and mature neurons. We found that transcriptional dysregulation was particularly pronounced in cortical neurons and widely affected synapse activity pathways. This was consistent with highly specific alterations of spontaneous network-bursts patterns evidenced by Micro-electrode-array (MEA)-based neural network. Profiling of H3K4me1 unveiled the almost complete uncoupling between this chromatin mark and the effects on transcription, which is instead reflected by defects in H3K27ac. Finally, we identified the direct targets of KMT2D in mature cortical neurons, uncovering TEAD2 as the main mediator of KMT2D haploinsufficiency. Our results uncover the multi-tissue architecture of KS1 dysregulation and define a unique electrical phenotype and its molecular underpinnings for the cortical neuronal lineage.

The growth factor TGFβ and the mechanosensitive calcium-permeable cation channel TRPV4 are both important for the development and maintenance of many tissues. Although TRPV4 and TGFβ both affect core cellular functions, how their signals are integrated is unknown. Here we show that pharmacological activation of TRPV4 significantly increased the canonical response to TGFβ stimulation in chondrocytes. Critically, this increase was only observed when TRPV4 was activated after, but not before TGFβ stimulation. The increase was prevented by pharmacological TRPV4 inhibition or knockdown and is calcium/CamKII dependent. RNA-seq analysis after TRPV4 activation showed enrichment for the TGFβ signalling pathway and identified JUN and SP1 as key transcription factors involved in this response. TRPV4 modulation of TGFβ signalling represents an important pathway linking mechanical signalling to tissue development and homeostasis.

BackgroundHeterozygous variants in the cardiac ryanodine receptor gene (RYR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Most pathogenic RYR2 variants are missense variants which result in a gain of function, causing ryanodine receptors to be increasingly sensitive to activation by calcium, have an increased open probability and an increased propensity to develop calcium waves. However, some RYR2 variants can lead to arrhythmias by a loss of function mechanism.ObjectiveTo understand the mechanism by which a novel nonsense variant in RYR2 p.(Arg4790Ter) leads to ventricular arrhythmias.MethodsHuman induced pluripotent stem cells (hiPSCs) harbouring the novel nonsense variant in RYR2 were differentiated into cardiomyocytes (RYR2-hiPSC-CMs) and molecular and calcium handling properties were studied.ResultsRYR2-hiPSC-CMs displayed significant calcium handling abnormalities at baseline and following treatment with isoproterenol. Treatment with carvedilol and nebivolol resulted in a significant reduction in calcium handling abnormalities in the RYR2-hiPSC-CMs. Expression of the mutant RYR2 allele was confirmed at the mRNA level and partial silencing of the mutant allele resulted in a reduction in calcium handling abnormalities at baseline.ConclusionThe nonsense variant behaves similarly to other gain of function variants in RYR2. Carvedilol and nebivolol may be suitable treatments for patients with gain of function RYR2 variants.

Embryo implantation begins with blastocyst trophectoderm (TE) attachment to the endometrial epithelium, followed by the breaching of this barrier by TE-derived trophoblast. Dynamic protein modification with O-linked &beta, N-acetylglucosamine (O-GlcNAcylation) is mediated by O-GlcNAc transferase and O-GlcNAcase (OGA), and couples cellular metabolism to stress adaptation. O-GlcNAcylation is essential for blastocyst formation, but whether there is a role for this system at implantation remains unexplored. Here, we used OGA inhibitor thiamet g (TMG) to induce raised levels of O-GlcNAcylation in mouse blastocysts and human trophoblast cells. In an in vitro embryo implantation model, TMG promoted mouse blastocyst breaching of the endometrial epithelium. TMG reduced expression of TE transcription factors Cdx2, Gata2 and Gata3, suggesting that O-GlcNAcylation stimulated TE differentiation to invasive trophoblast. TMG upregulated transcription factors OVOL1 and GCM1, and cell fusion gene ERVFRD1, in a cell line model of syncytiotrophoblast differentiation from human TE at implantation. Therefore O-GlcNAcylation is a conserved pathway capable of driving trophoblast differentiation. TE and trophoblast are sensitive to physical, chemical and nutritive stress, which can occur as a consequence of maternal pathophysiology or during assisted reproduction, and may lead to adverse neonatal outcomes and associated adult health risks. Further investigation of how O-GlcNAcylation regulates trophoblast populations arising at implantation is required to understand how peri-implantation stress affects reproductive outcomes.

At implantation, trophoblast derived from the trophectoderm of the blastocyst-stage embryo invades the endometrium to establish pregnancy. To understand how embryos breach the endometrial epithelium, we modelled human implantation using blastocysts or trophoblast stem cell spheroids cultured with endometrial epithelial cells (EEC). Blastocyst invasion of the EEC layer was initiated by multinuclear syncytiotrophoblast. Spheroids also invaded the epithelium with syncytiotrophoblast, and EEC induced upregulation of syncytiotrophoblast markers. Modelling implantation in silico using blastocyst and EEC transcriptomes revealed gene networks that exhibited greater connectivity and organisation in trophectoderm of the polar region of the embryonic axis. However, gene ontologies and machine learning suggested that EEC drives syncytiotrophoblast differentiation in polar and mural trophectoderm. This is the first evidence for endometrial epithelium-induced trophectoderm differentiation to invasive syncytiotrophoblast as the cellular mechanism of embryonic breaching of the endometrium in humans, with implications for reproductive medicine and our understanding of human embryonic development.

Background: Kidney disease causes major suffering and premature mortality worldwide. With no cure for kidney failure currently available, and with limited options for treatment, there is an urgent need to develop effective pharmaceutical interventions to slow or prevent kidney disease progression. Summary: In this review, we consider the feasibility of using human pluripotent stem cell-derived kidney tissues, or organoids, to model genetic kidney disease. Notable successes have been made in modelling genetic tubular diseases (e.g., cystinosis), polycystic kidney disease, and medullary cystic kidney disease. Organoid models have also been used to test novel therapies that ameliorate aberrant cell biology. Some progress has been made in modelling congenital glomerular disease, even though glomeruli within organoids are developmentally immature. Less progress has been made in modelling structural kidney malformations, perhaps because sufficiently mature metanephric mesenchyme-derived nephrons, ureteric bud-derived branching collecting ducts, and a prominent stromal cell population are not generated together within a single protocol. Key Messages: We predict that the field will advance significantly if organoids can be generated with a full complement of cell lineages and with kidney components displaying key physiological functions, such as glomerular filtration. The future economic upscaling of reproducible organoid generation will facilitate more widespread research applications, including the potential therapeutic application of these stem cell-based technologies.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44–HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment.

Summary MicroRNAs (miRNAs) are gene expression regulators and they have been implicated in acquired kidney diseases and in renal development, mostly through animal studies. We hypothesized that the miR-199a/214 cluster regulates human kidney development. We detected its expression in human embryonic kidneys by in situ hybridization. To mechanistically study the cluster, we used 2D and 3D human embryonic stem cell (hESC) models of kidney development. After confirming expression in each model, we inhibited the miRNAs using lentivirally transduced miRNA sponges. This reduced the WT1+ metanephric mesenchyme domain in 2D cultures. Sponges did not prevent the formation of 3D kidney-like organoids. These organoids, however, contained dysmorphic glomeruli, downregulated WT1, aberrant proximal tubules, and increased interstitial capillaries. Thus, the miR-199a/214 cluster fine-tunes differentiation of both metanephric mesenchymal-derived nephrons and kidney endothelia. While clinical implications require further study, it is noted that patients with heterozygous deletions encompassing this miRNA locus can have malformed kidneys., Graphical Abstract, Highlights • miR-199a/214 is expressed in the developing human kidney • Its function can be modeled using hESC-derived kidney organoids • Inhibiting these miRNAs results in dysmorphic glomeruli and proximal tubules • Their inhibition also leads to a denser vascular network between tubules, Bantounas and colleagues studied the role of the miR-199a/214 cluster in hESC-kidney development because they found it was expressed in native embryonic human kidneys. Using hESC-derived 3D kidney organoids, they found that blocking the miRNAs' function resulted in aberrations in glomeruli and proximal tubules and increased vascular density between tubules. Thus, the cluster fine-tunes kidney 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, at different development stages. Activation of SIRT1 during 3D-pellet culture led to significant increases in 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 ACAN –10 enhancer. Unexpectedly, while ACAN was enhanced, GAG retention in the matrix was reduced when SIRT1 was activated. Significantly, ARID5B and COL2A1 were positively correlated, with Co-IP indicating association of ARID5B with SIRT1 suggesting that COL2A1 expression is promoted by an ARID5B and SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, whilst altering ECM composition and suppressing GAG content during cartilage development. These results suggest that SIRT1 activity can be beneficial to cartilage development and matrix protein synthesis but tailored by addition of other positive GAG mediators.

STUDY QUESTION Does embryo transfer medium containing hyaluronate (HA) promote the attachment phase of human embryo implantation? SUMMARY ANSWER HA-containing medium does not promote human blastocyst attachment to endometrial epithelial cells in vitro. WHAT IS KNOWN ALREADY Embryo transfer media containing high concentrations of HA are being used to increase implantation and live birth rates in IVF treatment, although the mechanism of action is unknown. STUDY DESIGN, SIZE, DURATION Expression of HA-interacting genes in frozen-thawed oocytes/embryos was assessed by microarray analysis (n = 21). Fresh and frozen human blastocysts (n = 98) were co-cultured with human endometrial epithelial Ishikawa cell layers. Blastocyst attachment and the effects of a widely used HA-containing medium were measured. PARTICIPANTS/MATERIALS, SETTING, METHODS Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Blastocyst-stage embryos were transferred at day 6 to confluent Ishikawa cell layers; some blastocysts were artificially hatched. Blastocyst attachment was monitored from 1 to 48 h, and the effects of blastocyst pre-treatment for 10 min with HA-containing medium were determined. MAIN RESULTS AND THE ROLE OF CHANCE Human embryos expressed the HA receptor genes CD44 and HMMR, hyaluronan synthase genes HAS1–3, and hyaluronidase genes HYAL1–3, at all stages of preimplantation development. Attachment of partially hatched blastocysts to Ishikawa cells at 24 and 48 h was related to trophectoderm grade (P = 0.0004 and 0.007, respectively, n = 34). Blastocysts of varying clinical grades that had been artificially hatched were all attached within 48 h (n = 21). Treatment of artificially hatched blastocysts with HA-containing medium did not significantly affect attachment at early (1–6 h) or late (24 and 48 h) time points, compared with control blastocysts (n = 43). LIMITATIONS, REASONS FOR CAUTION Using an adenocarcinoma-derived cell line to model embryo-endometrium attachment may not fully recapitulate in vivo interactions. The high levels of blastocyst attachment seen with this in vitro model may limit the sensitivity with which the effects of HA can be observed. WIDER IMPLICATIONS OF THE FINDINGS Morphological trophectoderm grade can be correlated with blastocyst attachment in vitro. HA-containing medium may increase pregnancy rates by mechanisms other than promoting blastocyst attachment to endometrium. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by a grant from the Wellbeing of Women, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the Department of Health Scientist Practitioner Training Scheme, and the Ministry of Higher Education, The State of Libya. None of the authors has any conflict of interest to declare.

Summary CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common form of genetic stroke and vascular dementia syndrome resulting from mutations in NOTCH3. To elucidate molecular mechanisms of the condition and identify drug targets, we established a patient-specific induced pluripotent stem cell (iPSC) model and demonstrated for the first time a failure of the patient iPSC-derived vascular mural cells (iPSC-MCs) in engaging and stabilizing endothelial capillary structures. The patient iPSC-MCs had reduced platelet-derived growth factor receptor β, decreased secretion of the angiogenic factor vascular endothelial growth factor (VEGF), were highly susceptible to apoptotic insults, and could induce apoptosis of adjacent endothelial cells. Supplementation of VEGF significantly rescued the capillary destabilization. Small interfering RNA knockdown of NOTCH3 in iPSC-MCs revealed a gain-of-function mechanism for the mutant NOTCH3. These disease mechanisms likely delay brain repair after stroke in CADASIL, contributing to the brain hypoperfusion and dementia in this condition, and will help to identify potential drug targets., Highlights • Generated an iPSC model for the genetic small vessel disease CADASIL • Patient iPSC-mural cells failed to support angiogenic capillary structure • Patient iPSC-mural cells reduced VEGF secretion contributing to capillary instability • Patient iPSC-mural cells have survival disadvantage and causing apoptosis of ECs, Wang, Kimber and colleagues established an iPSC model for the most common genetic stroke and vascular dementia syndrome CADASIL, and identified primary defects of iPSC-derived mural cells in stabilizing vascular capillary structures with reduced PDGFRβ expression and VEGF secretion. Supplementation of VEGF significantly rescued the capillary destabilization, suggesting a therapeutic target. siRNA knockdown of NOTCH3 suggests a gain-of-function mechanism.

In the future, stem cell-based technologies may be harnessed to replace conventional dialysis and transplantation in patients with diabetic nephropathy. Recently, there has been considerable effort to improve methods for the differentiation of human pluripotent stem cells (hPSCs) into kidney cells in culture. Here, we present a protocol for obtaining more advanced kidney structures than have currently been possible in vitro, including vascularized glomeruli and tubular elements. HPSCs are first differentiated in 2D culture to a kidney progenitor stage. These cells are then dissociated and injected subcutaneously into immunocompromised mice. Twelve weeks later, the cells have developed into mature kidney structures and are excised for further characterization. This method constitutes a significant improvement on protocols that involve either exclusively a 2D culture or placing the cells in 3D organoid culture at the air-liquid interface in vitro.

Human embryonic stem cells (hESCs) have great potential for the repair of damaged articular cartilage. We developed a serum-free 14-day protocol for hESC differentiation into chondrocyte progenitors, which surprisingly lacked strong cartilage matrix production in in vitro tests. In order to direct these progenitors to a more mature phenotype, we investigated substituting different members of the TGFβ family in the protocol. Initially, we supplemented, or substituted GDF5 (day 11–14), with combinations of BMP7 and TGFβ-1, or −3, but these modifications yielded no improvement in matrix gene expression. However, replacing BMP4 with BMP2 (days 3–10 of the protocol) resulted in a more rapid increase in SOX9 gene expression and increased expression of chondrogenic genes SOX5, ACAN and COL2A1. The replacement of BMP4 with BMP2 also enhanced the formation of chondrogenic cell aggregates, with greater deposition of type II collagen. This change was not accompanied by hypertrophic chondrocyte marker COL10A1 expression. The results demonstrate that BMP2 has greater specificity for the generation of chondrogenic cells from hESCs than BMP4 and this was consistent in two hESC lines (HUES1 and MAN7). hESC-chondrogenic cells derived with either BMP2 or BMP4 were tested in vivo by implanting them in fibrin into osteochondral defects in the femur of RNU rats. Repaired cartilage tissue, positive for Safranin O and type II collagen was detected at 6 and 12 weeks with both cell sources, but the BMP2 cells scored higher for tissue quality (Pineda score). Therefore, BMP2 is more effective at driving chondrogenic differentiation from human pluripotent stem cells than BMP4 and the effect on the resulting chondroprogenitors is sustained in an in vivo setting., Graphical abstract Unlabelled Image, Highlights • BMP2 stimulates increased chondrogenic gene expression from hESC chondroprogenitors compared to BMP4. • Other TGFΒ members were not effective. • BMP2 promoted COL2a1 positive aggregate formation. with no effect on culture expansion or apoptosis.

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.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo&ndash, epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoderm.

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.

Human embryonic stem cells (hESCs) derived from the pluripotent Inner cell mass (ICM) of the blastocyst are fundamental tools for understanding human development, yet are not identical to their tissue of origin. To investigate this divergence we compared the transcriptomes of genetically paired ICM and trophectoderm (TE) samples with three hESC lines: MAN1, HUES3 and HUES7 at similar passage. We generated inferred interactome networks using transcriptomic data unique to the ICM or TE, and defined a hierarchy of modules (highly connected regions with shared function). We compared network properties and the modular hierarchy and show that the three hESCs had limited overlap with ICM specific transcriptome (6%-12%). However this overlap was enriched for network properties related to transcriptional activity in ICM (p=0.016); greatest in MAN1 compared to HUES3 (p=0.048) or HUES7 (p=0.012). The hierarchy of modules in the ICM interactome contained a greater proportion of MAN1 specific gene expression (46%) compared to HUES3 (28%) and HUES7 (25%) (p=9.0×10−4).These findings show that traditional methods based on transcriptome overlap are not sufficient to identify divergence of hESCs from ICM. Our approach also provides a valuable approach to the quantification of differences between hESC lines.And Manchester Academic Health Sciences Centre

Summary Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application., Highlights • We designed a hepatocyte differentiation process under defined conditions • The hepatocyte phenotype is improved and stabilized on two laminin isoforms • Laminin-supported differentiation suppresses inappropriate gene networks • Laminin surfaces deliver hepatocytes with greater similarity to adult hepatocytes, In this article, Hay and colleagues demonstrate that hepatocyte production is possible under defined conditions with GMP-grade hESC lines. The use of recombinant laminins significantly improved the stem cell-derived hepatocyte phenotype, which was closer in nature to primary adult hepatocytes.

Background Human embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos. Methods A comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines. Results We developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement. Conclusions The present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0561-y) contains supplementary material, which is available to authorized users.

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.

In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions.

Background: Interaction of stem cells with extracellular matrix (ECM) controls their fate. Results: MS reveals interacting ECM networks produced by human embryonic stem cells (hESCs) and their feeders; supportive and unsupportive hESC substrates comprise distinct ECM compositions. Conclusion: Several ECM molecules maintain hESC self-renewal. Significance: Better understanding of hESC self-renewal has applications in understanding development, generating cell therapies, and modeling diseases., 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.

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-mediatedMMP13gene 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-1β, 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-1β 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 humanOA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in humanOAchondrocytes,which appears to bemediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.

In health, the mature human kidney contains on average 1.4 million nephrons (1). Each of these epithelial tubes begins in the cortex of the organ with a glomerulus that ultrafilters blood, clearing water and small molecules but retaining large proteins and blood cells. The two kidneys receive around 20% of the cardiac output, a high pressure and high volume arterial flow needed to generate daily the 200 litres of ultrafiltrate. The nephron tubule segments modify this ultrafiltrate, for example reclaiming some sodium and water. The nascent urine then flows into collecting ducts that executes concentration and further acidification. The branched collecting ducts converge towards the papilla where urine, around one to two litres a day, enters the renal pelvis and then the ureters, in which it is propelled towards the urinary bladder by peristaltic waves.

Our limited understanding of the processes underlying steroid hormonal control of human endometrial receptivity is largely due to the lack of a relevant model system. To overcome scarcity of material, we have developed a model in which mouse embryos attach to human Ishikawa cells, which express functional steroid hormone receptors. Blastocysts flushed from day 4 pregnant superovulated mice were transferred to confluent Ishikawa cell monolayers. After 48 h of co-culture, 85% of the blastocysts had attached loosely, but only 40% attached stably to the epithelial cell surface. In contrast, 95% of the embryos attached stably to tissue culture plastic. Thus, weak attachment of a majority of the embryos was followed by stronger adhesion of a smaller proportion. Seventeen percent of the transferred blastocysts modified the epithelial cell surface with loss of MUC1 at the attachment site, extending variably to adjacent epithelial cells. Initially, stable attachment occurred without disruption to the integrity of the epithelial monolayer, but at later stages after the embryo had spread laterally, displacement of subjacent cells was observed. A modest increase in stable attachment, but no changes to MUC1 clearance, was observed after assisted hatching. After 24 h priming of Ishikawa cells by 17β-oestradiol (OE2) followed by 72-h incubation with medroxyprogesterone acetate and OE2, stable attachment increased from 40 to 70%. Initial attachment is efficient either in the presence or in the absence of hormone; steroid treatment increased the incidence of stable attachment. Implantation failure is predicted to occur in this model when embryos fail to progress from initial to stable attachment.

We report here the derivation of two new human embryonic stem cell lines, Man-1 and Man-2, and their full characterization as novel pluripotent stem cell lines. Man-1 was derived from an embryo surplus to requirement from routine IVF, while Man-2 was obtained from an oocyte classified as failed to fertilise and subsequently chemically activated. We report the characterisation of pluripotency and the differentiation potential of these lines. Work is in progress to establish novel methods of stem cell derivation and culture, which will avoid the use of xenobiotics and be relevant to clinical production of human embryonic stem cell lines. Both newly derived human embryonic stem cell lines will be available for the research community from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).

A fundamental step in embryonic development is cell differentiation whereby highly specialised cell types are developed from a single undifferentiated, fertilised egg. One of the earliest lineages to form in the mammalian conceptus is the trophoblast, which contributes exclusively to the extraembryonic structures that form the placenta. Trophoblast giant cells (TGCs) in the rodent placenta form the outermost layer of the extraembryonic compartment, establish direct contact with maternal cells, and produce a number of pregnancy-specific cytokine hormones. Giant cells differentiate from proliferative trophoblasts as they exit the cell cycle and enter a genome-amplifying endocycle. Normal differentiation of secondary TGCs is a critical step toward the formation of the placenta and normal embryonic development. Trophoblast development is also of particular interest to the developmental biologist and immunobiologist, as these cells constitute the immediate cellular boundary between the embryonic and maternal tissues. Abnormalities in the development of secondary TGCs results in severe malfunction of the placenta. Herein we review new information that has been accumulated recently regarding the molecular and cellular regulation of trophoblast and placenta development. In particular, we discuss the molecular aspects of murine TGC differentiation. We also focus on the role of growth and transcription factors in TGC development.

The metanephros is formed from the reciprocal inductive interaction of two precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB). The UB induces MM to condense and differentiate forming the glomerulus and renal tubules, whilst the MM induces the UB to differentiate into the collecting tubules of the mature nephron. Uninduced MM is considered the progenitor cell population of the developing metanephros because of its potential to differentiate into more renal cell types than the UB. Previous studies have identified the phenotype of renal precursor cells; however, expression of candidate marker genes have not been analysed in other tissues of the murine embryo. We have assayed up to 19 candidate genes in eight embryonic tissues at five gestation stages of the mouse embryo to identify markers definitively expressed by renal cells during metanephric induction and markers developmentally regulated during kidney maturation. We then analysed their expression in other developing tissues. Results show Dcn, Hoxc9, Mest, Wt1 and Ywhaq were expressed at moderate to high levels during the window of metanephric specification and early differentiation (E10.5-E12.5 dpc), and Hoxc9, Ren1 and Wt1 expression was characteristic of mature renal cells. We demonstrated Cd24a, Cdh11, Mest, Scd2 and Sim2 were regulated during brain development, and Scd2, Cd24a and Sip1 expression was enriched in developing liver. These markers may be useful negative markers of kidney development. Use of a combination of highly expressed and negative markers may aid in the identification and removal of non-renal cells from heterogeneous populations of differentiating stem cells.

Uteri ofLifnull mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is requiredin vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P≤0.0001) and was effective maximally between 0930 and 1230 h. Antagonism of LIF signalling was evidenced by a lack of phosphorylated STAT3 in the luminal epithelium (LE). Amphiregulin was absent from the LE on day 4 evening and H-type-1 antigen expression was retained in the LE on day 5 in inhibited uteri. Interleukin-1α and oncostatin M expression were reduced in the stroma on day 6, following LIF inhibition. Unexpectedly, PTGS2 expression in stroma was unaffected by LIF inhibitionin vivo, in contrast toLifnull mice. In summary, this suggests that LIF signalling is effective for implantation during a discrete time window on day 4 and antagonism of LIF signalling recapitulates many features exhibited inLifnull uteri. The data presented validates the use of antagonists to investigate tissue specific and temporal cytokine signalling in reproductive function.

Human embryonic stem cell (hESC) growth is dependent on various factors released by feeder cells. Some of them have already been elucidated, although much research is still needed to understand the biology of stem cell maintenance in culture. Traditionally, primary mouse embryonic fibroblasts (PMEFs) have been used as feeder layers, and both murine and human fibroblast cell lines have been shown to support pluripotency and self-renewal of hESC. Here we report the derivation of three new mouse embryonic fibroblast cell lines, MEFLU-M, MEFLU-T, and MEFLU-TB, with different properties regarding growth and support for undifferentiated hESCs. MEFLU-TB is able to support continuous growth of the newly derived Man-1, as well as H1, HUES-1, HUES-7, HUES-8, and HUES-9 human embryonic stem cell lines. After more than 50 passages and doublings, MEFLU-TB feeders compare to early passage primary mouse embryonic fibroblasts in their ability to support undifferentiated hESC growth. Our results contradict a previous paradigm that PMEFs tend to lose their capacity to support proliferation of hESCs with increasing passages, and show that the MEFLU-TB mouse embryonic fibroblast cell line and its conditioned medium have the potential to support the maintenance of hESC lines. Also, our results clearly show that spontaneous immortalization of primary fibroblasts can be achieved in culture without any chemical addition or genetic modification.

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

This study addresses the regulation of the interleukin 1 (IL1) system in the murine uterine luminal epithelium (LE) and stroma by leukemia inhibitory factor (LIF). Using RT-PCR we compared expression of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 during the pre- and peri-implantation periods of pregnancy in wild-type (WT) and LIF-null LE and stroma. In WT LE, Il1a transcripts were downregulated on Day 4 of pregnancy (D4), with renewed expression by the evening of D4 (D4 pm). In Lif � /� LE there was a gradual decrease in expression on D2, and expression became undetectable by D6. Il1b and Il1r1 expression were similar in WT and null mice, but Il1rn expression was almost completely lost during the peri-implantation period in Lif � /� LE. In the stroma, Il1a was sharply down-regulated on D4 and reappeared on D4 pm but was only expressed from D3 to D5 in the null mice. Stromal Il1r1 and Il1r2 were also misregulated. Il1rn showed constitutive expression in null stroma in contrast to the loss of expression on D4 in the WT mouse. In Lif-deficient mice, immunostaining indicated a reduction of endometrial IL1A at the time of implantation and of IL1B in stroma. LE-stromal coculture revealed that LIF stimulated the apical secretion of both IL1A and PTGES2 by LE cells without affecting basal secretion of IL1A and with only a small effect on basal PTGES2 secretion. We conclude that Il1a and Il1rn in LE and Il1a, Il1rn, and Il1r1 in stroma are regulated by LIF, which stimulates apical secretion of IL1A by LE. implantation, interleukin 1, leukemia inhibitory factor, prostaglandin, uterus

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 culturedin 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.

The differentiation of murine trophoblast giant cells (TGCs) is well characterised at the molecular level and, to some extent, the cellular level. Currently, there is a rudimentary understanding about factors regulating the cellular differentiation of secondary TGCs. Using day 8.5 p.c.-ectoplacental cone (EPC) explant in serum-free culture, we have found parathyroid hormone-related protein (PTHrP) to regulate cellular changes during TGC differentiation. PTHrP greatly stimulated the formation and organisation of actin stress fibres and actin expression in trophoblast outgrowth. This coincided with changing cell shape into a flattened/fibroblastic morphology, suppression of E-cadherin expression, and increased cell spreading in culture. PTHrP also increased the nuclear staining of beta-catenin and, similar to activator protein-2gamma (AP-2gamma), showed microtubule-dependent nuclear localisation in vitro. These cellular and behavioural changes correlated with changes in the expression of RhoGTPases and in both expression and phosphorylation of Eph/Ephrin kinases. The effects of PTHrP on trophoblast cellular differentiation were abolished after blocking its action. In conclusion, PTHrP provides an excellent example of the extrinsic factors that, through their network of activities, plays an important role in cellular differentiation of secondary TGCs.

Leukaemia inhibitory factor (LIF) is one of the most important cytokines in the reproductive tract. Without expression of LIF in the uterus, implantation of a blastocyst cannot begin. Yet, 13 years after publication of the phenotype of the LIF knockout mouse we are only just beginning to understand how LIF functions in the uterus. This review addresses our knowledge of the role of LIF in regulating implantation through its influence on the luminal epithelium and stromal decidualization, but also its influence on reproductive tract cells such as leukocytes and glandular epithelium, during the pre-implantation phase of pregnancy.

The murine trophoblast cell lineage represents an intriguing experimental cell model as it is composed of four trophoblast stem (TS)-derived cell types: trophoblast giant cells (TGCs), spongiotrophoblast, syncytotrophoblast, and glycogen trophoblast cells. To investigate the role of parathyroid hormone-related protein (PTHrP) in TGC differentiation, we analyzed the effect of exogenous PTHrP on secondary TGCs of day 8.5 p.c. ectoplacental cone explant culture. Secondary TGCs expressed PTHrP and PTHR1 receptor in vivo and in vitro. TGCs treated with PTHrP had reduced proliferation and decreased apoptosis starting from day 2 in culture, and enhanced properties of giant cell differentiation: increased DNA synthesis, number of cells with giant nuclei and expression of placental lactogen-II (PL-II). The induction of TGC formation by PTHrP correlated with downregulation of cyclin B1 and mSNA expression, but upregulation of cyclin D1, thus allowing mitotic-endocycle transition. Moreover, PTHrP treatment influenced TGC differentiation by inducing the expression of transcription factors known to stimulate giant cell formation: Stra13 and AP-2gamma, and inhibiting the formation of other trophoblast cell types by suppressing trophoblast progenitors and spongiotrophoblast-promoting factors, Eomes, Mash-2, and mSNA. Taken together with the spatial and temporal patterns of TGC formation and PTHrP synthesis in vivo, these findings indicate an important role for PTHrP in the differentiation of secondary TGCs during placentation.

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.