Aging results in a decline in cellular and molecular functions. One of the hallmarks of aging is stem cell exhaustion which impacts self-renewal and differentiation. We employ the Drosophila larval lymph gland (LG) to investigate the impact of genetic perturbation of cellular homeostasis on hematopoiesis. LG consists of a Posterior Signalling Center (PSC) - a stem cell niche that maintains Medullary Zone (MZ) prohemocytes whereas Cortical Zone (CZ) consists of differentiated hemocytes. We employ over-activation of Toll or Imd pathway for disrupting cellular homeostasis whereas over-express Foxo or Atg8 for balancing it. Genetic perturbation of cellular homeostasis displays hallmarks of aging. Induction of Toll or Imd pathway locally and systemically leads to a decreased niche size and increased differentiation whereas Foxo or Atg8 over-expression shows an opposite trend. We show that the Integrated Stress Response (ISR) pathway is induced upon Toll or Imd over-activation and LG's with ISR perturbation show increased hemocyte differentiation. Genetic epistasis shows that ectopic over-expression of ISR components upon Imd activation can rescue hematopoietic defects. Overall, our study explores how genetic perturbation of cellular homeostasis can impact hematopoiesis. Our research has implications in understanding how abrogation of cellular homeostatic mechanisms may lead to onset of malignancies.

Quantification of cellular lipids in a reproducible and high-throughput manner is a key step in the development of therapeutics for lipid storage diseases. Niemann-Pick Disease Type C (NPC) is a genetic disorder characterized by the accumulation of unesterified cholesterol in late endosomes/lysosomes, which is usually measured by the filipin fluorescence assay. However, the nonspecific binding of filipin to other sterol derivatives, multiple assay steps, and difficulty in quantitation present limitations for high-throughput screening and accurate cellular cholesterol quantification. We report the development of an integrated and semiautomated protocol to extract and quantify cellular cholesterol in 384-well plates by utilizing a liquid handling platform in conjunction with a high-throughput mass spectrometry (MS) system. The 384-well plate format enables seamless lipid extraction and subsequent MS analysis in less than 2 h from a cell culture plate to final MS data. Cholesterol was extracted from neural stem cells differentiated from NPC induced pluripotent stem cells using methyl tert-butyl ether (MTBE), with 13C-cholesterol serving as an internal standard for quantification and normalization of native cholesterol. This integrated platform showed excellent quantification linearity and reproducibility (intraday and interday, R2 > 0.99) with a recovery rate between 83 and 107%. We employed this integrated platform to screen a collection of 241 investigational compounds at seven concentrations each, benchmarking the method as an efficient, label-free cellular cholesterol quantification assay for high-throughput applications. Furthermore, we demonstrated the capability to multiplex extraction and quantification of sphingosine/cholesterol in a single MS run, extending the applicability of this integrated workflow to other lipid storage diseases.

Avascular necrosis (AVN) of the hip is a disease characterized by vascular disruptions, where 67% of untreated cases may lead to the collapse of the femoral head. None of the current approaches, such as core decompression (CD), vascularized bone grafting, osteotomy, tissue implantation, and other methods, have been proven fully effective in delaying the progression of osteonecrosis. Recent findings indicate that bone marrow stem cells (BMSCs) have significant potential to regenerate necrotic tissue and prevent the progression of AVN in the hip. This study aims to evaluate the efficacy, side effects, treatment failure rate, most effective treatment stage of AVN hip, and application technique to treat AVN hip.

We conducted a prospective randomized clinical trial to investigate the combination of post-transplant cyclophosphamide (PTCy) and abatacept for GVHD prophylaxis. Patients with hematologic malignancies undergoing an allogeneic transplant from an 8/8 matched related or unrelated donor were randomized 1:1 to tacrolimus and methotrexate (SOC arm) or PTCy on days +3 and +4 followed by abatacept on an extended schedule: days +5, +14, +28 and every 4 weeks up to D+168 (PTCy+aba arm). All patients received peripherally collected stem cells. The primary endpoint was moderate and severe chronic GVHD at one year. Following Food and Drug Administration (FDA) approval of abatacept for GVHD prophylaxis leading to change in SOC, we amended our trial and subsequently only enrolled onto the PTCy+aba arm. 25 patients were treated on PTCy+aba and 15 on SOC arm. The trial met its primary endpoint: Kaplan-Meier estimates of moderate/severe chronic GVHD were 0% on the PTCy+aba and 65.8% on the SOC arm (p < 0.0001). GVHD-Free, Relapse-Free Survival (GRFS) was 62.5% on PTCy+aba and 24.1% on SOC (p=0.010). There were no treatment related deaths on PTCy+aba and two on SOC. There was no difference in the overall survival rates (92% aba, 80% SOC, p=0.28), disease free survival (68% aba, 92.9% SOC, p=0.105) and infection at one year. Grade III/IV acute GVHD rate was 4.2% on PTCy+aba and 21.4% on SOC (p=0.092). Proliferation of regulatory T-cells was preserved on PTCy+aba compared to SOC and PTCy and abatacept treatment was associated with increased CD16+CD56dim cytotoxic NK cells. In conclusion, the combination of PTCy and abatacept is well tolerated and associated with reduced chronic GVHD and a favorable GRFS rate. This trial was registered at www.ClinicalTrials.gov as #NCT03680092.

Multipotent mesenchymal stromal cells are progenitors of the bone marrow stromal microenvironment that support hematopoiesis. Mitochondria, which can be transferred between cells via nanotubes or extracellular vesicles, play a key role in the functions of mesenchymal stromal cells. In a murine model, donor hematopoietic stem and progenitor cells transfer functional mitochondria to bone marrow mesenchymal stromal cells of the recipient. The aim of this study was to find out whether such transfer occurs in humans after allogeneic hematopoietic stem cell transplantation.

Approximately 7 % of men are suffering from infertility, accounting for 40 %-50 % of all cases of all infertility, and low testosterone levels are closely associated with male infertility. Our previous study indicated that transcriptional repressor GATA binding 1 (Trps1) could inhibit testosterone synthesis in Leydig cells. In the present study, to elucidate the molecular mechanism by which Trps1 regulates testosterone synthesis in Leydig cells, differentially expressed genes (DEGs) following Trps1 knockdown was conducted by RNA-sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Genes ontology (GO) analysis were utilized to investigate the pathways associated with Trps1. Gene-silencing technology, Real-Time PCR, and Western blot were performed to validate DEGs. In addition, testosterone and cellular cholesterol content were further observed. We found that GO analysis of DEGs associated with "cholesterol metabolism". Real-Time PCR and Western blot showed that the expression of steroidogenic factor-1 (Sf-1) and scavenger receptor class B, member 1 (Scarb1) were up-regulated after Trps1 silencing. Knockdown of Sf-1 and Scarb1 could revert the elevated testosterone and cellular cholesterol levels caused by Trps1 deficiency. Moreover, ChIP-seq and CUT&Tag-qPCR demonstrated that the promoter of Scarb1 has a binding site for SF-1. The present study revealed that TRPS1 exerts regulatory control over the expression of Scarb1 by modulating Sf-1 transcriptional activity, thereby enhancing cholesterol level and promoting the synthesis of testosterone in Leydig cells.

Diabetes mellitus may adversely affect the regenerative capacities of tissues, including skin healing and bone health (osteoporosis). Dipeptidyl peptidase-4 (DPP4) inhibitors (DPP4i) are antidiabetic drugs that increase the half-life of incretins, improving glucose levels. Several proteins that regulate important physiological processes are also substrates of DPP4. Therefore, DPP4i may have multiple effects. The aim of this work was to study if DPP4i sitagliptin affects regenerative capacities in a diabetic animal model. Thus, its effects on skin wound healing were evaluated. Likewise, if it produces changes in serum composition that might affect the viability and differentiation of endothelial cells and mesenchymal stem cells (MSC).

Caveolins (CAV), a family of integral membrane proteins, are involved in regulating stem cell fate, which are critical for tissue repair and regeneration. Drawing from scientometric studies and comprehensive research, this review investigates the mechanisms by which CAV regulates stem cell fate can improve the efficiency and accuracy of stem cell therapy in treating chronic degenerative diseases (CDD). For instance, CAV1 inhibits neuronal differentiation of neural stem/progenitor cells (NSCs/NPCs) by downregulating VEGF, p44/42MAPK phosphorylation and NeuroD1 signaling pathway following ischemic stroke, while CAV3 interacts with MG53 to enhance the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) in diabetic wound healing by activating the eNOS/NO signaling pathway.

The placenta serves as a vital interface for fetal-maternal exchange, relying on trophoblast differentiation for development. This process involves cytotrophoblasts (CTBs) transitioning into syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs), driving placental maturation. Focal adhesion kinase (FAK), a key cytoplasmic tyrosine kinase, regulates cellular processes such as proliferation, survival, and signaling. However, its role in trophoblast differentiation and metabolism remains unclear. Here, using human trophoblast stem cells (hTSCs) and trophoblast cell lines (BeWo, HTR8/SVneo), we investigated FAK signaling in trophoblast lineage differentiation. Inhibiting the FAK signaling pathway suppresses MAPK pathway activity, reduces glycolytic metabolism and impairs trophoblast syncytialization. Additionally, blocking FAK with the inhibitor Defactinib disrupts EVTs cytoskeletons and impairs migration, invasion, and differentiation potential. Notably, reduced FAK signaling is observed in patients with recurrent spontaneous abortion (RSA), suggesting a role in RSA pathogenesis. Our findings highlight FAK as a pivotal regulator of trophoblast lineage development, linking it to placental function and RSA. This study offers new insights into placental disorders and potential therapeutic targets.

The transdifferentiation of alveolar epithelial type II cells (AECIIs) to alveolar epithelial type I cells (AECIs) plays an important role in the epithelial repair in acute lung injury (ALI). Although transitional state cells have been reported to regenerate the alveolar epithelium surface and promote a repair process, the treatment of ALI based on transitional state cells has not been suggested. Here, we demonstrate that nebulized mesenchymal stem cell exosomes (MSC exosomes) can be used for ALI and MSC exosomes have the ability to promote the differentiation of transitional state cells into AECIs by regulating the STAT3/Krt8/AQP5 axis. In the in vivo study, immunohistochemistry and immunofluorescence staining results showed that MSC exosomes could reduce the expression of transitional state cells and promote transdifferentiation of AECIIs. In the in vitro study, western blotting (WB) results showed that MSC exosomes downregulated signal transducer and activator of transcription 3 (STAT3) phosphorylation and the expression of transitional state cell specific keratin 8 (Krt8), and upregulated the expression of AECIs specific aquaporin 5 (AQP5). The results indicate that MSC exosomes inhibit STAT3 phosphorylation through STAT3/Krt8/AQP5 axis, promote the transdifferentiation of transitional state cells to AECIs and accelerate the regeneration of alveolar epithelium after LPS-induced lung injury. Regulating transitional state cells to alleviate ALI is suggested for the first time.

Liver and peritoneum are the most common metastatic organs in colorectal cancer. However, the mechanisms of metastatic organotropism remain unexplored. Here we used single-cell RNA-sequencing to reveal the intermediate EMT state of tumor cells from colorectal cancer liver metastasis (CRLM) and colorectal cancer peritoneum metastasis (CRPM). There was a significant heterogeneity in expression profiles across multiple cell types in the tumor microenvironment between two groups. Although both groups of tumor cells obtained the activation of EMT, they were in different partial-EMT (pEMT) states and expressed distinct pEMT markers. Tumor cells in CRLM group were more in an epithelial-biased pEMT state, while a higher proportion of cells in CRPM group was in a mesenchymal-biased pEMT state. In addition, we observed differentially infiltrated myeloid and fibroblast sub-clusters, rendering the peritoneal metastasis group a more prominent inflammatory stimulus. Our study provides a comprehensive resource at single-cell level for in-depth exploration of metastatic organotropism in CRC.

Spinal cord injury (SCI) is a severe neurological and pathological disorder, but there are few effective treatments that can significantly promote functional recovery after SCI. Neural stem cell (NSC) transplantation therapy is considered a promising approach to repair neural connection and enhance functional recovery. However, a series of pathological changes at the injury site make the microenvironment unfavorable for NSC neuronal differentiation. In this study, black phosphorus nanosheets (BPNSs), a kind of nanomaterial which degraded into phosphate anions, exhibited good biocompatibility and effective function on regulating NSC differentiation. In vitro, BPNSs can promote neuronal differentiation of NSC by upregulating the p53 signaling pathway via activating membrane receptors and intracellular receptors mediated by its adhesin and cell pinocytosis. In vivo, BPNSs-treated NSCs transplantation could promote neural regeneration and functional recovery effectively. In conclusion, our results suggest that BPNSs have the potential to be a nanomedical strategy for the repair of SCI. STATEMENT OF SIGNIFICANCE: Black phosphorus nanosheets (BPNSs), which are composed of the single chemical element phosphorus that is a fundamental component of biological systems, exhibit good biocompatibility, as they predominantly degrade into phosphate anions. BPNSs are quickly internalized by neural stem cells (NSCs) within 6 hours and can promote NSC neuronal differentiation by upregulating the p53 signaling pathway. Transplantation of NSCs pre-treated with BPNSs effectively promotes nerve regeneration and facilitates significant functional recovery following spinal cord injury.

Dry eye disease negatively impacts the quality of life of many patients worldwide, and currently, there is no cure. Stem cell therapy may offer a potential new treatment option. Mesenchymal stem cells (MSCs)-derived exosomes have similar effects as MSCs but with fewer side effects. This study investigated the effects and mechanisms of human umbilical cord mesenchymal stem cells (HUCMSCs)-derived exosomes in cell and mouse models of dry eye disease. Exosomes were isolated from HUCMSCs (HUCMSCs-EXO) and characterized by measuring surface markers. Human corneal epithelial cells (HCECs) were cultured in hypertonic (500 mOsm) or isotonic medium (310 mOsm) and treated with HUCMSC-EXO or PBS. A mouse model of dry eye disease was generated by treating mice with benzalkonium chloride and confirmed by measuring tear secretion and performing H&E staining. Cell viability, apoptosis, RNA, and protein expression levels were assessed using CCK-8 assay, TUNEL staining, qPCR, and Western blotting. The regulation of SQSTM1 expression by miR-146a was evaluated using a dual luciferase reporting assay. Exposure to hyperosmotic pressure decreased cell viability, increased apoptosis and inflammation, decreased miR-146a expression, and increased SQSTM1 expression in HCECs. Treatment with HUCMSCs-EXO alleviated the effects of hyperosmotic pressure on inflammation and apoptosis. Overexpression of miR-146a increased cell viability and inhibited apoptosis and inflammation, suggesting that miR-146a mediates the beneficial effects of HUCMSCs-EXO. These findings were validated in the mouse model of dry eye disease. Further experiments revealed that miR-146a targets and promotes SQSTM1 expression. Overexpression of SQSTM1 increased cell viability and inhibited apoptosis and inflammation in HCECs. In conclusion, this study demonstrated that HUCMSCs-derived exosomal miR-146a targets SQSTM1 and promotes its expression, resulting in the alleviation of inflammation and apoptosis in cell and mouse models of dry eye disease.

Androgen receptor positive prostate cancer (PC), castration resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) invariably become resistant to treatment with targeted and cytotoxic agents. Multiple pathways have been identified as being responsible for these pleotropic mechanisms of resistance. The MUC1 gene is aberrantly expressed in CRPC/NEPC in association with poor clinical outcomes; whereas, it is not known if the oncogenic MUC1-C/M1C protein drives treatment resistance. We demonstrated that MUC1-C is necessary for resistance of (i) PC cells to enzalutamide (ENZ), and (ii) CRPC and NEPC cells to docetaxel (DTX). Our results showed that MUC1-C-mediated resistance is conferred by upregulation of aerobic glycolysis and suppression of reactive oxygen species necessary for self-renewal. Dependence of these resistant phenotypes on MUC1-C for the cancer stem cell (CSC) state identified a potential target for treatment. In this regard, we further demonstrated that targeting MUC1-C with a M1C antibody-drug conjugate (ADC) is highly effective in suppressing (i) self-renewal of drug-resistant CRPC/NEPC CSCs and (ii) growth of t-NEPC tumor xenografts derived from drug-resistant cells and a patient with refractory disease. These findings uncovered a common MUC1-C-dependent pathway in treatment-resistant CRPC/NEPC progression and identified MUC1-C as a target for their treatment with a M1C ADC.

Human embryonic stem cells (hESCs) can be classified as having naïve and primed pluripotency states. While several studies have reported different gene expression networks between these two pluripotency states, the role of alternative splicing (AS) in regulating these differences has not been well characterized. In this study, we performed RNA sequencing and identified differential AS events in 784 genes between naïve and primed hESCs. Among these, KIAA1522, whose function has not been well studied, has state-specific isoforms regulated by alternative first exon (AFE). This splicing event resulted in isoforms with distinct N-terminal domains and subcellular localization. Notably, the sequences and alternative isoform patterns of KIAA1522 were conserved between humans and mice. Further investigation using cleavage under targets and tagmentation (CUT&Tag) experiments in cells with specific-isoform overexpression or knockdown revealed the opposite activity of long terminal repeat retrotransposons (LTR-RTs) and motif enrichment profiles. The naïve-specific (N-P) isoform upregulated naïve marker gene expression and preferentially activated LTR-RTs by binding to the motifs enriched for POU and FOX family transcription factor binding sites. Conversely, the primed-specific (P-P) isoform promoted primed marker gene expression and suppressed LTR-RTs activity by binding to the motifs enriched for zinc finger protein binding sites. Collectively, KIAA1522 regulates the balance between naïve and primed pluripotency states through isoform-specific regulation of LTR-RTs activity and collaboration with distinct transcriptional regulators. In summary, our results characterize the splicing atlas of hESCs in naïve and primed states and reveal the regulatory function and mechanism of AFE usage by KIAA1522.

Glioblastoma (GBM) is an aggressive brain tumor associated with high post-therapy recurrence and very poor survival rates. One of the factors contributing to the aggressive nature of this disease is the level of heterogeneity seen at the phenotypic and genetic level. Glioma stem cells (GSCs) are stem-like cells within the tumor with the ability to self-renew and give rise to different types of cells within the tumor, hence giving rise to the heterogeneity found in GBM. GSCs are often implicated in the resistance of glioma to standard of care radiation and chemotherapy. The physical niche within a tumor mass supports stemness and aggressive characteristics of GSCs, hence, experimental systems providing a relevant tumor microenvironment (TME) are critical for adequate assessment of molecular mechanisms regulating GSC populations. Although mouse models continue to be an integral part of an in vivo experimental design, they are neither time- nor cost-efficient. Danio rerio (zebrafish) patient-derived xenografts (PDXs) overcome several of the obstacles of the mammalian systems. Zebrafish constitute a robust, easily reproducible experimental model allowing for relevant investigation of GSC populations with TME. This chapter describes methods required for generation of zebrafish PDXs to study aspects of GSC-mediated tumorigenesis and interactions with the TME.

Local therapies are in development for targeting of brain tumors and cancer stem cells, as well as non-brain tumors, due to significant transport barriers that hinder sufficient drug accumulation at the disease site. For chemotherapeutics, local delivery is already a well-established practice in the clinic for brain tumors. For immunotherapy applications, local therapies can include the delivery of immune cell engagers, adoptive cell therapies, or combination therapies to encourage adaptive responses. The duration of action of local therapies is important to extend and screen to identify efficacious formulations with a single or minimal administration. To this end, this chapter discusses the development of three-dimensional embedded multi-cellular spheroid models to track spheroid killing in real time and to screen the extended duration of action of local T cell engagers with new therapeutic scaffolds and drug eluting medical devices.

Immunohistochemistry (IHC) is a useful method to detect and visualize targeted proteins in tissues, with a high degree of specificity and clarity. Through the strategic use of chromogens and counterstains, it enables a contrast-rich and accurate evaluation of specific molecules, such as surface markers. Here, we outline a comprehensive IHC followed with counterstaining protocol for the positive selection of stem-like cells and immune cells in brain tumor tissue.

A defining characteristic of brain tumor stem cells (BTSCs) is their ability to migrate and invade the brain parenchyma. This invasive behavior is particularly critical in understanding how BTSCs originating from primary tumors contribute to the formation of secondary brain tumor lesions. In this chapter, we present a streamlined and unbiased method to assess BTSC migration capacity. By leveraging real-time imaging with the Incucyte system, we provide a standardized and efficient approach for studying BTSC motility under controlled conditions.

Successful containment of unwanted cell cycle progression in tumors such as glioblastoma (GBM) requires targeted therapeutic approaches, which rely on understanding cell cycle dynamics in response to microenvironmental stimuli. Glioma Stem Cells (GSCs) can drive tumor initiation, recurrence, therapy resistance, and are often attributed to the heterogeneity and plasticity of GBM. In vitro models, using patient-derived GSCs, provide a life relevant tool for exploration of complex molecular mechanisms underlying the aggressive characteristics of GBM. Introduction of 3D tissue culture systems permits the study of spatial complexity of the tumor mass and enables control over diverse conditions within the surrounding microenvironment. This chapter demonstrates detailed methods to study spatiotemporal changes to the cell cycle dynamics using available fluorescent cell cycle reporter systems in combination with bioinformatics-based signal intensity and localization analysis. We present a successful approach that investigates the 3D cell cycle dynamics of GSC populations. This approach utilizes GBM neurosphere and organoid cultures, which are assessed over time and under therapeutic pressure. These models can be further explored, manipulated, and customized to serve specific experimental designs.