Specification of primordial germ cells (PGCs) establishes germline development during early embryogenesis, yet the underlying mechanisms in humans remain largely unknown. Here, we reveal the functional roles of germline-specific RNA-binding protein (RBP) DND1 in human PGC (hPGC) specification. We discovered that DND1 forms a complex with another RBP, NANOS3, to restrict hPGC specification. Furthermore, by analyzing the mRNAs bound by DND1 and NANOS3, we found that DND1 facilitates the binding of NANOS3 to hPGC-like cells-related mRNAs. We identified SOX4 mRNAs as the key downstream factor for the DND1 and NANOS3 complex. Mechanistically, DND1 and NANOS3 function in processing bodies (P-bodies) to repress the translation of SOX4 mRNAs, with NANOS3 mediating the interaction between DND1 and the translational repressor 4E-T. Altogether, these findings identify the RBP complex formed by DND1 and NANOS3 functioning as a "braking system" to restrict the entry of germ cell fate in humans.

Genotoxic stress-induced stem cell maldifferentiation (GSMD) integrates DNA damage responses with loss of stemness and lineage-specific differentiation to prevent damaged stem cell propagation. However, molecular mechanisms governing GSMD remain unclear. Here, we identify the p53-induced long non-coding RNA LOC644656 as a key regulator of GSMD in human embryonic stem cells. LOC644656 accumulates in the nucleus upon DNA damage, disrupting pluripotency by interacting directly with POU5F1 and KDM1A/LSD1-NuRD complexes, repressing stemness genes, and activating TGF-β signaling. Additionally, LOC644656 mitigates DNA damage by binding DNA-PKcs and modulating the DNA damage response. In cancer, elevated LOC644656 correlates with poor patient survival and enhanced chemoresistance. Our findings demonstrate that LOC644656 mediates stemness suppression and resistance to genotoxic stress by coordinating DNA damage signaling and differentiation pathways. Thus, LOC644656 represents a potential therapeutic target for overcoming chemoresistance and advancing stem cell biology.

Acute myeloid leukemia (AML) is an aggressive hematological cancer with a 70% five-year mortality rate. Relapse occurs in approximately half of adults treated with intensive chemotherapy, while responses to targeted therapies are short-lasting. Frequent mutations in signaling pathways, such as FLT3 tyrosine kinase and RAS, lead to dysregulated mammalian target of rapamycin complex 1 (mTORC1)and mitogen-activated protein kinase (MAPK) signaling, increased protein synthesis, enhanced mitochondrial fitness, and metabolic adaptations that drive leukemic cell proliferation and survival. Here, emerging evidence supporting the unique role of eukaryotic initiation factor 4F as a key driver of the expression of proteins regulating leukemic cell metabolism and survival and the potential therapeutic benefit of targeting this pathway pharmacologically in AML are discussed.

The dysregulation of the inflammatory microenvironment following tendon injury significantly hinders regeneration. In this study, we developed an all-silk-derived functional scaffold (rKL@MPs-ASF) by integrating silk fibroin (SF) microspheres (MPs) loaded with the anti-inflammatory protein recombinant α-Klotho (rKL) into a biomimetic aligned SF (ASF) scaffold. This scaffold is designed to regulate the inflammatory microenvironment and facilitate tendon regeneration. Proteomic analysis revealed that rKL preserves the tenogenic differentiation potential of tendon stem/progenitor cells (TSPCs) by mitigating the oxidative stress response in a tumor necrosis factor-alpha-induced inflammatory microenvironment in vitro. The rKL@MPs-ASF scaffold demonstrated good drug loading/release capabilities and biocompatibility in vitro. In a rat full-thickness Achilles tendon defect model, the rKL@MPs-ASF scaffold reduced inflammatory cells infiltration and promoted fibroblast infiltration compared to the PBS@MPs-ASF group at 4 weeks post-operation. At 8 weeks post-operation, rKL@MPs-ASF-treated tendons showed increased collagen fiber deposition and reduced heterogeneous ossification, facilitating tendon regeneration and functional recovery. In conclusion, this all-silk-derived functional system creates a conducive microenvironment for tendon regeneration. STATEMENT OF SIGNIFICANCE: Regulation of the inflammatory microenvironment plays a crucial role in modulating the differentiation of TSPCs, thereby promoting tendon tissue regeneration. In this study, we demonstrate that rKL effectively preserves the tenogenic differentiation potential of TSPCs by mitigating oxidative stress within an inflammatory microenvironment. We developed an innovative, all-silk-based functional system (rKL@MPs-ASF), which integrates rKL-loaded SF MPs into an aligned silk fibroin scaffold. This system enables the controlled release of rKL, thereby modulating inflammation, promoting collagen fiber deposition, inhibiting heterotopic ossification, and ultimately improving tendon regeneration and functional recovery. Our findings highlight the potential of the rKL@MPs-ASF system, which combines structural and biological properties with a versatile drug-delivery platform, as a promising strategy for enhancing tendon repair and regenerative outcomes.

Decellularized extracellular matrix derived from specific organs represents a promising platform for organoid development, offering distinct advantages in tissue engineering. This matrix maintains the complex three-dimensional network of biological macromolecules secreted by tissue-specific cells, including collagen, glycoproteins, and proteoglycans. This extracellular matrix orchestrates cellular behaviors, such as proliferation, migration, and differentiation, while maintaining optimal tissue homeostasis. The organ-specific composition of decellularized extracellular matrix preserves native biological cues, including growth factors and cytokines, as well as mechanical properties, facilitating natural cell-matrix interactions and promoting appropriate stem cell development. These characteristics make organ-derived decellularized extracellular matrix an ideal scaffold for organoid construction. The implementation of decellularized extracellular matrix enhances the physiological relevance of organoid models, which is particularly valuable in drug development, personalized medicine, and the study of complex organ microenvironments. This advancement significantly improves the translational potential of organoid technology for organ transplantation while providing robust research tools. Consequently, decellularized extracellular matrix-based organoid models offer superior platforms for preclinical therapeutic evaluation. This review examines recent progress in decellularized extracellular matrix-based organoid development, beginning with current application strategies and proceeding to an analysis of existing decellularized extracellular matrix-derived organoid models.

Degenerative disc disease (DDD) is a significant cause of chronic low back pain, often leading to disability and high healthcare costs. Current treatments, including physical therapy, pain management, and surgical interventions such as spinal fusion and total disc replacement, do not reverse degeneration. Bioactive therapies offer a potential alternative by targeting the underlying degenerative process. Cell-based therapies, including the use of mesenchymal stem cells and platelet-rich plasma, aim to restore disc structure and function by promoting extracellular matrix production and reducing inflammation. Early studies show potential benefits in pain relief and disc regeneration, but long-term efficacy remains unclear. Nucleus pulposus augmentation and replacement strategies, such as the use of hydrogel implants and in situ curing polymers, are aimed at restoring disc height and biomechanical function. While these strategies are promising, issues such as implant durability and migration require further study. Total disc replacement preserves motion and avoids adjacent-segment disease, but outcomes depend on patient selection and implant design. Despite encouraging results, bioactive therapies still require research to establish long-term safety and effectiveness. Advancements in biomaterials, patient selection criteria, and clinical trials will determine their role in the future management of DDD.

Interactions between the liver and pancreas are key features of the carbohydrate and lipid homeostasis in healthy and pathological patients. To investigate the crosstalk between the two organs, we have developed an organ-on-chip coculture model derived from human induced pluripotent stem cells. The presence of pancreatic-derived tissue in the culture environment contributed to increase the CYP3A4 activity, the glycogen storage, and the expression of genes related to lipids, bile acids and sterol metabolism in the liver derived tissue. Concomitantly, the presence of liver cells led to increase the C-peptide secretion in pancreas. The coculture with liver modulated the pancreatic differentiation by increasing the activity of important transcription factors (REST, MAFB, PBX1) and by downregulating several hormone encoding genes (INS, GCG, TTR). The liver also stimulated the expression of genes involved in the response to inflammation in pancreas (via TGFβ/SMAD pathway). In parallel we observed a pancreatic cell reorganization coupled with the activation of the cell proliferation related transcription factor (SCRT1) and the upregulation of cellular remodeling genes (FLNA, FLNB, FN1, COL4A5). Finally, the pancreatic lipid genes were also upregulated in presence of the liver tissue. Overall, our results reflect a complex synergy between both tissues. We believe that those results are an encouraging step toward the development of relevant human model using advanced organ on chip technology and stem cells sources.

Male germline development is crucial for the proper establishment of spermatogonial stem cell pool and life-long production of spermatozoa, but the full-term developmental profiling of human male germline is not fully understood. Here, by integrating 92,488 human testicular cells spanning from six-week-old embryo to old men, we constructed a comprehensive human male germ cell atlas. Further analysis found that the precursor of undifferentiated spermatogonia underwent regulatory network reconfiguration starting from week 7 post-fertilization, accompanied by WNT6-FZD3/LRP6-JUN/MYC signaling axis. And JUN and MYC were revealed to be candidate core transcription factors that might inhibit spermatogonia differentiation. In addition, the activation of ANGPTL signaling played a role in the maintenance of human spermatogonial stem cell. Finally, by interrogating the scRNA-seq datasets from several types of idiopathic non-obstructive azoospermia (iNOA) patients, we identified several iNOA-dysregulated genes such as CAPN3, FTMT, IZUMO2 and LACE1, which were significantly down-regulated in round spermatids of iNOA patients. Collectively, our work constructed a comprehensive human male germ cell development atlas, revealing the factors that might regulate male germline development and providing iNOA-dysregulated genes for future clinical diagnosis.

In regenerative medicine, addressing the complex challenge of bone tissue regeneration demands innovative strategies. Exosomes, nanoscale vesicles rich in bioactive molecules, have shown great promise in tissue repair. This study focuses on exosomes derived from mineralized osteoblasts (MOBs), which play a pivotal role in bone formation. We investigated the therapeutic potential of exosomes isolated from osteoblasts cultured in osteogenic medium for 21 days, delivered via 3D-printed gyroid scaffolds composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). The exosomes were characterized through nanoparticle tracking analysis to determine size, morphology, and concentration, while proteomics revealed their cargo contents. In vitro, rabbit bone marrow mesenchymal stromal cells (rBMSCs) were cultured as monolayers and within ceramic scaffolds, where MOB-derived exosomes were shown to promote osteogenic differentiation. In vivo, their osteoconductive and bone augmentation capabilities were evaluated in two rabbit calvarial models, while the osteoinductive potential was further tested in a heterotopic mouse model. Neo-bone formation was assessed using µCT and histological analysis. Our findings demonstrated that MOB-derived exosomes upregulated bone-related gene expression and promoted mineralization in rBMSCs, even in the absence of osteogenic medium. Proteomics confirmed the presence of bone-associated proteins in these exosomes. In rabbit models, however, exosomes did not significantly enhance bone formation. In contrast, in the heterotopic mouse model, exosomes functionalized onto ceramic scaffolds exhibited strong osteoinductive activity. This study highlights the potential of MOB-derived exosomes to enhance 3D-printed ceramic scaffolds for bone regeneration, offering a promising avenue for bone healing without the need for additional growth factors or stem cells. STATEMENT OF SIGNIFICANCE: The here presented report of our project not only advances our understanding of the role of exosome-functionalized scaffolds in bone regeneration but also proposes a promising alternative to traditional growth factor- or cell-based approaches. We are confident that this study represents a novel and impactful contribution to the field.

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by aggressive proliferation and chemoresistance, leading to poor patient outcomes. Despite advances in chemotherapy, resistance mechanisms remain inadequately understood, particularly at the cellular and molecular level.

Proline-based cyclic dipeptides (CDPs) from lactic acid bacteria are stereochemically diverse molecules, possessing oral bioavailability and significant pharmacological potential. Herein, we developed a robust two-step ion-exchange purification platform to comprehensively isolate 16 structurally defined CDPs from 82-h culture filtrates (CFs) of Lactobacillus plantarum LBP-K10. Utilizing cation (Amberlite IRA-120) and anion (Amberlite IRA-67) exchange chromatography, we generated the K10-CCDP-IV fraction from 82-h acetate membrane-filtered CFs, followed by CH2Cl2 extraction to obtain K10-CCDP-IV-MC. Additional test agents included LBP-K10-CF (CH2Cl2-unextracted CF), LBP-K10-MC (CH2Cl2-extracted CF), and a single cis-cyclo(L-Phe-L-Pro). LC-MS-linked HPLC-based time-course quantification revealed peaks in total CDP concentration at 82 h, with notable increases in fractions F6, F7, F12, F16, and F17. In vitro studies using MDA-MB-231 breast cancer cells demonstrated that both K10-CCDP-IV-MC and LBP-K10-MC significantly suppressed cell proliferation by inducing G1-phase arrest and mitochondria-mediated apoptosis, as evidenced by the increased expression of cytochrome c, cleaved caspase-3, and BAD, along with the downregulation of Bcl-2. Furthermore, both treatments inhibited cancer stem cell characteristics, including a reduction in the CD133+ subpopulation, repression of Oct4, and inhibition of sphere formation. In vivo, oral administration of LBP-K10-MC in xenograft-bearing SCID mice resulted in a significant reduction in tumor volume without systemic toxicity or adverse effects. These findings underscore the therapeutic relevance and preclinical validation of CDP-based consortia as orally deliverable, bioavailable, and multifunctional anticancer agents derived from a single probiotics. This supports their translational potential in dietary and therapeutic applications, offering a scalable, food-grade platform for the production of functional CDPs targeting breast cancer stemness.

Retinal ganglion cell degeneration is the main cause of irreversible vision loss in optic neuropathies. Pigment epithelium-derived factor (PEDF) and its smaller peptide components (44-mer and 17-mer) have shown neuroprotective effects. In this study, using a stepwise protocol we investigated their effects on human-induced pluripotent stem cell differentiation to retinal ganglion cells. Various concentrations of PEDF, 44-mer and 17-mer were added at day 18. Investigated compounds significantly upregulated the expression of retinal ganglion cells-specific (Brn3b, Sncg), retinal progenitor (Pax6) and neuroaxonal markers (Tau, NFH). They also highly increased Brn3b expression, as well as neurite length and density, supporting their neurotrophic properties. Our findings suggest that PEDF and its smaller peptide components, 44-mer and 17-mer, can be suggested as neuroprotective agentd for the promotion of retinal ganglion cell differentiation from human-induced pluripotent stem cells. 44-mer and 17-mer have comparable or even higher effects to full-length PEDF and might also bypass PEDF usage limitations.

Glaucomatous optic neuropathy represents a prevalent optic nerve degenerative disease. Neuroinflammation is recognized as a significant mechanism underlying optic nerve damage in glaucoma; however, the precise mechanisms driving neuroinflammation remain largely elusive. Existing studies have indicated that microglia-driven neuroinflammation is pivotal for neuroinflammation onset and progression. Mitochondrial dysfunction, encompassing mitochondrial DNA (mtDNA) damage, metabolic deficiencies, and quality control impairments, is upstream of microglial activation and neuroinflammation. Thus, a deeper comprehension of the link between mitochondrial dysfunction and microglial activation in glaucoma may provide valuable insights into the underlying pathogenesis. As a result of these findings, promising avenues for developing effective interventions to mitigate optic nerve damage and preserve visual function in glaucoma patients have been identified.

The human brain has a very limited capacity for self-repair, presenting significant challenges in recovery following injuries such as ischemic stroke. Stem cell-based therapies have emerged as promising strategies to enhance post-stroke recovery. Building on a large body of preclinical evidence, clinical trials are currently ongoing to prove the efficacy of stem cell therapy in stroke patients. However, the mechanisms through which stem cell grafts promote neural repair remain incompletely understood. Key questions include whether these effects are primarily driven by (1) the secretion of trophic factors that stimulate endogenous repair processes, (2) direct neural cell replacement, or (3) a combination of both mechanisms. This review explores the latest advancements in neural stem cell therapy for stroke, highlighting research insights in brain repair mechanisms. Deciphering the fundamental mechanisms underlying stem cell-mediated brain regeneration holds the potential to refine therapeutic strategies and advance treatments for a range of neurological disorders.

The transcriptional regulation underlying eye field (retinal primordium) development requires precise control, yet the mechanisms guiding lineage-specific differentiation within the central nervous system (CNS) remain incompletely understood. Using neuroectoderm (NE) organoids derived from mouse embryonic stem cells, we investigate the role of PRDM13 in eye field specification. We demonstrate that Prdm13 expression inhibits RX+ eye field fate but permits non-eye field NE differentiation, an effect that depends on its first and second zinc-finger domains. Prdm13 activates the WNT/β-catenin signaling pathway during differentiation, leading to downregulation of key transcription factors crucial for establishing the eye field. Pharmacological inhibition of WNT signaling abolishes PRDM13-mediated suppression, restoring RX+ eye field differentiation. Our work reveals a previously undescribed PRDM13-WNT signaling axis that regulates lineage-specific neural differentiation of embryonic stem cells.

Human pluripotent stem cells (hPSCs) maintain diploid populations for generations despite frequent mitotic errors that cause aneuploidy or chromosome imbalances. Consequently, aneuploid hPSC propagation must be prevented to sustain genome stability, but how this is achieved is unknown. Surprisingly, we find that, unlike somatic cells, uniformly aneuploid hPSC populations with heterogeneous abnormal karyotypes proliferate. Instead, in mosaic populations, cell-non-autonomous competition between neighboring diploid and aneuploid hPSCs eliminates less fit aneuploid cells, regardless of specific chromosome imbalances. Aneuploid hPSCs with lower MYC or higher p53 levels relative to diploid neighbors are outcompeted but conversely gain an advantage when MYC and p53 relative abundance switches. Thus, MYC- and p53-driven cell competition preserves hPSC genome integrity despite their low mitotic fidelity and intrinsic capacity to proliferate with an aneuploid genome. These findings have important implications for using hPSCs in regenerative medicine and for how diploid human embryos form during development despite the prevalence of aneuploidy.

Ceiling culture-derived preadipocytes (ccdPAs) are fibroblast-like cells believed to originate from mature adipocytes after the loss of lipid droplets. Unlike adipose-derived stem cells (ASCs), which are heterogeneous, ccdPAs are considered a more homogeneous population. However, their response to adipogenic differentiation stimuli at the single-cell level has not been fully characterized. In this study, we performed single-cell RNA sequencing (scRNA-seq) and epigenetic analyses to investigate early transcriptomic changes following adipogenic induction in ASCs and ccdPAs. scRNA-seq revealed that, in addition to preadipocytes/adipocytes, a substantial population of regulatory or structural cells, characterized by the expression of genes associated with extracellular matrix organization, structural support, and cell-cell interactions, such as F3 and MGP, emerged in both ASCs and ccdPAs. Notably, ATOH8, a transcription factor with limited prior characterization in adipogenesis, showed markedly higher expression in ccdPAs than in ASCs. This was supported by epigenetic analyses demonstrating lower CpG methylation and higher H3K4me3 levels at the ATOH8 locus in ccdPAs. Our findings suggest that adipogenic induction of ccdPAs generates diverse cell populations. This study provides the first single-cell level insight into the adipogenic response of primary cultured human ccdPAs.

Preeminent human existence in space raises concerns about bone health due to the effect of microgravity on bone tissue degeneration. Space experiments pose logistical challenges, but ground-based research using microgravity simulation provides information about bone loss mechanisms. This review compiles and evaluates data from astronaut, animal, and cellular experiments, emphasizing microgravity-induced skeletal deconditioning. These findings contribute to creating treatment approaches for preventing bone loss risks in space and potentially on Earth. Astronauts experience notable bone loss, up to 1 %-2 % per month in a gravity-less environment, predominantly influencing weight-bearing bones. These necessitate finding efficient treatment approaches for preventing bone loss risks in space and potentially on Earth. There is a significant need to investigate microgravity's impact on various bone compartments and skeletal recovery processes. The current review explores the stages of bone remodeling and the fundamental causes of bone loss in microgravity, including effects on osteoblasts, osteocytes, osteoclasts, hematopoietic stem cells, and bone marrow stromal cells, as well as the impact on calcium levels. The article also explores various treatment options, including general management, recent therapies, supportive therapies, and emerging therapies such as BP-NELL-PEG, melatonin, calcitonin, and molecular therapies, highlighting their therapeutic potential against microgravity-induced bone loss.

Chromophobe renal cell carcinoma (ChRCC) is the third most common type of RCC. There are no proven therapies for patients with metastatic ChRCC, with a median survival of 27 months. KIT (CD117) is a membrane-associated tyrosine kinase receptor. Antibody-drug conjugates (ADC) targeting KIT were previously found to be safe and effective in preclinical models of KIT-positive cancers but have not been tested in ChRCC.

The inability of most human organs to regenerate themselves after injury underlies the lifelong morbidity of numerous diseases. As we continue to seek solutions for these intractable conditions, the liver emerges as an inspiring and informative exception. The liver is the only solid organ that can completely regenerate itself. At the core of this extraordinary feat of organ physiology lie two equally exceptional features of cell biology. First, liver regeneration is driven not by stem cells, but rather by the proliferation of the liver's differentiated cells. Second, many of these liver cells are polyploid, yet still able to execute proper cell division. Understanding how liver cells maintain proliferative capacity as differentiated cells and how they execute mitosis faithfully in a polyploid state could offer powerful insights toward engineering regenerative capacity in other organs. The liver thus offers not only proof that mammalian organ regeneration is possible, but also a blueprint for achieving this long-standing goal of regenerative medicine.