Iron deficiency in astrocytes alters cellular status and impacts on oligodendrocyte differentiation.

Iron deficiency (ID) has been shown to affect central nervous system (CNS) development and induce hypomyelination. Previous work from our laboratory in a gestational ID model showed that both oligodendrocyte (OLG) and astrocyte (AST) maturation was impaired. To explore the contribution of AST iron to the myelination process, we generated an in vitro ID model by silencing divalent metal transporter 1 (DMT1) in AST (siDMT1 AST) or treating AST with Fe3+ chelator deferoxamine (DFX; DFX AST). siDMT1 AST showed no changes in proliferation but remained immature. Co-cultures of oligodendrocyte precursors cells (OPC) with siDMT1 AST and OPC cultures incubated with siDMT1 AST-conditioned media (ACM) rendered a reduction in OPC maturation. These findings correlated with a decrease in the expression of AST-secreted factors IGF-1, NRG-1, and LIF, known to promote OPC differentiation. siDMT1 AST also displayed increased mitochondrial number and reduced mitochondrial size as compared to control cells. DFX AST also remained immature and DFX AST-conditioned media also hampered OPC maturation in culture, in keeping with a decrease in the expression of AST-secreted growth factors IGF-1, NRG-1, LIF, and CNTF. DFX AST mitochondrial morphology and number showed results similar to those observed in siDMT1 AST. In sum, our results show that ID, induced through two different methods, impacts AST maturation and mitochondrial functioning, which in turn hampers OPC differentiation.

p-Smad3 differentially regulates the cytological behavior of osteoclasts before and after osteoblasts maturation.

BACKGROUND: A series of previous investigations have revealed that p-Smad3 plays a facilitative role in the differentiation and maturation of osteoblasts, while also regulating the expression of certain intercellular communication factors. However, the effects of p-Smad3 in osteoblasts before and after maturation on the proliferation, migration, differentiation, apoptosis and other cellular behaviors of osteoclasts have not been reported. METHODS: MC3T3-E1 cells were cultured in osteogenic induction medium for varying durations, After that, the corresponding conditioned medium was collected and the osteoclast lineage cells were treated. To elucidate the regulatory role of p-Smad3 within osteoblasts, we applied the activator TGF-ß1 and inhibitor SIS3 to immature and mature osteoblasts and collected corresponding conditioned media for osteoclast intervention. RESULTS: We observed an elevation of p-Smad3 and Smad3 during the early stage of osteoblast differentiation, followed by a decline in the later stage. we discovered that as osteoblasts mature, their conditioned media inhibit osteoclasts differentiation and the osteoclast-coupled osteogenic effect. However, it promotes apoptosis in osteoclasts and the angiogenesis coupled with osteoclasts. p-Smad3 in immature osteoblasts, through paracrine effects, promotes the migration, differentiation, and osteoclast-coupled osteogenic effects of osteoclast lineage cells. For mature osteoblasts, p-Smad3 facilitates osteoclast apoptosis and the angiogenesis coupled with osteoclasts. CONCLUSIONS: As pre-osteoblasts undergo maturation, p-Smad3 mediated a paracrine effect that transitions osteoclast cellular behaviors from inducing differentiation and stimulating bone formation to promoting apoptosis and coupling angiogenesis.

Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

INTRODUCTION: Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. MATERIALS AND METHODS: We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. RESULTS: Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. CONCLUSIONS: These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Proliferation Patterns of MCF-7 Breast Cancer Cells in Lipoaspirate Conditioned Media.

INTRODUCTION: Autologous fat grafting (AFG) is a common technique used to enhance aesthetic outcomes in postmastectomy breast reconstruction patients. Adipokines are hormones secreted by adipose tissue that play a critical role in regulating metabolic processes and the immune system. However, dysregulated adipokine secretion and signaling can contribute to the development and progression of cancer by promoting angiogenesis, altering the immune response, and inducing the epithelial mesenchymal transition. We aimed to assess how breast cancer cells behave in conditioned media derived from fat grafting lipoaspirates and gain a better understanding of the potential interactions that may occur within the tumor microenvironment. METHODS: Patients who were undergoing AFG as a part of breast reconstruction at NY-Presbyterian/Weill Cornell Medical Center between March 2021 and July 2023 were consented and enrolled in the study. This study was approved by the Weill Cornell Medicine Institutional Review Board (#20-10022850-14). Conditioned media is created using 20% of patient lipoaspirate secretome and 80% starving media. The growth of MCF-7, a human ER/PR+ breast cancer cell line, in conditioned media is assessed using CyQUANT. RESULTS: The breast cancer cells incubated in conditioned media displayed similar growth trends as those in complete media, which is enriched for cell growth (P > 0.05). MCF-7 cell behavior in conditioned media differed significantly from their proliferation patterns when serum starved in 100% starving media (P < 0.05). DISCUSSION: Our results suggest that there may be inherent factors within the lipoaspirate that may promote MCF-7 proliferation. One potential implication is that AFG used for breast reconstruction should be delayed until local-regional disease control has been established. In addition, based on the in vitro proliferation patterns of breast cancer cells in conditioned media, the safety profile of AFG may be enhanced if the procedure is performed after attaining negative margins and the completion breast cancer treatment.

Conditioned medium-enriched umbilical cord mesenchymal stem cells: a potential therapeutic strategy for spinal cord injury, unveiling transcriptomic and secretomic insights.

INTRODUCTION: Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. MATERIALS AND METHODS: In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. RESULTS: The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. CONCLUSIONS: In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.

Identification of the role of DAB2 and CXCL8 in uterine spiral artery remodeling in early-onset preeclampsia.

Aberrant remodeling of uterine spiral arteries (SPA) is strongly associated with the pathogenesis of early-onset preeclampsia (EOPE). However, the complexities of SPA transformation remain inadequately understood. We conducted a single-cell RNA sequencing analysis of whole placental tissues derived from patients with EOPE and their corresponding controls, identified DAB2 as a key gene of interest and explored the mechanism underlying the communication between Extravillous trophoblast cells (EVTs) and decidual vascular smooth muscle cells (dVSMC) through cell models and a placenta-decidua coculture (PDC) model in vitro. DAB2 enhanced the motility and viability of HTR-8/SVneo cells. After exposure to conditioned medium (CM) from HTR-8/SVneoshNC cells, hVSMCs exhibited a rounded morphology, indicative of dedifferentiation, while CM-HTR-8/SVneoshDAB2 cells displayed a spindle-like morphology. Furthermore, the PDC model demonstrated that CM-HTR-8/SVneoshDAB2 was less conducive to vascular remodeling. Further in-depth mechanistic investigations revealed that C-X-C motif chemokine ligand 8 (CXCL8, also known as IL8) is a pivotal regulator governing the dedifferentiation of dVSMC. DAB2 expression in EVTs is critical for orchestrating the phenotypic transition and motility of dVSMC. These processes may be intricately linked to the CXCL8/PI3K/AKT pathway, underscoring its central role in intricate SPA remodeling.

Fibroblast matrix implants-a better alternative for incisional hernia repair?

The standard surgical procedure for abdominal hernia repair with conventional prosthetic mesh still results in a high recurrence rate. In the present study, we propose a fibroblast matrix implant (FMI), which is a three-dimensional (3D) poly-L-lactic acid scaffold coated with collagen (matrix) and seeded with fibroblasts, as an alternative mesh for hernia repair. The matrix was seeded with fibroblasts (cellularized) and treated with a conditioned medium (CM) of human Umbilical Cord Mesenchymal Stem Cells (hUC-MSC). Fibroblast proliferation and function were assessed and compared between treated with CM hUC-MSC and untreated group, 24 h after seeding onto the matrix (n= 3). To study the matricesin vivo,the hernia was surgically created on male Sprague Dawley rats and repaired with four different grafts (n= 3), including a commercial mesh (mesh group), a matrix without cells (cell-free group), a matrix seeded with fibroblasts (FMI group), and a matrix seeded with fibroblasts and cultured in medium treated with 1% CM hUC-MSC (FMI-CM group).In vitroexamination showed that the fibroblasts' proliferation on the matrices (treated group) did not differ significantly compared to the untreated group. CM hUC-MSC was able to promote the collagen synthesis of the fibroblasts, resulting in a higher collagen concentration compared to the untreated group. Furthermore, thein vivostudy showed that the matrices allowed fibroblast growth and supported cell functionality for at least 1 month after implantation. The highest number of fibroblasts was observed in the FMI group at the 14 d endpoint, but at the 28 d endpoint, the FMI-CM group had the highest. Collagen deposition area and neovascularization at the implantation site were observed in all groups without any significant difference between the groups. FMI combined with CM hUC-MSC may serve as a better option for hernia repair, providing additional reinforcement which in turn should reduce hernia recurrence.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Angiotensin II Is Involved in MLKL Activation During the Development of Heart Failure Following Myocardial Infarction in Rats.

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT1 receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Detection of Interleukin-1 ß (IL-1ß) in Single Human Blastocyst-Conditioned Medium Using Ultrasensitive Bead-Based Digital Microfluidic Chip and Its Relationship with Embryonic Implantation Potential.

The implantation of human embryos is a complex process involving various cytokines and receptors expressed by both endometrium and embryos. However, the role of cytokines produced by a single embryo in successful implantation is largely unknown. This study aimed to investigate the role of IL-1ß expressed in a single-embryo-conditioned medium (ECM) in embryo implantation. Seventy samples of single ECM were analyzed by a specially designed magnetic-beads-based microfluidic chip from 15 women. We discovered that IL-1ß level increased as the embryo developed, and the difference was significant. In addition, receiver operator characteristic (ROC) curves analysis showed a higher chance of pregnancy when the IL-1ß level on day 5 ECM was below 79.37 pg/mL and the difference between day 5 and day 3 was below 24.90 pg/mL. Our study discovered a possible association between embryonic proteomic expression and successful implantation, which might facilitate single-embryo transfer in the future by helping clinicians identify the embryo with the greatest implantation potential.

Evaluation of the efficacy of mesenchymal stem cells derived conditioned medium in the treatment of striae distensae: a double blind randomized clinical trial.

BACKGROUND: Striae distensae is a disfiguring atrophic skin condition that impairs the body's aesthetic image. Despite the variety of conducted studies, there is controversy regarding the best modalities. Human mesenchymal stem cells are considered a rich source for scar treatment. Skin needling is among the most efficient and safe aesthetic and therapeutic devices. This study aimed to evaluate the efficacy of the combination of needling and intradermal injection of mesenchymal stem cells compared to skin needling alone for treating striae distensae. METHOD: This study was a randomized, double-blind clinical trial involving 10 women aged 18-60. Each striae lesion was divided into two parts, with one side receiving needling and intradermal injection of conditioned medium, while the other side received needling and intradermal injection of normal saline. This treatment was administered in three sessions with three-week intervals. Patients were evaluated before the first intervention and three months after the final session. Three months after the completion of the intervention, patients' lesions were evaluated using biometric criteria, physician evaluation, and patient self-assessment. RESULTS: The results demonstrated a significant improvement in dermal and complete thickness and skin density in patients treated with microneedling. All skin ultrasound parameters improved significantly in patients receiving the combination of needling and conditioned medium. When comparing the two groups, significantly higher physician and patient satisfaction was observed in the combination group. However, the comparison of biometric indices improvement wasn't significant between these groups. CONCLUSION: The combination of human mesenchymal stem cells with microneedling could be considered a novel effective option for stretch marks.

[Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage].

OBJECTIVE: To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. METHODS: The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. RESULTS: Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). CONCLUSIONS: LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

Human umbilical cord/placenta mesenchymal stem cell conditioned medium attenuates intestinal fibrosis in vivo and in vitro.

BACKGROUND: A significant unmet need in inflammatory bowel disease is the lack of anti-fibrotic agents targeting intestinal fibrosis. This study aimed to investigate the anti-fibrogenic properties and mechanisms of the conditioned medium (CM) from human umbilical cord/placenta-derived mesenchymal stem cells (UC/PL-MSC-CM) in a murine intestinal fibrosis model and human primary intestinal myofibroblasts (HIMFs). METHODS: UC/PL-MSC-CM was concentrated 15-fold using a 3 kDa cut-off filter. C57BL/6 mice aged 7 weeks old were randomly assigned to one of four groups: (1) control, (2) dextran sulfate sodium (DSS), (3) DSS + CM (late-phase treatment), and (4) DSS + CM (early-phase treatment). Chronic DSS colitis and intestinal fibrosis was induced by three cycles of DSS administration. One DSS cycle consisted of 7 days of oral DSS administration (1.75%, 2%, and 2.5% DSS), followed by 14 days of drinking water. UC/PL-MSC-CM was intraperitoneally administered in the late phase (from day 50, 10 times) or early phase (from day 29, 10 times) of DSS cycles. HIMFs were treated with TGF-ß1 and co-treated with UC/PL-MSC-CM (10% of culture media) in the cellular model. RESULTS: In the animal study, UC/PL-MSC-CM reduced submucosa/muscularis propria thickness and collagen deposition, which improved intestinal fibrosis in chronic DSS colitis. The UC/PL-MSC-CM significantly reduced the expressions of procollagen1A1 and α-smooth muscle actin, which DSS significantly elevated. The anti-fibrogenic effect was more apparent in the UC-MSC-CM or early-phase treatment model. The UC/PL-MSC-CM reduced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs in the cellular model. The UC/PL-MSC-CM downregulated fibrogenesis by suppressing RhoA, MRTF-A, and SRF expression. CONCLUSIONS: Human UC/PL-MSC-CM inhibits TGF-ß1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and chronic DSS colitis-induced intestinal fibrosis. Thus, it may be regarded as a novel candidate for stem cell-based therapy of intestinal fibrosis.

Isolation and Characterization of Extracellular Vesicles Through Orthogonal Approaches for the Development of Intraocular EV Therapy.

Purpose: Isolating extracellular vesicles (EVs) with high yield, replicable purity, and characterization remains a bottleneck in the development of EV therapeutics. To address these challenges, the current study aims to establish the necessary framework for preclinical and clinical studies in the development of stem cell-derived intraocular EV therapeutics. Methods: Small EVs (sEVs) were separated from the conditioned cell culture medium (CCM) of the human embryogenic stem cell-derived fully polarized retinal pigment epithelium (hESC-RPE-sEV) by a commercially available microfluidic tangential flow filtration (TFF) device ExoDisc (ED) or differential ultracentrifugation (dUC). The scaling and concentration capabilities and purity of recovered sEVs were assessed. Size, number, and surface markers of sEVs were determined by orthogonal approaches using multiple devices. Results: ED yielded higher numbers of sEVs, ranging from three to eight times higher depending on the measurement device, compared to dUC using the same 5 mL of CCM input. Within the same setting, the purity of ED-recovered hESC-RPE-sEVs was higher than that for dUC-recovered sEVs. ED yielded a higher concentration of particles, which is strongly correlated with the input volume, up to 10 mL (r = 0.98, P = 0.016). Meanwhile, comprehensive characterization profiles of EV surface markers between ED- and dUC-recovered hESC-RPE-sEVs were compatible. Conclusions: Our study supports TFF as a valuable strategy for separating sEVs for the development of intraocular EV therapeutics. However, there is a growing need for diverse devices to optimize TFF for use in EV preparation. Using orthogonal approaches in EV characterization remains ideal for reliably characterizing heterogeneous EV.

Secretome from Magnetically Stimulated Muscle Exhibits Anticancer Potency: Novel Preconditioning Methodology Highlighting HTRA1 Action.

Briefly (10 min) exposing C2C12 myotubes to low amplitude (1.5 mT) pulsed electromagnetic fields (PEMFs) generated a conditioned media (pCM) that was capable of mitigating breast cancer cell growth, migration, and invasiveness in vitro, whereas the conditioned media harvested from unexposed myotubes, representing constitutively released secretome (cCM), was less effective. Administering pCM to breast cancer microtumors engrafted onto the chorioallantoic membrane of chicken eggs reduced tumor volume and vascularity. Blood serum collected from PEMF-exposed or exercised mice allayed breast cancer cell growth, migration, and invasiveness. A secretome preconditioning methodology is presented that accentuates the graded anticancer potencies of both the cCM and pCM harvested from myotubes, demonstrating an adaptive response to pCM administered during early myogenesis that emulated secretome-based exercise adaptations observed in vivo. HTRA1 was shown to be upregulated in pCM and was demonstrated to be necessary and sufficient for the anticancer potency of the pCM; recombinant HTRA1 added to basal media recapitulated the anticancer effects of pCM and antibody-based absorption of HTRA1 from pCM precluded its anticancer effects. Brief and non-invasive PEMF stimulation may represent a method to commandeer the secretome response of muscle, both in vitro and in vivo, for clinical exploitation in breast and other cancers.

Oviduct and endometrial epithelium improve in vitro produced bovine embryo developmental kinetics.

In brief: Standard in vitro produced (IVP) bovine embryo culture media limit embryonic development. Culturing IVP bovine embryos in standard IVP bovine embryo culture media conditioned with oviduct and/or endometrial cells improves blastocyst formation and reduces the time to formation. Abstract: In vitro embryo production in cattle greatly impacts blastomere biochemistry, embryo rate of development and pre- and post-transfer survival. In vivo, the bovine embryo migrates through the oviduct isthmus before entering the uterus on approximately day 4 of development where it remains unattached within the uterine lumen until day 20 of gestation. During this time, the embryo is sequentially exposed to oviduct followed by endometrial secretions that support embryonic development. Considering this, we tested the effect of culturing in vitro produced (IVP) bovine embryos sequentially in oviduct epithelial- (OEp; days 1-3) followed by endometrial epithelial- (EEp) or EEp and fibroblast cell (EEp/F; days 4-8)-conditioned media on embryonic development using a time-lapse monitoring system. Compared to control, culturing IVP embryos in EEp- or EEp/F-conditioned media without prior culture in OEp-conditioned media increased blastocyst formation (P < 0.05) and reduced the time to blastocyst formation (P < 0.05). Culturing IVP bovine embryos in OEp-conditioned media followed by EEp- or EEp/F-conditioned media, however, had the greatest impact on embryo developmental kinetics and increased morula and blastocyst formation (P < 0.05) and reduced time to formation (P < 0.05). Day 8 blastocyst cell numbers, diameter and quality were not significantly different, although, blastocyst quality scores were less (indicative of better quality) for all cell-conditioned media compared to control. In conclusion, IVP bovine embryo development may be improved using a sequential embryo culture system involving bovine oviduct followed by endometrial cell-conditioned media.

The interplay between alterations in esophageal microbiota associated with Th17 immune response and impaired LC20 phosphorylation in achalasia.

BACKGROUND: Achalasia is an esophageal motility disorder with an unknown etiology. We aimed to determine the pathogenesis of achalasia by studying alterations in esophageal smooth muscle contraction and the associated inflammatory response, and evaluate the role of esophageal microbiota in achalasia development. METHODS: We analyzed esophageal mucosa and lower esophageal sphincter (LES) samples, obtained from patients with type II achalasia who underwent peroral endoscopic myotomy. Esophageal conditioned media obtained from patients were transferred into the mouse esophagus to determine whether the esophageal intraluminal environment is associated with achalasia. RESULTS: Approximately 30% of 20-kDa myosin light chains (LC20) was phosphorylated in LES from the control group under resting and stimulated conditions, whereas less than 10% of LC20 phosphorylation was detected in achalasia under all conditions. The hypophosphorylation of LC20 in achalasia was associated with the downregulation of the myosin phosphatase-inhibitor protein CPI-17. Th17-related cytokines, including IL-17A, IL-17F, IL-22, and IL-23A, were significantly upregulated in achalasia. α-Diversity index of esophageal microbiota and the proportion of several microbes, including Actinomyces and Dialister, increased in achalasia. Actinomyces levels positively correlated with IL-23A levels, whereas Dialister levels were positively associated with IL-17A, IL-17F, and IL-22 levels. Esophageal IL-17F levels increased in mice after oral administration of the conditioned media. CONCLUSIONS: In LES of patients with achalasia, hypophosphorylation of LC20, a possible cause of impaired contractility, was associated with CPI-17 downregulation and an increased Th17-related immune response. The esophageal intraluminal environment, represented by the esophageal microbiota, could be associated with the development and exacerbation of achalasia.

Conditioned Media from Deer Antler Stem Cells Effectively Alleviate Type 1 Diabetes Mellitus Possibly via Inhibiting the NF-κB Signaling Pathway.

BACKGROUND: Type 1 diabetes mellitus (T1D) represents a severe threat to human health. Persistent hyperglycemia and dyslipidemia can lead to damaged liver function, while effective interventions for these complications are currently lacking. Deer antler stem cells (AnSCs), a novel type of adult stem cells, significantly reduced liver injury, which was speculated to be achieved through the paracrine pathway. METHODS: In this study, AnSC-conditioned medium (AnSC-CM) was used to treat C57BL/6 mice with T1D symptoms induced by streptozotocin (STZ). The therapeutic effects of AnSC-CM on T1D were evaluated, and the underlying mechanism was investigated. RESULTS: It was shown that AnSC-CM alleviated the T1D symptom: decreased body weight, increased blood glucose levels and islet lesions, and reduced insulin secretion. Moreover, AnSC-CM treatment improved liver function and mitigated liver injury in T1D mice. Impressively, the therapeutic effects of AnSC-CM on T1D were better than those of bone marrow mesenchymal stem cell-CM (BMSC-CM). The mechanistic study revealed that AnSC-CM significantly downregulated the NF-κB signaling pathway in both pancreatic and liver tissues. CONCLUSIONS: Therapeutic effects of AnSC-CM on STZ-induced T1D and liver injury may be achieved through targeting the NF-κB signaling pathway.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.