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Circulating tumor cells (CTCs) are noninvasive biomarkers that can indicate the therapeutic response and prognosis. The study aimed to investigate the cellular characteristics of CTCs focusing on monitoring during atezolizumab and bevacizumab (Atezo-Bev) therapy in patients with hepatocellular carcinoma (HCC). Peripheral blood samples were collected from 10 healthy controls and 40 patients with HCC. CTCs enriched using RosetteSep™ Human CD45 depletion cocktail were analyzed by multiparametric flow cytometry. CTC isolation was based on PanCK(+)CD45(-) cells, and CTCs exhibiting markers CD90, CD133, EpCAM, or vimentin. The total number of CTCs and the number of CTCs expressing CD90, CD133, EpCAM, and vimentin were correlated with the BCLC stage of HCC. The change in total CTC count accurately reflected the initial response to Atezo-Bev therapy. The numbers and mean fluorescence intensity of the CTC subsets expressing CD90 and EpCAM molecules decreased in patients with partial response/stable disease, and increased in patients with progressive disease and were markedly correlated with overall survival. CD90(+) and EpCAM(+) CTCs may be candidate biomarkers for the early prediction of the treatment response and the overall survival of patients with HCC receiving Atezo-Bev therapy.

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The combination of anti-angiogenesis agents with immune-checkpoint inhibitors is a promising treatment for patients with advanced hepatocellular carcinoma (HCC); however, therapeutic resistance caused by cancer stem cells present in tumor microenvironments remains to be overcome. In this study, we report for the first time that the Kringle 1 domain of human hepatocyte growth-factor α chain (HGFK1), a previously described anti-angiogenesis peptide, repressed the sub-population of CD90+ cancer stem cells (CSCs) and promoted their differentiation and chemotherapy sensitivity mainly through downregulation of pre-Met protein expression and inhibition of Wnt/ß-catenin and Notch pathways. Furthermore, we showed that the i.p. injection of PH1 (a tumor-targeted and biodegradable co-polymer), medicated plasmids encoding Endostatin (pEndo), HGFK1 genes (pEndo), and a combination of 50% pEndo + 50% pHGFK1 all significantly suppressed tumor growth and prolonged the survival of the HCC-bearing mice. Importantly, the combined treatment produced a potent synergistic effect, with 25% of the mice showing the complete clearance of the tumor via a reduction in the microvessel density (MVD) and the number of CD90+ CSCs in the tumor tissues. These results suggest for the first time that HGFK1 inhibits the CSCs of HCC. Furthermore, the combination of two broad-spectrum anti-angiogenic factors, Endo and HGFK1, is the optimal strategy for the development of effective anti-HCC drugs.

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BACKGROUND: Over the past 25 years, acquired equine polyneuropathy (AEP) has emerged as a neurological disease in Scandinavian horses. This condition is characterized by histopathological features including the presence of Schwann cell (SC) inclusions. Cultivated equine SCs would serve as a valuable resource for investigations of factors triggering this Schwannopathy. Ideally, cells should be sampled for cultivation from fresh nerves immediately after death of the animal, however the availability of fresh material is limited, due to the inconsistent case load and the inherent technical and practical challenges to collection of samples in the field. This study aimed to cultivate SCs from adult equine peripheral nerves and assess their ability to survive in sampled nerve material over time to simulate harvesting of SCs in field situations. NEW METHODS: Peripheral nerves from five non-neurological horses were used. After euthanasia, both fresh and non-fresh nerve samples were harvested from each horse. Flow cytometry was employed to confirm the cellular identity and to determine the SC purity. RESULTS: The results revealed successful establishment of SC cultures from adult equine peripheral nerves, with the potential to achieve high SC purity from both fresh and non-fresh nerve samples. COMPARISON WITH EXISTING METHOD: While most SC isolation methods focus on harvest of cells from fresh nerve materials from laboratory animals, our approach highlights the possibility of utilizing SC cultures from field-harvested and transported nerve samples from horses. CONCLUSIONS: We describe a method for isolating SCs with high purity from both fresh and non-fresh peripheral nerves of adult horses.

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PURPOSE: On the new era of stem cell therapy, the present experimental study was conducted to investigate renal regenerative capacity related to kidney stem cell reserve in different nephrectomy (Nx) models. METHODS: Three- and eight-week-old rats (n = 168) were randomly divided into four groups to include control and three Nx subgroups (1/6 Nx, 1/2 Nx, and 5/6 Nx) (Fig. 1). On post-Nx days 15, 30 and 60, kidney specimens were obtained to determine renal regenerative capacity. The specimens were examined with immunofluorescence. CD90/CD105 and Ki-67 expressions were determined as stem cell and cellular proliferation markers, respectively. Fig. 1 Intraoperative photographs showing three different types of nephrectomies (unilateral total Nx has not been shown in 5/6 Nx group) RESULTS: CD90 and CD105 expressions were stronger in glomeruli, but Ki-67 expressions were present only in tubuli. When all Nx types and post-Nx days were considered, both 3- and 8-week-old rats undergone 5/6 Nx had the highest glomerular CD90 and CD105 double expressions. While the expressions gradually increased toward the day 60 in 3-weeks old rats, 8-week-old rats had almost stable double expressions. The strongest tubular Ki-67 expressions were seen in 5/6 Nx groups of both in 3- and 8-week-old rats. The expressions were strongest on day 15 and then gradually decreased. Ipsilateral 1/6 Nx groups had stronger Ki-67 expression than contralateral ones in both age groups. CONCLUSIONS: Kidneys may pose a regenerative response to tissue/volume loss through its own CD90- and CD105-related stem cell reserve which mainly takes place in glomeruli and seems to have some interactions with Ki-67-related tubular proliferative process. This response supports that kidney stem cells may have a potential to overcome tissue/volume loss-related damage.

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Cell adhesion and migration depend on the assembly and disassembly of adhesive structures known as focal adhesions. Cells adhere to the extracellular matrix (ECM) and form these structures via receptors, such as integrins and syndecans, which initiate signal transduction pathways that bridge the ECM to the cytoskeleton, thus governing adhesion and migration processes. Integrins bind to the ECM and soluble or cell surface ligands to form integrin adhesion complexes (IAC), whose composition depends on the cellular context and cell type. Proteomic analyses of these IACs led to the curation of the term adhesome, which is a complex molecular network containing hundreds of proteins involved in signaling, adhesion, and cell movement. One of the hallmarks of these IACs is to sense mechanical cues that arise due to ECM rigidity, as well as the tension exerted by cell-cell interactions, and transduce this force by modifying the actin cytoskeleton to regulate cell migration. Among the integrin/syndecan cell surface ligands, we have described Thy-1 (CD90), a GPI-anchored protein that possesses binding domains for each of these receptors and, upon engaging them, stimulates cell adhesion and migration. In this review, we examine what is currently known about adhesomes, revise how mechanical forces have changed our view on the regulation of cell migration, and, in this context, discuss how we have contributed to the understanding of signaling mechanisms that control cell adhesion and migration.

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The senescence of adipose stem cells (ASCs) impairs healthy adipose tissue remodeling, causing metabolic maladaptation to energy surplus. The intrinsic molecular pathways and potential therapy targets for ASC senescence are largely unclear. Here, we showed that visceral ASCs were prone to senescence that was caused by reactive oxygen species (ROS) overload, especially mitochondrial ROS. These senescent ASCs failed to sustain efficient glucose influx, pentose phosphate pathway (PPP) and redox homeostasis. We showed that CD90 silence restricted the glucose uptake by ASCs and thus disrupted their PPP and anti-oxidant system, resulting in ASC senescence. Notably, fibroblast growth factor 21 (FGF21) treatment significantly reduced the senescent phenotypes of ASCs by augmenting CD90 protein via glycosylation, which promoted glucose influx via the AKT-GLUT4 axis and therefore mitigated ROS overload. For diet-induced obese mice, chronic administration of low-dose FGF21 relieved their visceral white adipose tissue (VAT) dysfunction and systemic metabolic disorders. In particular, VAT homeostasis was restored in FGF21-treated obese mice, where ASC repertoire was markedly recovered, accompanied by CD90 elevation and anti-senescent phenotypes in these ASCs. Collectively, we reveal a molecular mechanism of ASC senescence by which CD90 downregulation interferes glucose influx into PPP and redox homeostasis. And we propose a FGF21-based strategy for healthy VAT remodeling, which targets CD90 glycosylation to correct ASC senescence and therefore combat obesity-related metabolic dysfunction.

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Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34+ hematopoietic stem and progenitor cells containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. We have previously identified the CD34+CD90+ subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells ex vivo. Most important, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. Our novel HSC-targeted viral vectors have the potential to significantly enhance the feasibility of ex vivo gene therapy and pave the way for future in vivo applications.

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Introduction: Engineered bone graft substitutes are a promising alternative and supplement to autologous bone grafts as treatments for bone healing impairment. Advances in human medicine extend an invitation to pursue these biomimetic strategies in animal patients, substantiated by the theory that specialized scaffolds, multipotent cells, and biological cues may be combined into a bioactive implant intended for the enhancement of tissue regeneration. Methods: This proof-of-concept study was designed to evaluate and validate the feasibility of beta-tricalcium phosphate foam scaffolds seeded with canine mesenchymal stem cells derived from adipose tissue. Cell-inoculated samples and sham controls were cultured statically for 72 hours in complete growth medium to evaluate seeding capacity, while a subset of loaded scaffolds was further induced with osteogenic culture medium for 21 days. Produced implants were characterized and validated with a combination of immunofluorescence and reflection confocal microscopy, scanning electron microscopy, and polymerase chain reaction to confirm osteogenic differentiation in tridimensional-induced samples. Results: After 72 hours of culture, all inoculated scaffolds presented widespread yet heterogeneous surface seeding, distinctively congregating stem cells around pore openings. Furthermore, at 21 days of osteogenic culture conditions, robust osteoblastic differentiation of the seeded cells was confirmed by the change of cell morphology and evident deposition of extra-cellular matrix, accompanied by mineralization and scaffold remodeling; furthermore, all induced cell-loaded implants lost specific stemness immunophenotype expression and simultaneously upregulated genomic expression of osteogenic genes Osterix and Ostecalcin. Conclusions: ß-TCP bio-ceramic foam scaffolds proved to be suitable carriers and hosts of canine adipose-derived MSCs, promoting not only surface attachment and proliferation, but also demonstrating strong in-vitro osteogenic potential. Although this research provides satisfactory in-vitro validation for the conceptualization and feasibility of a canine bio-active bone implant, further testing such as patient safety, large-scale reproducibility, and quality assessment are needed for regulatory compliance in future commercial clinical applications.

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OBJECTIVES: Thy-1 (CD90) is a glycosylphosphatidyl-anchored protein belonging to the immunoglobulin family and is known to control mesenchymal stromal cells differentiation into osteoblasts or adipocytes. The study aimed to investigate the salivary levels of Thy-1 in health, periodontitis, obesity, and any potential association. MATERIALS AND METHODS: Seventy-one participants were divided into four groups: healthy (H), subjects with periodontitis (P), obese individuals (O), and obese individuals having periodontitis (PO). Unstimulated whole saliva was collected from participants who were evaluated for periodontal parameters. The levels of Thy-1 were measured with a commercially available ELISA kit. The data were statistically analyzed. RESULTS: A significant difference in salivary Thy-1 levels among different groups was observed. Periodontitis patients had the maximum, and obese individuals had the minimum Thy-1 levels. Significant differences between H and P, H and PO, P and O, and O and PO were observed. Overall correlations between Thy-1 and periodontal parameters and a positive correlation with pocket depth in group PO were noted. CONCLUSION: Thy-1 could be detected in the saliva of all study participants. It is implied that a local inflammatory condition like periodontitis elevates the salivary levels of Thy-1 with, and without obesity.

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Chronic muscle injuries, such as massive rotator cuff tears, are associated with progressive muscle wasting, fibrotic scarring, and intramuscular fat accumulation. While progenitor cell subsets are usually studied in culture conditions that drive either myogenic, fibrogenic, or adipogenic differentiation, it is still unknown how combined myo-fibro-adipogenic signals, which are expected to occur in vivo, modulate progenitor differentiation. We therefore evaluated the differentiation potential of retrospectively generated subsets of primary human muscle mesenchymal progenitors in multiplexed conditions in the presence or absence of 423F drug, a modulator of gp130 signaling. We identified a novel CD90+CD56- non-adipogenic progenitor subset that maintained a lack of adipogenic potential in single and multiplexed myo-fibro-adipogenic culture conditions. CD90-CD56- demarcated fibro-adipogenic progenitors (FAP) and CD56+CD90+ progenitors were typified as myogenic. These human muscle subsets exhibited varying degrees of intrinsically regulated differentiation in single and mixed induction cultures. Modulation of gp130 signaling via 423F drug mediated muscle progenitor differentiation in a dose-, induction-, and cell subset-dependent manner and markedly decreased fibro-adipogenesis of CD90-CD56- FAP. Conversely, 423F promoted myogenesis of CD56+CD90+ myogenic subset, indicated by increased myotube diameter and number of nuclei per myotube. 423F treatment eliminated FAP-derived mature adipocytes from mixed adipocytes-FAP cultures but did not modify the growth of non-differentiated FAP in these cultures. Collectively, these data demonstrate that capability of myogenic, fibrogenic, or adipogenic differentiation is largely dependent on the intrinsic features of cultured subsets, and that the degree of lineage differentiation varies when signals are multiplexed. Moreover, our tests performed in primary human muscle cultures reveal and confirm the potential triple-therapeutic effects of 423F drug which simultaneously attenuates degenerative fibrosis, fat accumulation and promotes myo-regeneration.

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Parkinson's disease with cognitive impairment (PD-CI) results in several clinical outcomes for which specific treatment is lacking. Although the pathogenesis of PD-CI has not yet been fully elucidated, it is related to neuronal plasticity decline in the hippocampus region. The dopaminergic projections from the substantia nigra to the hippocampus are critical in regulating hippocampal plasticity. Recently, aerobic exercise has been recognized as an effective therapeutic strategy for enhancing plasticity through the secretion of various muscle factors. The exact role of FNDC5-an upregulated, newly identified myokine produced after exercise-in mediating hippocampal plasticity and regional dopaminergic projections in PD-CI remains unclear. In this study, the effect of treadmill exercise on hippocampal synaptic plasticity was evaluated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced chronic PD models. The results showed that treadmill exercise substantially alleviated the motor dysfunction, cognition disorder, and dopaminergic neuron degeneration induced by MPTP. Here, we discovered that the quadriceps, serum, and brain FNDC5 levels were lower in PD mice and that intervention with treadmill exercise restored FNDC5 levels. Moreover, treadmill exercise enhanced the synaptic plasticity of hippocampal pyramidal neurons via increased dopamine levels and BDNF in the PD mice. The direct protective effect of FNDC5 is achieved by promoting the secretion of BDNF in the hippocampal neurons via binding the integrin αVß5 receptor, thereby improving synaptic plasticity. Regarding the indirect protection effect, FNDC5 promotes the dopaminergic connection from the substantia nigra to the hippocampus by mediating the interaction between the integrin αVß5 of the hippocampal neurons and the CD90 molecules on the membrane of dopaminergic terminals. Our findings demonstrated that treadmill exercise could effectively alleviate cognitive disorders via the activation of the FNDC5-BDNF pathway and enhance the dopaminergic synaptic connection from SNpc to the hippocampus in the MPTP-induced chronic PD model.

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BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

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Huge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts in T cell biology. As such, flow cytometry gating strategies and markers, such as CD90, have been applied to indentify ILC. Here, we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly a sub-population of cells exhibit low or even no expression of this marker. CD90-negative and CD90-low CD127+ ILC were present amongst all ILC subsets in the gut. The frequency of CD90-negative and CD90-low CD127+ ILC was dependent on stimulatory cues in vitro and enhanced by dysbiosis in vivo. CD90-negative and CD90-low CD127+ ILC were a potential source of IL-13, IFNγ and IL-17A at steady state and upon dysbiosis- and dextran sulphate sodium-elicited colitis. Hence, this study reveals that, contrary to expectations, CD90 is not constitutively expressed by functional ILC in the gut.

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We had previously shown that THY1 (CD90) is a tumor suppressor in nasopharyngeal carcinoma (NPC) and that its down-regulation and loss of expression are associated with tumor metastasis, yet the mechanism leading to such effects remains unknown. In this study we show that tumor invasion could be suppressed by THY1 via adherens junction formation in a few NPC cell lines, and knockdown of THY1 would disrupt this cell-cell adhesion phenotype. Mechanistically, the activity of the SRC family kinase (SFK) member, SRC, and canonical Wnt signaling were dramatically reduced when THY1 was constitutively expressed. Previous studies by others have found that high levels of SRC activity in NPCs are associated with EMT and a poor prognosis. We hypothesized that THY1 can suppress tumor invasion in NPC via inhibition of SRC. By gene silencing of SRC, we found that the in vitro NPC cell invasion was significantly reduced and adherens junctions were restored. Through proteomic analysis, we identified that platelet-derived growth factor receptor ß (PDGF-Rß) and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) are novel and potential binding partners of THY1, which were subsequently verified by co-immunoprecipitation (co-IP) analysis. The ligand of PDGF-Rß (PDGF-BB) could highly induce SRC activation and NPC cell invasion, which could be almost completely suppressed by THY1 expression. On the other hand, the PTPN22 siRNA could enhance both the SRC activities and the cell invasion and could also disrupt the adherens junctions in the THY1-expressing NPC cells; the original THY1-induced phenotypes were reverted when the PTPN22 expression was reduced. Together, our results identified that PTPN22 is essential for THY1 to suppress cell invasion and SRC activity, maintain tight adherens junctions, and prevent NPC metastasis. These results suggested that PDGF-Rß and SRC can be used as drug targets for suppressing NPC metastasis. Indeed, our in vivo assay using the SRC inhibitor KX2-391, clearly showed that inhibition of SRC signaling can prevent the metastasis of NPC, indicating that targeting SRC can be a promising approach to control the NPC progression.

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Studying the expression of hematopoietic stem cell markers from different sources might be useful in understanding stem cell biology in different niche conditions. The study aimed to assess the difference in cell surface markers (CD44, CD90, CD96) on hematopoietic stem cells in three different niche conditions; umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow samples from idiopathic (immune) thrombocytopenic purpura (IBM). This study was conducted on 300 cases divided into three study groups; 100 umbilical cord blood units collected from mothers undergoing cesarian section in gynecology and obstetrics department, 100 bone marrow samples from idiopathic (immune) thrombocytopenic purpura patients collected from university children hospital and 100 normal bone marrow samples with no evidence of disease in bone marrow tissue. CD44 was significantly elevated in UCB and NBM groups compared to IBM group (<0.001). There was also a significant elevation of CD90 and CD96 in IBM group compared to NBM group and UCB (<0.001). CD90 and CD96 play a role in the pathogenesis of ITP disorder and could be applied as a targeted therapy to improve the outcome of this disease.

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Fibroblasts are highly heterogeneous mesenchymal stromal cells, and different fibroblast subpopulations play different roles. A subpopulation of fibroblasts expressing CD90, a 25-37 kDa glycosylphosphatidylinositol anchored protein, plays a dominant role in the fibrotic and pro-inflammatory state. In this review, we focused on CD90+ fibroblasts, and their roles and possible mechanisms in disease processes. First, the main biological functions of CD90+ fibroblasts in inducing angiogenesis and maintaining tissue homeostasis are described. Second, the role and possible mechanism of CD90+ fibroblasts in inducing pulmonary fibrosis, inflammatory arthritis, inflammatory skin diseases, and scar formation are introduced, and we discuss how CD90+ cancer-associated fibroblasts might serve as promising cancer biomarkers. Finally, we propose future research directions related to CD90+ fibroblasts. This review will provide a theoretical basis for the diagnosis and treatment CD90+ fibroblast-related disease.

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Mesenchymal stem cells are expected to be a cell source for stem cell therapy of various diseases in veterinary medicine. However, donor-dependent cell heterogenicity has been a cause of inconsistent therapeutic efficiency. Therefore, we established immortalized cells from canine adipose tissue-derived mesenchymal stem cells (ADSCs) to minimize cellular heterogeneity by reducing the number of donors, evaluated their properties, and compared them to the primary cells with RNA-sequencing. Immortalized canine ADSCs were established by transduction with combinations of the R24C mutation of human cyclin-dependent kinase 4 (CDKR24C), canine cyclin D1, and canine TERT. The ADSCs transduced with CDK4R24C, cyclin D1, and TERT (ADSC-K4DT) or with CDK4R24C and cyclin D1 (ADSC-K4D) showed a dramatic increase in proliferation (population doubling level >100) without cellular senescence compared to the primary ADSCs. The cell surface markers, except for CD90 of the ADSC-K4DT and ADSC-K4D cells, were similar to those of the primary ADSCs. The ADSC-K4DT and ADSC-K4D cells maintained their trilineage differentiation capacity and chromosome condition, and did not have a tumorigenic development. The ability to inhibit lymphocyte proliferation by the ADSC-K4D cells was enhanced compared with the primary ADSCs and ADSC-K4DT cells. The pathway analysis based on RNA-sequencing revealed changes in the pathways mainly related to the cell cycle and telomerase. The ADSC-K4DT and ADSC-K4D cells had decreased CD90 expression, but there were no obvious defects associated with the decreased CD90 expression in this study. Our results suggest that ADSC-K4DT and ADSC-K4D cells are a potential novel cell source for mesenchymal stem cell therapy.

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The preclinical development of hematopoietic stem cell (HSC) gene therapy/editing and transplantation protocols is frequently performed in large animal models such as nonhuman primates (NHPs). Similarity in physiology, size, and life expectation as well as cross-reactivity of most reagents and medications allows for the development of treatment strategies with rapid translation to clinical applications. Especially after the adverse events of HSC gene therapy observed in the late 1990s, the ability to perform autologous transplants and follow the animals long-term make the NHP a very attractive model to test the efficiency, feasibility, and safety of new HSC-mediated gene-transfer/editing and transplantation approaches.This protocol describes a method to phenotypically characterize functionally distinct NHP HSPC subsets within specimens or stem cell products from three different NHP species. Procedures are based on the flow-cytometric assessment of cell surface markers that are cross-reactive in between human and NHP to allow for immediate clinical translation. This protocol has been successfully used for the quality control of enriched, cultured, and gene-modified NHP CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as sort-purified CD34 subsets for transplantation in the pig-tailed, cynomolgus, and rhesus macaque. It further allows the longitudinal assessment of primary specimens taken during the long-term follow-up post-transplantation in order to monitor homing, engraftment, and reconstitution of the bone marrow stem cell compartment.

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During aging, the proliferation and differentiation ability of mesenchymal stem/stromal cells (MSCs) gets affected, and hence, aged MSCs are not preferred for regenerative purposes. Rapid identification of aging-associated changes within MSCs and the mechanistic pathways involved are necessary to determine optimal cell sources to treat musculoskeletal disorders in older patients. In the present study, we have identified a set of phenotypic markers, namely downregulated expression of CD90 and upregulated expression of CD45, as age-defining markers for the bone marrow-derived MSCs. We also show that these phenotypic changes in aged MSCs correlate with their aging-mediated differentiation defects. We find that oxidative stress signaling leading to the activation of nuclear factor kappa B (NF-κB) plays an essential role in altering the phenotype and differentiation ability of the aged MSCs. We further show that treatment of aged MSCs with the conditioned medium (CM) derived from young MSCs (young-CM) restored their phenotype and differentiation potential to the young-like by ameliorating activation of NF-κB signaling in them. Similar changes could also be achieved by using an inhibitor of NF-κB signaling, showing that oxidative stress-induced NF-κB activation is the causative factor in the aging of MSCs. Additionally, we show that treating young MSCs with hydrogen peroxide mimics all the aging-mediated changes in them, underscoring the involvement of oxidative stress in the aging of MSCs. Overall, our data suggest that the altered expression of CD90 and CD45 surface markers can be used as a primary screen to identify the onset of aging in the MSCs, which can be quickly reversed by their in vitro treatment with young-CM or NF-κB inhibitor. Our study also puts the phenotypic characterization of MSCs in a clinical perspective.

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INTRODUCTION: Human adipose tissue contains a heterogeneous and synergistic mixture of cells called stromal vascular fraction (SVF) with highly proliferative and angiogenic properties, conferring promising applicability in the field of regenerative medicine. This study aims to investigate if age, body mass index (BMI), history of obesity and massive weight loss, and harvest site are related to SVF cell marker expression. METHODS: A total of 26 samples of subcutaneous adipose tissue were harvested from patients admitted to the Plastic and Reconstructive department in University Hospital Center of São João, Porto, Portugal, for body contouring surgery. The percentage of cells expressing CD31, CD34, CD45, CD73, CD90, and CD105 was assessed and compared with patient's age, BMI, history of obesity and massive weight loss (ex-obese group), and harvest site. RESULTS: In the ex-obese group, a significantly higher number of cells expressing CD90 (P = 0.002) was found. BMI, harvest site, and age appear to have no association with SVF subpopulations. CONCLUSIONS: This study suggests that ex-obese patients have a higher percentage of SVF cells expressing CD90, which correlates with higher proliferative and angiogenic rates. The effect of former obesity and massive weight loss on the expression of CD90 is a new and relevant finding because it makes this population a suitable candidate for reconstructive and aesthetic surgery and other fields of regenerative medicine. The use of SVF appears also promising in older patients because no negative correlation between increasing age and different cell markers expression was found.

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