Extraembryonic mesoderm (ExM) is crucial for human development, yet its specification is poorly understood. Human embryonic stem cell (hESC)-based models, including embryoids and differentiated derivatives, are emerging as promising tools for studying ExM development. Despite this, the signaling mechanisms and developmental dynamics that underlie ExM specification from hESCs remain challenging to study. Here, we report that the modulation of BMP, WNT, and Nodal signaling pathways can rapidly (4-5 days) and efficiently ( ~90%) induce the differentiation of both naive and primed hESCs into ExM-like cells (ExMs). We reveal that ExM specification from hESCs predominantly proceeds through intermediates exhibiting a primitive streak (PS)-like gene expression pattern and delineate the regulatory roles of WNT and Nodal signaling in this process. Furthermore, we find that the initial pluripotent state governs hESC-based ExM specification by influencing signal response, cellular composition, developmental progression, and transcriptional characteristics of the resulting ExMs. Our study provides promising models for dissecting human ExM development and sheds light on the signaling principles, developmental dynamics, and influences of pluripotency states underlying ExM specification from hESCs.

Gonadotrophs are the essential pituitary endocrine cells for reproduction. They produce both luteinizing (LH) and follicle-stimulating (FSH) hormones that act on the gonads to promote germ cell maturation and steroidogenesis. Their secretion is controlled by the hypothalamic gonadotrophin-releasing hormone (GnRH), and gonadal steroid feedback. Gonadotrophs first appear in the embryonic pituitary, along with other endocrine cell types, and all expand after birth. While gonadotrophs may display heterogeneity in their response to GnRH, they appear, at least transcriptionally, as a homogenous population. The pituitary also contains a population of stem cells (SCs), whose contribution to postnatal growth is unclear, in part because endocrine cells maintain the ability to proliferate. Here we show an unsuspected dual origin of the murine adult gonadotroph population, with most gonadotrophs originating from postnatal pituitary stem cells starting early postnatally and up to puberty, while embryonic gonadotrophs are maintained. We further demonstrate that postnatal gonadotroph differentiation happens independently of gonadal signals and is not affected by impairment of GnRH signalling. The division of gonadotrophs based on separate origins has implications for our understanding of the establishment and regulation of reproductive functions, both in health and in disease.

Homeobox transcription factors CDX1 and CDX2 (hereafter, CDX1/2) play key roles in determining the identity of intestinal epithelial cells and regulating their stem cell functions. However, the role of CDX1/2 in regulating colon cancer stemness and the underlying mechanisms are unclear. Here, we show that complete loss of Cdx1 or concurrent loss of Cdx1/2 increased the stemness and malignancy of intestinal tumors. Consistently, CDX1/2 reduced the expression of cancer stemness-related genes, including LGR5. CDX1/2 bound to the downstream region of the LGR5 transcription start site (TSS), a region where β-catenin also binds. Despite increased H3 acetylation and an open chromatin structure, CDX1/2 reduced the occupancy of DRB sensitivity-inducing factor (DSIF), RNA polymerase II-associated factor 1 (PAF1), and RNA polymerase II (Pol II) complexes around the LGR5 TSS. Through their homeodomains, CDX1/2 inhibited the β-catenin-facilitated formation of active Pol II complexes containing DSIF and PAF1 complexes by preventing the interaction between β-catenin and these complexes, in an additive manner. Our findings suggest that CDX1/2 cooperatively suppressed colonic tumorigenesis and cancer stemness by antagonizing β-catenin via the DSIF and PAF1 complexes. Additionally, DSIF and PAF1 complexes acted as transcriptional platforms that integrated and funneled both tumor-suppressive and oncogenic signals into the expression of genes that control colon cancer stemness.

Glioblastomas (GBMs) are aggressive brain tumors and challenging cancers for diagnosis and treatment. Therapeutic options include surgery followed by chemotherapy with the DNA alkylator temozolomide (TMZ) and radiotherapy. However, the patient's prognosis remains poor due to tumor heterogeneity, cell infiltration and intrinsic or acquired resistance to therapy. Understanding the resistance mechanisms together with identifying new biomarkers are crucial for developing novel therapeutic strategies. MiRNAs play an important role in the biology of gliomas, they modulate tumorigenesis and therapy response. We recently identified the diagnostic/prognostic miR-1-3p, miR-26a-1-3p and miR-487b-3p signature that displays an oncosuppressive role on several glioma biological functions. In this study, we investigated the effects of the therapeutic potential of this three-miRNA signature as a regulator of response to TMZ. We found that ectopic expression of the miRNA signature in patient-derived GBM neurospheres treated with TMZ impaired cell proliferation and viability by necroptosis induction. Moreover, we identified WT1 and FOXA1, two transcription factors specifically involved in TMZ resistance, as novel direct targets of the miRNA signature. Of note, the repression of WT1 and FOXA1, elicited by the signature, caused a downregulation of the Androgen Receptor (AR) expression, an impairment of tumor-spheroid formation and reversed cancer cell stemness. These results were recapitulated using the AR inhibitor enzalutamide, confirming the involvement of the AR pathway. Our data indicate that the miR-1-3p/miR-26a-1-3p/miR-487b-3p signature, which has an impact on treatment response and cell stemness, may pave the way for miRNA-based complementary therapies in GBM patients.

Mutations in genes affecting mitochondrial complex I (CI) can lead to mitochondrial cardiomyopathy (MCM) yet no effective treatment. This study sought to determine whether adeno-associated virus 9 (AAV9)-based gene therapy could prevent or rescue Ndufs6 deficiency-induced MCM at different disease stages. Using Ndufs6gt/gt mice to mimic MCM, cardiac dysfunction was evident at week 4 post-birth, showing reduced ejection fraction, CI activity, increased fibrosis, mitochondrial fission, and disrupted cristae. Neonatal and adult mice were intravenously given AAV9-hNdufs6 (1e14 vg kg-1). AAV9-hNdufs6 therapy effectively prevented neonatal mice's cardiac dysfunction onset, preserving CI activity and cristae structure for 11 months. In contrast, therapy in adult mice post-disease onset failed to reverse or halt progression of heart dilation and failure after 3 months, showing mitochondrial abnormalities and cardiomyocyte apoptosis. Mechanistically, adult mouse Kupffer cells demonstrated enhanced phagocytic capabilities compared to neonatal mice, with higher expression levels of AAV9 cell surface receptors observed in neonatal mouse hearts, rendering neonatal mice more responsive to AAV9-mediated gene therapy for heart tissue. Additionally, AAV9-hNdufs6 gene therapy initiated at an early stage increased Ndufs6 expression in cardiac tissue, preserved mitochondrial structure and function, prevented cardiomyocyte fibrosis through modulation of the AMPK/Drp1 signaling pathway. In conclusion, early intervention with AAV9-hNdufs6 gene therapy can effectively prevent the onset of MCM, but intervention after disease onset has limited efficacy.

The establishment of the enthesis unit remains a significant challenge due to the inability to reconstitute the native histological zones postrepair. In this study, the authors investigated the effect of 3-dimensional hyaluronan-based scaffold implanted with Wharton jelly-derived mesenchymal stem cells on enthesis healing in rat model.

This study evaluated the cytotoxicity of clinically available dental cements containing resin: conventional adhesive resin cement (ARC), self-adhesive resin cement (SARC), and resin-modified glass-ionomer cement (RMGIC), focusing on their degree of conversion (DC) and effects on cellular responses.

Cardiac magnetic resonance (CMR) imaging provides critical insight into the prognosis of Fontan patients, enhancing our understanding of their long-term outcomes. This study aimed to investigate the prognostic role of CMR in a carefully selected cohort of Fontan patients with the highest initial likelihood of survival.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe genetic mucocutaneous fragility disorder characterised by chronic blistering, slow wound healing and increased risk of squamous cell carcinoma. Current management options are very limited.

Male infertility poses a substantial clinical challenge, affecting 8-12% of couples worldwide. The underlying causes of this condition range from cancer treatments to congenital disorders. The present approach to fertility preservation entails the cryopreservation of sperm for post-pubertal males. However, this method offers limited options for prepubertal patients and those with non-obstructive azoospermia. This review examines emerging stem cell-based therapies, including testicular tissue grafting, organ culture, cell reconstitution, spermatogonial stem cell transplantation, and pluripotent stem cell-derived in vitro gametogenesis. While these approaches have demonstrated notable efficacy in animal models, particularly in mice, translating these technologies into clinical applications is confronted by significant scientific, ethical, and regulatory challenges. These innovative stem cell strategies offer promising pathways to restore fertility potential and provide hope for patients currently without reproductive options.

Reconstruction using cartilage tissue is necessary to address deformities of the nose, ears, and maxillofacial region in several cases. However, autologous cartilage tissue transplantation is limited in the amount that can be harvested owing to invasiveness to the human body.
Moreover, artificial materials such as implants cannot be used in many situations, given their potential to induce reactions to foreign bodies. Therefore, there is a growing demand for biomaterials that are less likely to cause foreign body reactions. Given that a tissue with a functionally superior three-dimensional structure can replace autologous tissue and artificial materials, we have developed a three-dimensional cultured cartilage tissue without scaffolding material and are working toward its practical application. To achieve an off-theshelf product that allows prolonged storage, the tissue was fixed with glutaraldehyde to maintain high strength for subsequent processing and management. Although tissue fixation with glutaraldehyde may cause calcification due to the deposition of calcium phosphate, calcification can be prevented by washing with high-concentration ethanol. We generated three-dimensional cultured cartilage tissues using induced pluripotent stem cell-derived limb bud mesenchymal cells and an original cell self-culture aggregation method. The generated tissues were subjected to an anti-calcification treatment with glutaraldehyde and 80% ethanol.
The treated tissue had improved stability and strength with minimal calcification. The tissue retained its physical properties that were effectively processable and could be processed into an ear-like shape.
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Chronic kidney disease affects 10% of the global population and often progresses to end-stage renal disease, where dialysis or renal transplant are the only therapies, though neither is a permanent solution. Regenerative medicine, particularly the use of organoids, offers a po-tential solution. Organoids are valuable for studying organ development, diseases, and re-generation, and are suitable for drug screening. However, their limited ability to replicate adult organs' maturation, complexity, and functions restricts their application. Additionally, manual production of organoids causes variability, affecting scalability and reproducibility. Automation techniques like bioprinting could enhance organoid maturation and complexity by depositing cells and biomaterials in a controlled manner.
In this study, we established differentiation protocols to obtain human induced plu-ripotent stem cell-derived metanephric mesenchyme, ureteric bud progenitors, and the com-bination of these was used to form organoids. A microfluidic bioprinter capable of producing core-shell filaments was used to bioprint single cell progenitors in combination with gelatin in the core wrapped with an alginate shell. These filament constructs were cultured with an optimized mix of growth factors for two weeks. Within one week, renal vesicles were visi-ble, and after two weeks post-bioprinting the kidney organoids were functional and respond to the nephrotoxic drug doxorubicin. In conclusion, a bioprinted method was developed to generate in an automated way functional renal organoids from progenitors, offering a foun-dation for future kidney disease treatment.&#xD.

O6-methylguanine-DNA methyltransferase (MGMT) reverses alkylating-agent-induced methylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) at the O6 position of guanine. MGMT is irreversibly inhibited by O6-benzylguanine (O6BG), while the Pro140Lys (P140K) variant is resistant. Combining the use of O6BG/BCNU with gene transfer of MGMT P140K into hematopoietic stem cells (HSCs) has enabled in vivo enrichment of gene-modified HSCs for therapeutic effect in preclinical studies. However, the P140K substitution cannot reliably be made using currently available gene-editing approaches. Identifying functional MGMT variants that are resistant to inhibitors and amenable to gene editing would enable in vivo enrichment of HSCs edited at both MGMT and a therapeutic locus. We used computational analyses to select putative variants and generated a library of MGMT variant-expressing plasmids (pMGMTs). For our functional screen, we treated MGMT-deficient U251 cells with O6BG and co-transfected them with pMGMT together with a plasmid cocktail including a fluorescent host cell reactivation reporter plasmid (mPlum_O6MeG) for MGMT activity. Flow cytometric analysis of MGMT activity identified active and O6BG-resistant MGMT variants. Treatment with a second MGMT inhibitor, PaTrin-2, confirmed these results. We also found MGMT variants that are detectable in the general population and tumors to be active and O6BG sensitive. Taken together, our findings establish a functional database for MGMT variants and a cell-based platform for screening DNA-repair proteins for unknown functional properties.

Dr. Felicia Pagliuca is currently Senior Vice President, Cell and Genetic Therapy Research at Vertex Pharmaceuticals. She received a BS from Duke University and a PhD from Cambridge University where she was a Marshall Scholar. She completed her postdoctoral fellowship with Dr. Douglas Melton at the Harvard Stem Cell Institute, where she worked with Dr. Melton to discover how to generate stem cell-derived pancreatic beta cells in the lab. She co-founded Semma Therapeutics with Dr. Melton in 2014. While at Semma, she served as Vice President of Cell Biology Research and Development, where she led the development of investigational cell therapies for the treatment of type 1 diabetes. She joined Vertex Pharmaceuticals in 2019, when Semma was acquired by Vertex, and served as the company's Vice President and Disease Area Executive for type 1 diabetes for 5 years before moving into her current role.

Osteoblast differentiation is essential for fracture healing and bone regeneration. miR-324-5p has been implicated in osteoporosis, but its precise role in osteogenic differentiation remains unclear. We investigated the function and regulatory mechanisms of miR-324-5p in bone marrow mesenchymal stem cells (BMSCs).

Osteoarthritis (OA) is a degenerative joint disease that causes pain and reduces quality of life. Exosomes, dental pulp stem cells, and kappa carrageenan are arising as potential therapies for enhancing cartilage regeneration.

Stem cell-based embryo models have taken the scientific community by storm as they enable investigation of previously inaccessible stages of human development. Here, we discuss how stem cell-based embryo and placenta models can shine a light on two elusive and intertwined aspects of human development that are critical for successful pregnancy: the implantation of the blastocyst into the endometrium and the subsequent invasion of placental villi deep inside the maternal tissues. Both of these processes are mediated by the trophoblast lineage, which is specified in the preimplantation embryo and can be modeled using naïve pluripotent stem cells. We review how embryo and placenta models built from naïve stem cells can be leveraged to obtain mechanistic insights into human implantation and trophoblast invasion.

Allergic asthma (AA) is a prevalent chronic respiratory disease characterized by airway hyperresponsiveness (AHR) and chronic inflammation, significantly impairing patients' quality of life.

Calreticulin (CALR) is primarily an endoplasmic reticulum chaperone protein that also plays a key role in facilitating programmed cell removal (PrCR) by acting as an "eat-me" signal for macrophages, directing their recognition and engulfment of dying, diseased, or unwanted cells. Recent findings have demonstrated that macrophages can transfer their own CALR onto exposed asialoglycans on target cells, marking them for PrCR. Despite the critical role CALR plays in this process, the molecular mechanisms behind its secretion by macrophages and the formation of binding sites on target cells remain unclear. Our findings show that CALR undergoes C-terminal cleavage upon secretion, producing a truncated form that functions as the active eat-me signal detectable on target cells. We identify cathepsins as potential proteases involved in this cleavage process. Furthermore, we demonstrate that macrophages release neuraminidases, which modify the surface of target cells and facilitate CALR binding. These insights reveal a coordinated mechanism through which lipopolysaccharide (LPS)-activated macrophages regulate CALR cleavage and neuraminidase activity to mark target cells for PrCR. How they recognize the cells to be targeted remains unknown.

Z-DNA, a left-handed DNA conformation, plays critical roles in transcriptional regulation, genetic recombination, genomic instability, immunity, and human diseases. In 2019, a stable LR-chimera containing Z-DNA (Lk = 0) under physiological ionic conditions was prepared by hybridizing two complementary circular ssDNAs. However, the difficulty in preparing circular ssDNA precursors and the excessively long Z-DNA segment in the chimera limit its applications. In this study, using a splint-free circularization method, we prepared two circular ssDNAs (each with a hairpin structure). Hybridization of these two circles whose loops are complementary (but not the two hairpins) yielded a Stem-LR chimera containing short Z-DNA and B-DNA and two hairpins that could not hybridize with each other. Stability analysis revealed that the 18-34 bp Z-DNA segment with only unmodified nucleotides in the Stem-LR chimera remained stable under physiological conditions (10 mM Mg2+, 37 °C). When hairpins were far apart (180°), multiple Stem-LR chimera isomers (varying in B-Z junction numbers and Z-DNA lengths) formed. Intriguingly, higher hybridization temperatures (60 °C) favored continuous B-DNA and Z-DNA segments (minimal B-Z junctions). When hairpins were adjacent (0° orientation), exclusively continuous B-DNA/Z-DNA was obtained, even for hybridization at 10 °C. As expected, Stem-LR chimeras exhibited enhanced resistance to topoisomerase I compared to chimeras without hairpins. This approach holds promise for delivery into cells or organisms to investigate the impact of Z-DNA and its biological functions under physiological conditions.