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Spontaneous abortion is the most common complication in early pregnancy, the exact etiology of most cases cannot be determined. Emerging studies suggest that mutations in ciliary genes may be associated with progression of pregnancy loss. However, the involvement of primary cilia on spontaneous abortion and the underlying molecular mechanisms remains poorly understood. We observed the number and length of primary cilia were significantly decreased in decidua of spontaneous abortion in human and lipopolysaccharide (LPS)‐induced abortion mice model, accompanied with increased expression of proinflammatory cytokines interleukin (IL)−1β, IL‐6, and tumor necrosis factor (TNF)‐α. The length of primary cilia in human endometrial stromal cell (hESC) was significantly shortened after TNF‐α treatment. Knocking down intraflagellar transport 88 (IFT88), involved in cilia formation and maintenance, promoted the expression of TNF‐α. There was a reverse regulatory relationship between cilia shortening and TNF‐α expression. Further research found that shortened cilia impair decidualization in hESC through transforming growth factor (TGF)‐β/SMAD2/3 signaling. Primary cilia were impaired in decidua tissue of spontaneous abortion, which might be mainly caused by inflammatory injury. Primary cilia abnormalities resulted in dysregulation of TGF‐β/SMAD2/3 signaling transduction and decidualization impairment, which led to spontaneous abortion. [ABSTRACT FROM AUTHOR]

In this study, we investigate the effects of miRNA-138-5p and probable G-protein coupled receptor 124 (GPR124)- regulated inflammasome and downstream leukemia inhibitory factor (LIF)–STAT and adhesion molecule signaling in human decidual stromal cells. After informed consent was obtained from women aged 25–38 years undergoing surgical termination of the normal pregnancy and spontaneous miscarriage after 6–9 weeks of gestation, human decidual stromal cells were extracted from the decidual tissue. Extracellular vesicles (EVs) with microRNA (miRNA) between cells have been regarded as critical factors for embryo–maternal interactions on embryo implantation and programming of human pregnancy. MicroRNA-138-5p acts as the transcriptional regulator of GPR124 and the mediator of downstream inflammasome. LIF-regulated STAT activation and expression of integrins might influence embryo implantation. Hence, a better understanding of LIF–STAT and adhesion molecule signaling would elucidate the mechanism of microRNA-138-5p- and GPR124-regulated inflammasome activation on embryo implantation and pregnancy. Our results show that microRNA-138-5p, purified from the EVs of decidual stromal cells, inhibits the expression of GPR124 and the inflammasome, and activates the expression of LIF–STAT and adhesion molecules in human decidual stromal cells. Additionally, the knockdown of GPR124 and NLRP3 through siRNA increases the expression of LIF–STAT and adhesion molecules. The findings of this study help us gain a better understanding the role of EVs, microRNA-138-5p, GPR124, inflammasomes, LIF–STAT, and adhesion molecules in embryo implantation and programming of human pregnancy. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that promotes adipogenesis, lipid uptake and storage, insulin sensitivity, and glucose metabolism. Hence, defects in PPARγ have been associated to the development of metabolic disorders. Sex hormone-binding globulin (SHBG) is a glycoprotein primarily produced in the liver that regulates the bioavailability of sex hormones. Alike PPARγ, low SHBG levels have been correlated with insulin resistance and associated endocrine abnormalities. Therefore, this study aimed to verify whether SHBG may restore depleted PPARγ functions and thus serve as a new candidate for the management of metabolic conditions. A model of equine adipose-derived stromal cells (EqASCs) has been used, in which a PPARγ silencing and SHBG treatment have been achieved to determine the changes in cell viability, premature senescence, oxidative stress, and mitochondrial functions. Obtained data demonstrated that loss in PPARγ triggers cell apoptosis which is not reversed by SHBG application. Moreover, PPARγ knockdown cells exhibited premature senescence, which has been substantially alleviated by SHBG concomitantly to increased BAX/BCL2 ratio, suggesting a possible effect on senescence-induced apoptosis resistance. Interestingly, PPARγ silencing induced a significant alteration in mitochondrial membrane potential as well as the expression of dynamics and metabolism-related markers. SHBG treatment enabled to ameliorate the transmembrane potential, to normalize the expression levels of key dynamics and metabolism mediators, and to restore the protein levels of PINK, which is critically involved in mitochondria recycling machinery. Presented data suggest that SHBG may provide new mechanistic insights into the regulation of PPARγ functions, and thus offers a preliminary picture on a possible SHBG-PPARγ metabolic crosstalk. Key messages: PPARγ is a transcription factor that tightly regulates cell metabolism. Low SHBG levels correlate with insulin resistance and associated endocrine abnormalities. PPARγ silencing reduces cell viability, triggers premature senescence and profound mitochondrial failure in equine ASCs. SHBG protein reverses senescent phenotype and apoptosis resistance of PPARγ- ASCs. SHBG improves mitochondrial dynamics and metabolism following PPARγ knockdown. SHBG might serve as a PPARγ potential mimicking agent for the modulation of ASCs metabolic processes. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory potential and may be used to treat injured tissues. Pregnancy has been associated with increased MSCs in the peripheral circulation in multiple species, but to date, there are no reports on this matter in horses. This study aimed to evaluate the effect of pregnancy on isolation efficiency and proliferation capacity of equine MSCs derived from the peripheral blood (PB) of mares. Venous blood samples were collected at the 11th month of gestation and 1 month after delivery from clinically healthy Arabian mares that presented normal pregnancies. Blood samples were processed for in vitro cellular culture and hormonal and metabolic profiles. MSCs were isolated and characterized by trilineage differentiation potential, immunophenotyping, analyzed by gene sequencing and proliferation assays. The isolation of peripheral blood mononuclear cells (PBMCs) of pregnant mares were associated with higher isolation efficiency and proliferative capacity of MSCs derived from peripheral blood (PB-MSCs) recovered pre-partum than those isolated post-partum. Although fetal gender, parity, 5α-reduced pregnanes, insulin, and cortisol were shown to affect cellular proliferation, individual factors and the small population studied must be considered. This study suggests that PB-MSCs from pregnant mares could be a valuable alternative source of MSCs for therapeutic purposes. • Lack of reports onthe isolation of mesenchymal stromal cells (MSCs) from peripheral blood (PB-MSCs) in pregnant mares. • Pregnancy in Arabian mares was associated with higher isolation efficiency and proliferative capacity of PB-MSCs. • Fetal gender, parity, and levels of 5α-reduced pregnanes, insulin and cortisol were shown to affect cellular proliferation. • Pregnant mares could be a valuable, readily available source of MSCs for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) and their released factors (secretome) are intriguing options for regenerative medicine approaches based on the management of inflammation and tissue restoration, as in joint disorders like osteoarthritis (OA). Production strategy may modulate cells and secretome fingerprints, and for the latter, the effect of serum removal by starvation used in clinical-grade protocols has been underestimated. In this work, the effect of starvation on the molecular profile of interleukin 1 beta (IL1β)-primed adipose-derived MSCs (ASCs) was tested by assessing the expression level of 84 genes related to secreted factors and 84 genes involved in defining stemness potential. After validation at the protein level, the effect of starvation modulation in the secretomes was tested in a model of OA chondrocytes. IL1β priming in vitro led to an increase in inflammatory mediators' release and reduced anti-inflammatory potential on chondrocytes, features reversed by subsequent starvation. Therefore, when applying serum removal-based clinical-grade protocols for ASCs' secretome production, the effects of starvation must be carefully considered and investigated. [ABSTRACT FROM AUTHOR]

Simple Summary: Much interest has arisen around adipose-derived stem cells (ASCs) due to their multifunctional activities in the tumor microenvironment (TME). Mitochondrial dynamics and mitochondrial transfer are critical processes that promote tumor progression through fission, fusion, and transfer from stromal cells, such as ASCs. This perspective focuses on the connection between ASCs and tumor cells, leveraging the idea that inhibiting their possible pro-tumorigenic effect can interfere with these processes and limit the ability of tumor cells to survive. Unfortunately, the use of ASC/MSCs in cancer therapy has some limitations, as many variables must be considered; however, bridging the gap between preclinical studies and clinical applications could lead to new therapeutic strategies. Adipose-derived stem cells (ASCs) significantly influence tumor progression within the tumor microenvironment (TME). This review examines the pro-tumorigenic roles of ASCs, focusing on paracrine signaling, direct cell–cell interactions, and immunomodulation. ASC-mediated mitochondrial transfer through tunneling nanotubes (TNTs) and gap junctions (GJs) plays a significant role in enhancing cancer cell survival and metabolism. Cancer cells with dysfunctional mitochondria acquire mitochondria from ASCs to meet their metabolic needs and thrive in the TME. Targeting mitochondrial transfer, modulating ASC function, and influencing metabolic pathways are potential therapeutic strategies. However, challenges like TME complexity, specificity, safety concerns, and resistance mechanisms must be addressed. Disrupting the ASC–cancer cell–mitochondria axis offers a promising approach to cancer therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Head and neck cancer disparities in outcomes for Black Americans have been well recognized. However, the specific drivers of the inferior outcomes remain poorly understood. We investigated the biologic features of patient oral cancers and performed a follow-up study of the patient post-surgery recurrences and metastases aiming to explore potential mechanisms that might underpin the poorer outcomes among Black American patients. It was found that high levels of tumor stemness and tumor-promoting stromal characteristics were linked to patient recurrence and metastasis. There were more cases of Black American than White American exhibiting high stemness traits and strong tumor-promoting stromal features associated with tumor recurrences and metastases, although the investigated cases displayed comparable clinical diagnoses. Our findings revealed that the differences in tumor stemness and stromal property among cancers with similar diagnoses contribute to patient outcome disparities. Black Americans (BAs) with head and neck cancer (HNC) have worse survival outcomes compared to the White patients. While HNC disparities in patient outcomes for BAs have been well recognized, the specific drivers of the inferior outcomes remain poorly understood. Here, we investigated the biologic features of patient tumor specimens obtained during the surgical treatment of oral cancers and performed a follow-up study of the patients' post-surgery recurrences and metastases with the aim to explore whether tumor biologic features could be associated with the poorer outcomes among BA patients compared with White American (WA) patients. We examined the tumor stemness traits and stromal properties as well as the post-surgery recurrence and metastasis of oral cancers among BA and WA patients. It was found that high levels of tumor self-renewal, invasion, tumorigenesis, metastasis, and tumor-promoting stromal characteristics were linked to post-surgery recurrence and metastasis. There were more BA than WA patients demonstrating high stemness traits and strong tumor-promoting stromal features in association with post-surgery tumor recurrences and metastases, although the investigated cases displayed clinically comparable TNM stages and histological grades. These findings demonstrated that the differences in tumor stemness and stromal property among cancers with comparable clinical diagnoses contribute to the outcome disparity in HNCs. More research is needed to understand the genetic and molecular basis of the biologic characteristics underlying the inferior outcomes among BA patients, so that targeting strategies can be developed to reduce HNC disparity. [ABSTRACT FROM AUTHOR]

Women are more prone to develop rheumatoid arthritis, with peak incidence occurring around menopause. Estrogen has major effects on the immune system and is protective against arthritis. We have previously shown that treatment with estrogen inhibits inflammation and joint destruction in murine models of arthritis, although the mechanisms involved remain unclear. Fibroblastic reticular cells (FRCs) are specialized stromal cells that generate the three‐dimensional structure of lymph nodes (LNs). FRCs are vital for coordinating immune responses from within LNs and are characterized by the expression of the chemokine CCL19, which attracts immune cells. The aim of this study was to determine whether the influence of estrogen on innate and adaptive immune cells in arthritis is mediated by estrogen signaling in FRCs. Conditional knockout mice lacking estrogen receptor α (ERα) in CCL19‐expressing cells (Ccl19‐CreERαfl/fl) were generated and tested. Ccl19‐CreERαfl/fl mice and littermate controls were ovariectomized, treated with vehicle or estradiol and subjected to the 28‐day‐long antigen‐induced arthritis model to enable analyses of differentiated T‐ and B‐cell populations and innate cells in LNs by flow cytometry. The results reveal that while the response to estradiol treatment in numbers of FRCs per LN is significantly reduced in mice lacking ERα in FRCs, estrogen does not inhibit joint inflammation or markedly affect immune responses in this arthritis model. Thus, this study validates the Ccl19‐CreERαfl/fl strain for studying estrogen signaling in FRCs within inflammatory diseases, although the chosen arthritis model is deemed unsuitable for addressing this question. [ABSTRACT FROM AUTHOR]

The senescence of bone marrow mesenchymal stromal cells (MSCs) leads to the impairment of stemness and osteogenic differentiation capacity. In a previous study, we screened out stearoyl-CoA desaturase 2 (SCD2), the most evidently changed differential gene in lipid metabolism, using combined transcriptomic and metabolomic analyses, and verified that SCD2 could mitigate MSC senescence. However, the underlying molecular mechanism by which the rate-limiting enzyme of lipogenesis SCD2 manipulates MSC senescence has not been completely understood. In this study, we demonstrate that SCD2 over-expression alleviates MSC replicative senescence and ameliorates their osteogenic differentiation through the regulation of lipogenesis. Furthermore, SCD2 expression is reduced, whereas miR-200c-3p expression is elevated in replicative senescent MSCs. SCD2 is the direct target gene of miR-200c-3p, which can bind to the 3′-UTR of SCD2. MiR-200c-3p replenishment in young MSCs is able to diminish SCD2 expression levels due to epigenetic modulation. In addition, SCD2-rescued MSC senescence and enhanced osteogenic differentiation can be attenuated by miR-200c-3p repletion via suppressing lipogenesis. Taken together, we reveal the potential mechanism of SCD2 influencing MSC senescence from the perspective of lipid metabolism and epigenetics, which provides both an experimental basis for elucidating the mechanism of stem cell senescence and a novel target for delaying stem cell senescence. [ABSTRACT FROM AUTHOR]

Adipose tissue dysfunction, which is associated with an increased risk of colorectal cancer (CRC), is a significant factor in the pathophysiology of obesity. Obesity-related inflammation and extracellular matrix (ECM) remodeling promote colorectal cancer metastasis (CRCM) by shaping the tumor microenvironment (TME). When CRC occurs, the metabolic symbiosis of tumor cells recruits adjacent adipocytes into the TME to supply energy. Meanwhile, abundant immune cells, from adipose tissue and blood, are recruited into the TME, which is stimulated by pro-inflammatory factors and triggers a chronic local pro-inflammatory TME. Dysregulated ECM proteins and cell surface adhesion molecules enhance ECM remodeling and further increase contractibility between tumor and stromal cells, which promotes epithelial-mesenchymal transition (EMT). EMT increases tumor migration and invasion into surrounding tissues or vessels and accelerates CRCM. Colorectal symbiotic microbiota also plays an important role in the promotion of CRCM. In this review, we provide adipose tissue and its contributions to CRC, with a special emphasis on the role of adipocytes, macrophages, neutrophils, T cells, ECM, and symbiotic gut microbiota in the progression of CRC and their contributions to the CRC microenvironment. We highlight the interactions between adipocytes and tumor cells, and potential therapeutic approaches to target these interactions. [ABSTRACT FROM AUTHOR]

In patients with endometriosis, refluxed endometrial fragments evade host immunosurveillance, developing into endometriotic lesions. However, the mechanisms underlying this evasion have not been fully elucidated. N-Myc and STAT Interactor (NMI) have been identified as key players in host immunosurveillance, including interferon (IFN)-induced cell death signaling pathways. NMI levels are markedly reduced in the stromal cells of human endometriotic lesions due to modulation by the Estrogen Receptor beta/Histone Deacetylase 8 axis. Knocking down NMI in immortalized human endometrial stromal cells (IHESCs) led to elevated RNA levels of genes involved in cell-to-cell adhesion and extracellular matrix signaling following IFNA treatment. Furthermore, NMI knockdown inhibited IFN-regulated canonical signaling pathways, such as apoptosis mediated by Interferon Stimulated Gene Factor 3 and necroptosis upon IFNA treatment. In contrast, NMI knockdown with IFNA treatment activated non-canonical IFN-regulated signaling pathways that promote proliferation, including β-Catenin and AKT signaling. Moreover, NMI knockdown in IHESCs stimulated ectopic lesions' growth in mouse endometriosis models. Therefore, NMI is a novel endometriosis suppressor, enhancing apoptosis and inhibiting proliferation and cell adhesion of endometrial cells upon IFN exposure. [ABSTRACT FROM AUTHOR]

Previous research has unveiled a stem cell-like transcriptome enrichment in the aldehyde dehydrogenase-expressing (ALDHhigh) mesenchymal stromal cell (MStroC) fraction. However, considering the heterogeneity of MStroCs, with only a fraction of them presenting bona fide stem cells (MSCs), the actual potency of ALDH as an MSC-specific selection marker remains an issue. To address this, the proliferative and differentiation potential of individual ALDHhigh and ALDHlow MStroCs incubated at low oxygen concentrations, estimated to mimic stem cell niches (0.1% O 2), were assayed using single-cell clonal analysis, compared to standard conditions (20% O 2). We confirm that a high proliferative capacity and multi-potent MSCs are enriched in the ALDHhigh MStroC population, especially when cells are cultured at 0.1% O 2. Measurements of reduced/oxidized glutathione and mitochondrial superoxide anions with MitoSoX (MSX) indicate that this advantage induced by low oxygen is related to a decrease in the oxidative and reactive oxygen species (ROS) levels in the stem cell metabolic setup. However, ALDH expression is neither specific nor exclusive to MSCs, as high proliferative capacity and multi-potent cells were also found in the ALDHlow fraction. Furthermore, single-cell assays performed after combined cell sorting based on ALDH and MSX showed that the MSXlow MStroC population is enriched in stem/progenitor cells in all conditions, irrespective of ALDH expression or culture oxygen concentration. Importantly, the ALDHhighMSXlow MStroC fraction exposed to 0.1% O 2 was almost exclusively composed of genuine MSCs. In contrast, neither progenitors nor stem cells (with a complete absence of colony-forming ability) were detected in the MSXhigh fraction, which exclusively resides in the ALDHlow MStroC population. Our study reveals that ALDH expression is not exclusively associated with MSCs. However, cell sorting using combined ALDH expression and ROS content can be utilized to exclude MStroCs lacking stem/progenitor cell properties. [Display omitted] [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is characterized by an overactive immune system, with limited treatment options beyond immunosuppressive drugs or biological response modifiers. Human embryonic stem cell–derived mesenchymal stromal cells (hESC-MSCs) represent a novel alternative, possessing diverse immunomodulatory effects. In this study, we aimed to elucidate the therapeutic effects and underlying mechanisms of hESC-MSCs in treating RA. MSC-like cells were differentiated from hESC (hESC-MSCs) and cultured in vitro. Cell proliferation was assessed using Cell Counting Kit-8 assay and Ki-67 staining. Flow cytometry was used to analyze cell surface markers, T-cell proliferation and immune cell infiltration. The collagen-induced arthritis (CIA) mouse model and bleomycin-induced model of lung fibrosis (BLE) were established and treated with hESC-MSCs intravenously for in vivo assessment. Pathological analyses, reverse transcription-quantitative polymerase chain reaction and Western blotting were conducted to evaluate the efficacy of hESC-MSCs treatment. Intravenous transplantation of hESC-MSCs effectively reduced inflammation in CIA mice in this study. Furthermore, hESC-MSC administration enhanced regulatory T cell infiltration and activation. Additional findings suggest that hESC-MSCs may reduce lung fibrosis in BLE mouse models, indicating their potential to mitigate complications associated with RA progression. In vitro experiments revealed a significant inhibition of T-cell activation and proliferation during co-culture with hESC-MSCs. In addition, hESC-MSCs demonstrated enhanced proliferative capacity compared with traditional primary MSCs. Transplantation of hESC-MSCs represents a promising therapeutic strategy for RA, potentially regulating T-cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Adenomyosis and endometriosis are distinct gynecological disorders characterized by ectopic growth of endometrial tissue. Their etiology remains unclear, but stem cells have been implicated in both. The aim of this study was to investigate and compare the quantity of NOTCH1+ and CD117+ stem cells in endometriosis and adenomyosis lesions. Immunohistochemical staining of ectopic endometrium biopsies using antibodies against NOTCH1 and CD117 was performed. The quantity and spatial distribution of endometrial stromal cells positive for these markers were determined and compared between endometriosis and adenomyosis lesions. Additionally, their quantities were compared between endometriosis lesion types. Mann–Whitney U test showed that the median percentages of both NOTCH1+ and CD117+ cells in the endometriosis lesions were significantly higher than those in the adenomyosis lesions (2.26% vs. 0.13%, p = 0.002 and 0.44% vs. 0.26%, p = 0.016, respectively). Spearman's test showed a positive correlation between NOTCH1+ and CD117+ cells in endometriosis lesions (R = 0.45, p = 0.027) but no significant correlation in adenomyosis lesions (R = −0.11, p = 0.69). The quantity of both stem cell types was highest in extragenital endometriotic lesions. Unlike adenomyosis, endometriosis lesions are associated with higher quantities of NOTCH1+ and CD117+ stem cells and a coordinated increase in their number. These findings support the distinct origin of the two conditions. [ABSTRACT FROM AUTHOR]

Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks. [ABSTRACT FROM AUTHOR]

Head and neck cancer (HNC) entails a heterogenous neoplastic disease that arises from the mucosal epithelium of the upper respiratory system and the gastrointestinal tract. It is characterized by high morbidity and mortality, being the eighth most common cancer worldwide. It is believed that the mesenchymal/stem stromal cells (MSCs) present in the tumour milieu play a key role in the modulation of tumour initiation, development and patient outcomes; they also influence the resistance to cisplatin-based chemotherapy, the gold standard for advanced HNC. MSCs are multipotent, heterogeneous and mobile cells. Although no MSC-specific markers exist, they can be recognized based on several others, such as CD73, CD90 and CD105, while lacking the presence of CD45, CD34, CD14 or CD11b, CD79α, or CD19 and HLA-DR antigens; they share phenotypic similarity with stromal cells and their capacity to differentiate into other cell types. In the tumour niche, MSC populations are characterized by cell quiescence, self-renewal capacity, low reactive oxygen species production and the acquisition of epithelial-to-mesenchymal transition properties. They may play a key role in the process of acquiring drug resistance and thus in treatment failure. The present narrative review examines the links between MSCs and HNC, as well as the different mechanisms involved in the development of resistance to current chemo-radiotherapies in HNC. It also examines the possibilities of pharmacological targeting of stemness-related chemoresistance in HNSCC. It describes promising new strategies to optimize chemoradiotherapy, with the potential to personalize patient treatment approaches, and highlights future therapeutic perspectives in HNC. [ABSTRACT FROM AUTHOR]

Degenerative disorders like osteoarthritis (OA) might impair the ability of tissue-resident mesenchymal stem/stromal cells (MSCs) for tissue regeneration. As primary cells with MSC-like properties are exploited for patient-derived stem cell therapies, a detailed evaluation of their in vitro properties is needed. Here, we aimed to compare synovium-derived and bone-derived MSCs in early hip OA with those of patients without OA (non-OA). Tissues from three synovial sites of the hip (paralabral synovium, cotyloid fossa, inner surface of peripheral capsule) were collected along with peripheral trabecular bone from 16 patients undergoing hip arthroscopy (8 early OA and 8 non-OA patients). Primary cells isolated from tissues were compared using detailed in vitro analyses. Gene expression profiling was performed for the skeletal stem cell markers podoplanin (PDPN), CD73, CD164 and CD146 as well as for immune-related molecules to assess their immunomodulatory potential. Synovium-derived and bone-derived MSCs from early OA patients showed comparable clonogenicity, cumulative population doublings, osteogenic, adipogenic and chondrogenic potential, and immunophenotype to those of non-OA patients. High PDPN/low CD146 profile (reminiscent of skeletal stem cells) was identified mainly for non-OA MSCs, while low PDPN/high CD146 mainly defined early OA MSCs. These data suggest that MSCs from early OA patients are not affected by degenerative changes in the hip. Moreover, the synovium represents an alternative source of MSCs for patient-derived stem cell therapies, which is comparable to bone. The expression profile reminiscent of skeletal stem cells suggests the combination of low PDPN and high CD146 as potential biomarkers in early OA. [ABSTRACT FROM AUTHOR]

Chronic asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Previous studies have shown that mesenchymal stromal/stem cells (MSCs) exert anti-inflammatory effects on asthma via regulation of the immune cells. However, the therapeutic mechanism of MSCs, especially the mechanism of airway remodeling in chronic asthma, remains to be elucidated. Here, we aimed to investigate the therapeutic effect of MSCs on airway remodeling in chronic asthma and explored the mechanisms by analyzing the polarization phenotype of macrophages in the lungs. We established a mouse model of chronic asthma induced by ovalbumin (OVA) and evaluated the effect of MSCs on airway remodeling. The data showed that MSCs treatment before the challenge exerted protective effects on OVA-induced chronic asthma, i.e., decreased the inflammatory cell infiltration, Th2 cytokine levels, subepithelial extracellular matrix deposition, and transforming growth factor β (TGF-β)/Smad signaling. Additionally, we found that MSCs treatment markedly suppressed macrophage M2 polarization in lung tissue. At the same time, MSCs treatment inhibited NF-κB p65 nuclear translocation, ER stress, and oxidative stress in the OVA-induced chronic allergic airway remodeling mice model. In conclusion, these results demonstrated that MSCs treatment prevents OVA-induced chronic airway remodeling by suppressing macrophage M2 polarization, which may be associated with the dual inhibition of ER stress and oxidative stress. This discovery may provide a new theoretical basis for the future clinical application of MSCs. [ABSTRACT FROM AUTHOR]

Endometriosis is a chronic hormone-dependent disease characterized by the spread of endometrial cells outside the uterus, which form endometriotic lesions and disrupt the functions of the affected organs. The etiopathogenesis of endometriosis is still unclear, and thus it is important to examine the genes that may contribute to the establishment of endometriotic lesions. The aim of this study was to investigate the expression of new potential candidate gene latexin (LXN), an inhibitor of carboxypeptidases, in endometrium and endometriotic lesions to elucidate its possible role in endometriosis development. LXN expression in tissues was assessed using quantitative reverse transcription PCR (qRT–PCR) analysis and immunohistochemical staining (IHC). The functions of LXN were examined using Transwell and MTT assays. qRT–PCR analysis revealed that LXN expression in endometrium was menstrual cycle-dependent, being lowest in the early-secretory phase and highest in the late-secretory phase and was significantly upregulated in endometriotic lesions. IHC confirmed LXN expression in endometrial stromal cells, and in vitro assays demonstrated that knockdown of LXN effectively reduced the migratory capacity of endometrial stromal cells while promoting cell viability. In conclusion, our results showed that LXN can be involved in the pathogenesis of endometriosis by regulating the proliferation and migration activity of endometriotic stromal cells. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been recognized as promising cytotherapeutics due to their demonstrated immunomodulatory effects in various preclinical models. The immunomodulatory capabilities of EVs stem from the proteins and genetic materials they carry from parent cells, but the cargo contents of EVs are significantly influenced by MSC tissues and donors, cellular age and culture conditions, resulting in functional variations. However, there are no surrogate assays available to validate the immunomodulatory potency of MSC‐EVs before in vivo administration. In previous work, we discovered that microcarrier culture conditions enhance the immunomodulatory function of MSC‐EVs, as well as the levels of immunosuppressive molecules such as TGF‐β1 and let‐7b in MSC‐EVs. Building on these findings, we investigated whether TGF‐β1 levels in MSC‐EVs could serve as a surrogate biomarker for predicting their potency in vivo. Our studies revealed a strong correlation between TGF‐β1 and let‐7b levels in MSC‐EVs, as well as their capacity to suppress IFN‐γ secretion in stimulated splenocytes, establishing biopotency and surrogate assays for MSC‐EVs. Subsequently, we validated MSC‐EVs generated from monolayer cultures (ML‐EVs) or microcarrier cultures (MC‐EVs) using murine models of experimental autoimmune uveoretinitis (EAU) and additional in vitro assays reflecting the Mode of Action of MSC‐EVs in vivo. Our findings demonstrated that MC‐EVs carrying high levels of TGF‐β1 exhibited greater efficacy than ML‐EVs in halting disease progression in mice with EAU as well as inducing apoptosis and inhibiting the chemotaxis of retina‐reactive T cells. Additionally, MSC‐EVs suppressed the MAPK/ERK pathway in activated T cells, with treatment using TGF‐β1 or let‐7b showing similar effects on the MAPK/ERK pathway. Collectively, our data suggest that MSC‐EVs directly inhibit the infiltration of retina‐reactive T cells toward the eyes, thereby halting the disease progression in EAU mice, and their immunomodulatory potency in vivo can be predicted by their TGF‐β1 levels. [ABSTRACT FROM AUTHOR]

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV‐derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow‐derived MSCs (BMSCs) and adipose‐derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC‐EVs and ASC‐EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2‐macrophage genes (IL‐10, Arg1, TGFβ) than M1‐macrophage genes (IL‐6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade‐offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies. [ABSTRACT FROM AUTHOR]

Introduction: The immunomodulatory properties of mesenchymal stromal cells (MSC) have been well-characterized in in-vitro and in-vivo models. We have previously shown that liver MSC (L-MSC) are superior inhibitors of T-cell activation/proliferation, NK cell cytolytic function, and macrophage activation compared to adipose (A-MSC) and bone marrow MSC (BM-MSC) in-vitro. Method: To test these observations in-vivo, we infused these types of MSC into mice with unilateral renal artery stenosis (RAS), an established model of kidney inflammation. Unilateral RAS was induced via laparotomy in 11-week-old, male 129-S1 mice under general anesthesia. Control mice had sham operations. Human L-MSC, AMSC, and BM-MSC (5x105 cells each) or PBS vehicle were injected intra-arterially 2 weeks after surgery. Kidney morphology was studied 2 weeks after infusion using micro-MRI imaging. Renal inflammation, apoptosis, fibrosis, and MSC retention were studied ex-vivo utilizing western blot, immunofluorescence, and immunohistological analyses. Results: The stenotic kidney volume was smaller in all RAS mice, confirming significant injury, and was improved by infusion of all MSC types. All MSC-infused groups had lower levels of plasma renin and proteinuria compared to untreated RAS. Serum creatinine improved in micetreated with BM- and L-MSC. All types of MSC located to and were retained within the stenotic kidneys, but L-MSC retention was significantly higher than A- and BM-MSC. While all groups of MSC-treated mice displayed reduced overall inflammation and macrophage counts, L-MSC showed superior potency in-vivo at localizing to the site of inflammation and inducing M2 (reparative) macrophage polarization to reduce inflammatory changes. Discussion: These in-vivo findings extend our in-vitro studies and suggest that LMSC possess unique anti-inflammatory properties that may play a role in liverinduced tolerance and lend further support to their use as therapeutic agents for diseases with underlying inflammatory pathophysiology. [ABSTRACT FROM AUTHOR]

Aims: In an era of evolving diagnostic possibilities, existing diagnostic systems are not fully sufficient to promptly recognize patients with early‐stage hypertrophic cardiomyopathy (HCM) without symptomatic and instrumental features. Considering the sudden death of HCM, developing a novel diagnostic model to clarify the patients with early‐stage HCM and the immunological characteristics can avoid misdiagnosis and attenuate disease progression. Methods and results: Three hundred eighty‐five samples from four independent cohorts were systematically retrieved. The weighted gene co‐expression network analysis, differential expression analysis (|log2(foldchange)| > 0.5 and adjusted P < 0.05), and protein–protein interaction network were sequentially performed to identify HCM‐related hub genes. With a machine learning algorithm, the least absolute shrinkage and selection operator regression algorithm, a stable diagnostic model was developed. The immune‐cell infiltration and biological functions of HCM were also explored to characterize its underlying pathogenic mechanisms and the immune signature. Two key modules were screened based on weighted gene co‐expression network analysis. Pathogenic mechanisms relevant to extracellular matrix and immune pathways have been discovered. Twenty‐seven co‐regulated genes were recognized as HCM‐related hub genes. Based on the least absolute shrinkage and selection operator algorithm, a stable HCM diagnostic model was constructed, which was further validated in the remaining three cohorts (n = 385). Considering the tight association between HCM and immune‐related functions, we assessed the infiltrating abundance of various immune cells and stromal cells based on the xCell algorithm, and certain immune cells were significantly different between high‐risk and low‐risk groups. Conclusions: Our study revealed a number of hub genes and novel pathways to provide potential targets for the treatment of HCM. A stable model was developed, providing an efficient tool for the diagnosis of HCM. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) continue to be proposed for clinical investigation to treat myriad diseases given their purported potential to stimulate endogenous regenerative processes, such as angiogenesis. However, MSC functional heterogeneity has hindered clinical success and still poses a substantial manufacturing challenge from a product quality control perspective. Here, a quantitative bioassay based on an enhanced‐throughput is described, microphysiological system (MPS) to measure the specific bioactivity of MSCs to stimulate angiogenesis as a potential measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages are co‐cultured with human umbilical vein endothelial cells and exhibit significant heterogeneity in angiogenic potency between donors and cell passage. Depending on donor source and cellular passage number, MSCs varied in their ability to stimulate tip cell dominant or stalk cell dominant phenotypes in angiogenic sprout morphology which correlated with expression levels of hepatocyte growth factor (HGF). These findings suggest that MSC angiogenic bioactivity may be considered as a possible potency attribute in MSC quality control strategies. Development of a reliable and functionally relevant potency assay for measuring clinically relevant potency attributes of MSCs will help to improve consistency in quality and thereby, accelerate clinical development of these cell‐based products. [ABSTRACT FROM AUTHOR]

Background and aims: Monocyte recruitment in the lamina propria and inflammatory phenotype driven by the mucosal microenvironment is critical for the pathogenesis of inflammatory bowel disease. However, the stimuli responsible remain largely unknown. Recent works have focused on stromal cells, the main steady-state cellular component in tissue, as they produce pro-inflammatory chemokines that contribute to the treatment-resistant nature of IBD. Methods: We studied the regulation of these processes by examining the communication patterns between stromal and myeloid cells in ileal Crohn's disease (CD) using a complete single-cell whole tissue sequencing analysis pipeline and in vitro experimentation in mesenchymal cells. Results: We report expansion of S4 stromal cells and monocyte-like inflammatory macrophages in the inflamed mucosa and describe interactions that may establish sustained local inflammation. These include expression of CCL2 by S1 fibroblasts to recruit and retain monocytes and macrophages in the mucosa, where they receive signals for proliferation, survival, and differentiation to inflammatory macrophages from S4 stromal cells through molecules such as MIF, IFNγ, and FN1. The overexpression of CCL2 in ileal CD and its stromal origin was further demonstrated in vitro by cultured mesenchymal cells and intestinal organoids in the context of an inflammatory milieu. Conclusions: Our findings outline an extensive cross-talk between stromal and myeloid cells, which may contribute to the onset and progression of inflammation in ileal Crohn's disease. Understanding the mechanisms underlying monocyte recruitment and polarization, as well as the role of stromal cells in sustaining inflammation, can provide new avenues for developing targeted therapies to treat IBD. [ABSTRACT FROM AUTHOR]

Human mesenchymal stromal cells (hMSCs), whether used alone or together with three-dimensional scaffolds, are the best-studied postnatal stem cells in regenerative medicine. In this study, innovative composite scaffolds consisting of a core–shell architecture were seeded with bone-marrow-derived hMSCs (BM-hMSCs) and tested for their biocompatibility and remarkable capacity to promote and support bone regeneration and mineralization. The scaffolds were prepared by grafting three different amounts of gelatin–chitosan (CH) hydrogel into a 3D-printed polylactic acid (PLA) core (PLA-CH), and the mechanical and degradation properties were analyzed. The BM-hMSCs were cultured in the scaffolds with the presence of growth medium (GM) or osteogenic medium (OM) with differentiation stimuli in combination with fetal bovine serum (FBS) or human platelet lysate (hPL). The primary objective was to determine the viability, proliferation, morphology, and spreading capacity of BM-hMSCs within the scaffolds, thereby confirming their biocompatibility. Secondly, the BM-hMSCs were shown to differentiate into osteoblasts and to facilitate scaffold mineralization. This was evinced by a positive Von Kossa result, the modulation of differentiation markers (osteocalcin and osteopontin), an expression of a marker of extracellular matrix remodeling (bone morphogenetic protein-2), and collagen I. The results of the energy-dispersive X-ray analysis (EDS) clearly demonstrate the presence of calcium and phosphorus in the samples that were incubated in OM, in the presence of FBS and hPL, but not in GM. The chemical distribution maps of calcium and phosphorus indicate that these elements are co-localized in the same areas of the sections, demonstrating the formation of hydroxyapatite. In conclusion, our findings show that the combination of BM-hMSCs and PLA-CH, regardless of the amount of hydrogel content, in the presence of differentiation stimuli, can provide a construct with enhanced osteogenicity for clinically relevant bone regeneration. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer that accounts for over 90% of all pancreatic cancer cases. With a 5-year survival rate of only 13%, PDAC has proven to be extremely desmoplastic and immunosuppressive to most current therapies, including chemotherapy and surgical resection. In recent years, focus has shifted to understanding the tumor microenvironment (TME) around PDAC, enabling a greater understanding of biological pathways and intercellular interactions that can ultimately lead to potential for future drug targets. In this study, we leverage a combination of single-cell and spatial transcriptomics to further identify cellular populations and interactions within the highly heterogeneous TME. We demonstrate that SPP1+APOE+ tumor-associated macrophages (TAM) and CTHRC1+GREM1+ cancer-associated myofibroblasts (myCAF) not only act synergistically to promote an immune-suppressive TME through active extracellular matrix (ECM) deposition and epithelial mesenchymal transition (EMT), but are spatially colocalized and correlated, leading to worse prognosis. Our results highlight the crosstalk between stromal and myeloid cells as a significant area of study for future therapeutic targets to treat cancer. [ABSTRACT FROM AUTHOR]

Background: Recurrent spontaneous abortion (RSA) is a challenging condition that affects the health of women both physically and mentally, but its pathogenesis and treatment have yet to be studied in detail. In recent years, Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) have been shown to be effective in treating various diseases. Current understanding of RSA treatment using WJ-MSCs is limited, and the exact mechanisms of WJ-MSCs action in RSA remains largely unclear. In this study, we explored the decidual deficiencies in RSA and the therapeutic potential of WJ-MSCs at single-cell resolution. Methods: Three mouse models were established: a normal pregnancy group, an RSA group, and a WJ-MSC treatment group. Decidual tissue samples were collected for single-cell RNA sequencing (scRNA-seq) and functional verification, including single-cell resolution in situ hybridization on tissues (SCRINSHOT) and immunofluorescence. Results: We generated a single-cell atlas of decidual tissues from normal pregnant, RSA, and WJ-MSC-treated mice and identified 14 cell clusters in the decidua on day 14. Among these cell populations, stromal cells were the most abundant cell clusters in the decidua, and we further identified three novel subclusters (Str_0, Str_1, and Str_2). We also demonstrated that the IL17 and TNF signaling pathways were enriched for upregulated DEGs of stromal cells in RSA mice. Intriguingly, cell–cell communication analysis revealed that Str_1 cell-related gene expression was greatly reduced in the RSA group and rescued in the WJ-MSC treatment group. Notably, the interaction between NK cells and other cells in the RSA group was attenuated, and the expression of Spp1 (identified as an endometrial toleration-related marker) was significantly reduced in the NK cells of the RSA group but could be restored by WJ-MSC treatment. Conclusion: Herein, we implemented scRNA-seq to systematically evaluate the cellular heterogeneity and transcriptional regulatory networks associated with RSA and its treatment with WJ-MSCs. These data revealed potential therapeutic targets of WJ-MSCs to remodel the decidual subpopulations in RSA and provided new insights into decidua-derived developmental defects at the maternal–foetal interface. [ABSTRACT FROM AUTHOR]

Background: Ferroptosis is associated with the pathological progression of hemorrhagic injury and ischemia–reperfusion injury. According to our previous study, exosomes formed through bone marrow mesenchymal stem cells modified with miR-340-3p (MB-exos) can restore damaged endometrium. However, the involvement of ferroptosis in endometrial injury and the effect of MB-exos on ferroptosis remain elusive. Methods: The endometrial injury rat model was developed. Exosomes were obtained from the supernatants of bone marrow mesenchymal stromal cells (BMSCs) and miR-340/BMSCs through differential centrifugation. We conducted RNA-seq analysis on endometrial tissues obtained from the PBS and MB-exos groups. Ferroptosis was induced in endometrial stromal cells (ESCs) by treating them with erastin or RSL3, followed by treatment with B-exos or MB-exos. We assessed the endometrial total m6A modification level after injury and subsequent treatment with B-exos or MB-exos by methylation quantification assay. We performed meRIP-qPCR to analyze m6A modification-regulated endogenous mRNAs. Results: We reveal that MB-exos facilitate the injured endometrium to recover by suppressing ferroptosis in endometrial stromal cells. The injured endometrium showed significantly upregulated N6-methyladenosine (m6A) modification levels; these levels were attenuated by MB-exos through downregulation of the methylase METTL3. Intriguingly, METTL3 downregulation appears to repress ferroptosis by stabilizing HMOX1 mRNA, thereby potentially elucidating the mechanism through which MB-exos inhibit ferroptosis in ESCs. We identified YTHDF2 as a critical m6A reader protein that contributes to HMOX1 mRNA degradation. YTHDF2 facilitates HMOX1 mRNA degradation by identifying the m6A binding site in the 3′-untranslated regions of HMOX1. In a rat model, treatment with MB-exos ameliorated endometrial injury-induced fibrosis by inhibiting ferroptosis in ESCs. Moreover, METTL3 short hairpin RNA-mediated inhibition of m6A modification enhanced the inhibitory effect of MB-exos on ferroptosis in endometrial injury. Conclusions: Thus, these observations provide new insights regarding the molecular mechanisms responsible for endometrial recovery promotion by MB-exos and highlight m6A modification-dependent ferroptosis inhibition as a prospective therapeutic target to attenuate endometrial injury. [ABSTRACT FROM AUTHOR]

Introduction: Fibroblast activation protein (FAP) overexpression on cancerassociated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. Methods: In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAPpositive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results: Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Discussion: Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells. [ABSTRACT FROM AUTHOR]

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells – menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-β1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems. [ABSTRACT FROM AUTHOR]

Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC‐to‐iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage‐specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage‐specific iMSCs, and six source‐specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo. [ABSTRACT FROM AUTHOR]

Background: Squamous cell carcinomas (SCCs) across different anatomical locations possess common molecular features. Recent studies showed that stromal cells may contribute to tumor progression and metastasis of SCCs. Limited by current sequencing technology and analysis methods, it has been difficult to combine stroma expression profiles with a large number of clinical information. Methods: With the help of transfer learning on the cell line, single-cell, and bulk tumor sequencing data, we identified and validated 2 malignant gene patterns (V1 and V5) expressed by stromal cells of SCCs from head and neck (HNSCC), lung (LUSC), cervix (CESC), esophagus, and breast. Results: Pattern V5 reflected a novel malignant feature that explained the mixed signals of HNSCC molecular subtypes. Higher expression of pattern V5 was related to shorter PFI with gender and cancer-type specificity. The other stromal gene pattern V1 was associated with poor PFI in patients after surgery in all the three squamous cancer types (HNSCC p = 0.0055, LUSC p = 0.0292, CESC p = 0.0451). Cancer-associated fibroblasts could induce HNSCC cancer cells to express pattern V1. Adjuvant radiotherapy may weaken the effect of high V1 on recurrence and metastasis, depending on the tumor radiosensitivity. Conclusion: Considering the prognostic value of stromal gene patterns and its universality, we suggest that the genetic subtype classification of SCCs may be improved to a new system that integrates both malignant and non-malignant components. [ABSTRACT FROM AUTHOR]

Current in vitro models struggle to balance the complexity of human diseases with suitability for large-scale drug tests. While 3D cultures simulate human tissues, they lack cellular intricacy, and integrating these models with high-throughput drug screening remains a challenge. Here, we introduce a method that uses self-assembling nucleic acid nanostructures decorated living cells, termed NACs, to create spheroids with a customizable 3D layout. To demonstrate its uniqueness, our method effectively creates designer 3D spheroids by combining parenchymal cells, stromal cells, and immune cells, leading to heightened physiological relevance and detailed modeling of complex chronic diseases and immune-stromal interactions. Our approach achieves a high level of biological fidelity while being standardized and straightforward to construct with the potential for large-scale drug discovery applications. By merging the precision of DNA nanotechnology with advanced cell culture techniques, we are streamlining human-centric models, striking a balance between complexity and standardization, to boost drug screening efficiency. [ABSTRACT FROM AUTHOR]

Reliable cell labeling and tracking techniques are imperative for elucidating the intricate and ambiguous interactions between mesenchymal stromal cells (MSCs) and tumors. Here, we explore fluorescent photoconvertible nanoengineered vesicles to study mMSC migration in brain tumors. These 3 μm sized vesicles made of carbon nanoparticles, Rhodamine B (RhB), and polyelectrolytes are readily internalized by cells. The dye undergoes photoconversion under 561 nm laser exposure with a fluorescence blue shift upon demand. The optimal laser irradiation duration for photoconversion was 0.4 ms, which provided a maximal blue shift of the fluorescent signal label without excessive laser exposure on cells. Vesicles modified with an extra polymer layer demonstrated enhanced intracellular uptake without remarkable effects on cell viability, motility, or proliferation. The optimal ratio of 20 vesicles per mMSC was determined. Moreover, the migration of individual mMSCs within 2D and 3D glioblastoma cell (EPNT-5) colonies over 2 days and in vivo tumor settings over 7 days were traced. Our study provides a robust nanocomposite platform for investigating MSC–tumor dynamics and offers insights into envisaged therapeutic strategies. Photoconvertible vesicles also present an indispensable tool for studying complex fundamental processes of cell–cell interactions for a wide range of problems in biomedicine. [ABSTRACT FROM AUTHOR]

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-β and IL-1β was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies. [ABSTRACT FROM AUTHOR]

Background: Endometriosis (EM) is a chronic painful condition that predominantly affects women of reproductive age. Currently, surgery or medication can only provide limited symptom relief. This study used a comprehensive genetic analytical approach to explore potential drug targets for EM in the plasma proteome. Methods: In this study, 2,923 plasma proteins were selected as exposure and EM as outcome for two-sample Mendelian randomization (MR) analyses. The plasma proteomic data were derived from the UK Biobank Pharmaceutical Proteomics Project (UKB-PPP), while the EM dataset from the FinnGen consortium R10 release data. Several sensitivity analyses were performed, including summary-data-based MR (SMR) analyses, heterogeneity in dependent instruments (HEIDI) test, reverse MR analyses, steiger detection test, and bayesian co-localization analyses. Furthermore, proteome-wide association study (PWAS) and single-cell transcriptomic analyses were also conducted to validate the findings. Results: Six significant (p < 3.06 × 10-5) plasma protein-EM pairs were identified by MR analyses. These included EPHB4 (OR = 1.40, 95% CI: 1.20 - 1.63), FSHB (OR = 3.91, 95% CI: 3.13 - 4.87), RSPO3 (OR = 1.60, 95% CI: 1.38 - 1.86), SEZ6L2 (OR = 1.44, 95% CI: 1.23 - 1.68) andWASHC3 (OR = 2.00, 95% CI: 1.54 - 2.59) were identified as risk factors, whereas KDR (OR = 0.80, 95% CI: 0.75 - 0.90) was found to be a protective factor. All six plasma proteins passed the SMR test (P < 8.33 × 10-3), but only four plasma proteins passed the HEIDI heterogeneity test (PHEIDI > 0.05), namely FSHB, RSPO3, SEZ6L2 and EPHB4. These four proteins showed strong evidence of co-localization (PPH4 > 0.7). In particular, RSPO3 and EPHB4 were replicated in the validated PWAS. Single-cell analyses revealed high expression of SEZ6L2 and EPHB4 in stromal and epithelial cells within EM lesions, while RSPO3 exhibited elevated expression in stromal cells and fibroblasts. Conclusion: Our study identified FSHB, RSPO3, SEZ6L2, and EPHB4 as potential drug targets for EM and highlighted the critical role of stromal and epithelial cells in disease development. These findings provide new insights into the diagnosis and treatment of EM. [ABSTRACT FROM AUTHOR]

Exosomes derived from mesenchymal stem cells (MSCs) have been confirmed to enhance cell proliferation and improve tissue repair. Exosomes release their contents into the cytoplasmic solution of the recipient cell to mediate cell expression, which is the main pathway through which exosomes exert therapeutic effects. The corresponding process of exosome internalization mainly occurs in the early stage of treatment. However, the therapeutic effect of exosomes in the early stage remains to be further studied. We report that the three-dimensional cell traction force can intuitively reflect the ability of exosomes to enhance the cytoskeleton and cell contractility of recipient cells, serving as an effective method to characterize the therapeutic effect of exosomes. Compared with traditional biochemical methods, we can visualize the early therapeutic effect of exosomes in real time without damage by quantifying the cell traction force. Through quantitative analysis of traction forces, we found that endometrial stromal cells exhibit short-term cell roundness accompanied by greater traction force during the early stage of exosome therapy. Further experiments revealed that exosomes enhance the traction force and cytoskeleton by regulating the Rac1/RhoA signaling pathway, thereby promoting cell proliferation. This work provides an effective method for rapidly quantifying the therapeutic effects of exosomes and studying the underlying mechanisms involved. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC). Methods: DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry. Results: DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages. Conclusions: Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC. [ABSTRACT FROM AUTHOR]

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders. The ability to maintain blood stem cells (HSCs) in vitro would allow us to provide better therapies for blood diseases. Here, the authors report that polymer-organised extracellular proteins, coupled to soft environments mimicking bone marrow stiffness, allow stromal cells to maintain HSCs in vitro. [ABSTRACT FROM AUTHOR]

The suppressive tumour microenvironment significantly hinders the efficacy of immunotherapy in treating solid tumors. In this context, stromal cells, such as tumour-associated fibroblasts, undergo changes that include an increase in the number and function of immunosuppressive cells. Adenosine, a factor that promotes tumour growth, is produced from ATP breakdown and is markedly elevated in the tumour microenvironment. It acts through specific binding to adenosine receptors, with A2A and A2B adenosine receptor being primary drivers of immunosuppression. This paper presents the roles of various adenosine receptors in different tumour microenvironments. This review focus on the function of adenosine receptors in the stromal cells and non-cellular components of the tumour microenvironment. Additionally, we summarize and discuss recent advances and potential trends in using adenosine receptor antagonists combined with immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. Methods: To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl−/−) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). Results: We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl−/− mice as compared to wild-type. Rankl−/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl−/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. Conclusions: We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance. [ABSTRACT FROM AUTHOR]

Mesenchymal stem and stromal cells (MSCs) hold potential to treat a broad range of clinical indications, but clinical translation has been limited to date due in part to challenges with batch-to-batch reproducibility of potential critical quality attributes (pCQAs) that can predict potency/efficacy. Here, we designed and implemented a microcarrier-microbioreactor approach to cell therapy manufacturing, specific to anchorage-dependent cells such as MSCs. We sought to assess whether increased control of the biochemical and biophysical environment had the potential to create product with consistent presentation and elevated expression of pCQAs relative to established manufacturing approaches in tissue culture polystyrene (TCPS) flasks. First, we evaluated total cell yield harvested from dissolvable, gelatin microcarriers within a microbioreactor cassette (Mobius Breez) or a flask control with matched initial cell seeding density and culture duration. Next, we identified 24 genes implicated in a therapeutic role for a specific motivating indication, acute respiratory distress syndrome (ARDS); expression of these genes served as our pCQAs for initial in vitro evaluation of product potency. We evaluated mRNA expression for three distinct donors to assess inter-donor repeatability, as well as for one donor in three distinct batches to assess within-donor, inter-batch variability. Finally, we assessed gene expression at the protein level for a subset of the panel to confirm successful translation. Our results indicated that MSCs expanded with this microcarrier-microbioreactor approach exhibited reasonable donor-to-donor repeatability and reliable batch-to-batch reproducibility of pCQAs. Interestingly, the baseline conditions of this microcarrier-microbioreactor approach also significantly improved expression of several key pCQAs at the gene and protein expression levels and reduced total media consumption relative to TCPS culture. This proof-of-concept study illustrates key benefits of this approach to therapeutic cell process development for MSCs and other anchorage-dependent cells that are candidates for cell therapies. [ABSTRACT FROM AUTHOR]

Cancer, as a complicated disease, is considered to be one of the major leading causes of death globally. Although various cancer therapeutic strategies have been established, however, some issues confine the efficacies of the treatments. In recent decades researchers for finding efficient therapeutic solutions have extensively focused on the abilities of stem cells in cancer inhibition. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can the most widely extracted from various sources such as the bone marrow (BM), placenta, umbilical cord (UC), menses blood, Wharton's jelly (WJ), adipose tissue and dental pulp (DP). These cells are capable of differentiating into the osteoblasts, chondrocytes, and adipocytes. Due to the unique characteristics of MSCs such as paracrine effects, immunomodulation, tumor-tropism, and migration, they are considered promising candidates for cancer therapeutics. Currently, MSCs are an excellent living carrier for delivery of therapeutic genes and chemical agents to target tumor sites. Also, exosomes, the most important extracellular vesicle released from MSCs, act as a strong cell-free tool for cancer therapeutics. MSCs can prevent cancer progression by inhibiting several signaling pathways, such as wnt/β-catenin and PI3K/AKT/mTOR. However, there are several challenges associated with the use of MSCs and their exosomes in the field of therapy that need to be considered. This review explores the significance of MSCs in cell-based therapy, focusing on their homing properties and immunomodulatory characteristics. It also examines the potential of using MSCs as carriers for delivery of anticancer agents and their role in modulating the signal transduction pathways of cancer cells. [ABSTRACT FROM AUTHOR]

Objective: Kienböck disease is a rare condition characterized by severe pain and restricted wrist movement. Various palliative methods have been proposed as therapeutic strategies for alleviating symptoms. Mesenchymal stromal cell transplantation has been suggested as an innovative and promising approach due to its potential for inducing regeneration and immunomodulation in the necrotic tissue. This study aims to evaluate the safety of autologous bone marrow derived mesenchymal stromal cells (BM-MSCs) transplantation after core decompression in Kienböck disease. Materials and Methods: In this phase I of an open-label clinical trial, three patients (one female and two males) with stage 2 Kienböck disease underwent autologous BM-MSCs transplantation following lunate core decompression. The patients were followed up for six months to assess safety as well as secondary clinical outcomes, including pain level, range of motion (ROM), and functional disability. Results: Safety of BM-MSCs injection following the core decompression was evaluated by recording post-treatment complications during the six-month follow-up. No adverse events (AEs) or severe AEs (SAEs) were reported, indicating that BM-MSCs injection after core decompression is a safe intervention. All patients showed a remarkable reduction in visual analog scale (VAS) scores and "Disabilities of the Arm, Shoulder, and Hand" (DASH) questionnaire scores, suggesting the therapeutic potential of this intervention. Moreover, an increase in the ROM indicated that BM-MSCs transplantation can improve wrist functionality. Additionally, radiographic assessments before and after cell infusion demonstrated a reduction in lunate sclerosis after six months of follow-up. Conclusion: The transplantation of autologous BM-MSCs following lunate core decompression seems to be a safe clinical intervention and may lead to pain relief in patients with Kienböck disease. Furthermore, this procedure may help prevent disease progression during the follow-up period (registration number: NCT02646007). [ABSTRACT FROM AUTHOR]

Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in various diseases and is gaining considerable attention in the realm of endometriosis. Considering the classical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased iron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective case–control study provides a comprehensive immunohistochemical assessment of the expression and tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis patients. The case group consisted of 38 women with laparoscopically and histologically confirmed endometriosis and the control group consisted of 18 women with other gynecological conditions. Our study revealed a significant downregulation of GPX4 in stromal cells of endometriosis patients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = −2.90, p = 0.005). This finding aligned with slightly, but not significantly, higher iron levels detected in the blood of endometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2–13.5) g/dL versus 12.5 (12.2–13.4) g/dL in the control group; t (54) = −0.897, p = 0.374). Interestingly, there was no concurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014), responsible for iron uptake into cells. Our empirical findings provide support for the involvement of ferroptosis in the context of endometriosis. However, variances in expression patterns within stromal and epithelial cellular subsets call for further in-depth investigations. [ABSTRACT FROM AUTHOR]

Mesenchymal stem/stromal cells (MSCs) are one of the most widely used cell types in advanced therapies due to their therapeutic potential in the regulation of tissue repair and homeostasis, and immune modulation. However, their use in cancer therapy is controversial: they can inhibit cancer cell proliferation, but also potentially promote tumour growth by supporting angiogenesis, modulation of the immune milieu and increasing cancer stem cell invasiveness. This opposite behaviour highlights the need for careful and nuanced use of MSCs in cancer treatment. To optimize their anti-cancer effects, diverse strategies have bioengineered MSCs to enhance their tumour targeting and therapeutic properties or to deliver anti-cancer drugs. In this review, we highlight the advanced uses of MSCs in cancer therapy, particularly as carriers of targeted treatments due to their natural tumour-homing capabilities. We also discuss the potential of MSC-derived extracellular vesicles to improve the efficiency of drug or molecule delivery to cancer cells. Ongoing clinical trials are evaluating the therapeutic potential of these cells and setting the stage for future advances in MSC-based cancer treatment. It is critical to identify the broad and potent applications of bioengineered MSCs in solid tumour targeting and anti-cancer agent delivery to position them as effective therapeutics in the evolving field of cancer therapy. [ABSTRACT FROM AUTHOR]

The current limitations of commercial microcarriers for mesenchymal stromal cell (MSC) expansion are driving the demand for novel tuneable microcarriers. Novel microcarriers are effective in improving MSC expansion under static and dynamic culture conditions without compromising their stemness and differentiation potential. Integrating microcarrier-based cultures within dynamic bioreactors provides an effective scalable approach for MSC expansion and harvest for downstream clinical translation. The development of good manufacturing practice (GMP)-grade systems, non-xenogenic culture media, and optimised bioreactors, which are compatible with novel microcarriers, is essential to improve cell yield and reproducibility. Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs. [ABSTRACT FROM AUTHOR]

Addressing the challenges in managing ischemic tissue repair and remodelling remains a prominent clinical concern. Current research is heavily concentrated on identifying innovative cell-based therapies with the potential to enhance revascularization in patients affected by these diseases. We have previously developed and validated a manufacturing process for human umbilical cord mesenchymal stromal cells (UC-MSCs)-based cell therapy medicinal product, according to Good Manufacturing Practices. In this study, we demonstrate that these UC-MSCs enhance the proliferation and migration of endothelial cells and the formation of capillary structures. Moreover, UC-MSCs and endothelial cells interact, allowing UC-MSCs to acquire a perivascular cell phenotype and consequently provide direct support to the newly formed vascular network. This characterization of the proangiogenic properties of this UC-MSCs based-cell therapy medicinal product is an essential step for its therapeutic assessment in the clinical context of vascular regeneration. [ABSTRACT FROM AUTHOR]

Summary: B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL) blasts strictly depend on the transport of extra‐cellular asparagine (Asn), yielding a rationale for L‐asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP‐ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V‐9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase‐sensitive RS4;11 cells and the relatively ASNase‐insensitive NALM‐6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP‐ALL. [ABSTRACT FROM AUTHOR]