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.

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.

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.

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.

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.

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.

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.

Comparative analysis of effects of conditioned mediums obtained from 2D or 3D cultured mesenchymal stem cells on kidney functions of diabetic rats: Early intervention could potentiate transdifferentiation of parietal epithelial cell into podocyte precursors.

AIM: The secretome of mesenchymal stem cells (MSCs) could be a potential therapeutic intervention for diabetes and associated complications like nephropathy. This study aims to evaluate the effects of conditioned mediums (CMs) collected from umbilical cord-derived MSCs incubated under 2-dimensional (2D) or 3D culture conditions on kidney functions of rats with type-I diabetes (T1D). MAIN METHODS: Sprague-Dawley rats were treated with 20 mg/kg streptozocin for 5 consecutive days to induce T1D, and 12 doses of CMs were applied intraperitoneally for 4 weeks. The therapeutic effects of CMs were comparatively investigated by biochemical, physical, histopathological, and immunohistochemical analysis. KEY FINDINGS: 3D-CM had significantly higher total protein concentration than the 2D-CM Albumin/creatinine ratios of both treatment groups were significantly improved in comparison to diabetes. Light microscopic evaluations showed that glomerular and cortical tubular damages were significantly ameliorated in only the 3D-CM applied group compared to the diabetes group, which were correlated with transmission electron microscopic observations. The nephrin and synaptopodin expressions increased in both treatment groups compared to diabetes. The WT1, Ki-67, and active caspase-3 expressions in glomeruli and parietal layers of the treatment groups suggest that both types of CMs suppress apoptosis and promote possible parietal epithelial cells' (PECs') transdifferentiation towards podocyte precursor cells by switching on WT1 expression in parietal layer rather than inducing new cell proliferation. SIGNIFICANCE: 3D-CM was found to be more effective in improving kidney functions than 2D-CM by ameliorating glomerular damage through the possible mechanism of transdifferentiation of PECs into podocyte precursors and suppressing glomerular apoptosis.

Evaluation of immunomodulatory potential of probiotic conditioned medium on murine macrophages.

Probiotics are a mixture of beneficial live bacteria and/or yeasts that naturally exist in our bodies. Recently, numerous studies have focused on the immunostimulatory effects of single-species or killed multi-species probiotic conditioned mediums on macrophages. This study investigates the immunostimulatory effect of commercially available active, multi-species probiotic conditioned medium (CM) on RAW264.7 murine macrophages. The probiotic CM was prepared by culturing the commercially available probiotic in a cell-culture medium overnight at 37 °C, followed by centrifugation and filter-sterilization to be tested on macrophages. The immunostimulatory effect of different dilution percentages (50%, 75%, 100%) of CM was examined using the MTT assay, proinflammatory cytokine (tumor necrosis factor TNF-alpha) production in macrophages, migration, and phagocytosis assays. For all the examined CM ratios, the percentages of cell viability were > 80%. Regarding the migration scratch, TNF-alpha and phagocytosis assays, CM demonstrated a concentration-dependent immunostimulatory effect. However, the undiluted CM (100%) showed a significant (p-value < 0.05) stimulatory effect compared to the positive and negative controls. The findings suggest that the secretions and products of probiotics, as measured in the CM, may be closely associated with their immune-boosting effects. Understanding this relationship between probiotic secretions and immune function is crucial for further exploring the potential benefits of probiotics in enhancing overall health and well-being.

Obesity-associated inflammatory macrophage polarization is inhibited by capsaicin and phytolignans.

Obesity is often accompanied by increased adipose tissue inflammation, a process that is partially driven by adipose tissue-resident macrophages. In this study, we explored the potential for plant-derived dietary compounds to exert anti-inflammatory effects in macrophages that alleviate obesity-associated adipocyte dysfunction. Capsaicin (CAP), schisandrin A (SA), enterodiol (END), and enterolactone (ENL) treatment polarized J774 macrophages to an "M2" or anti-inflammatory phenotype and inhibited responses to stimulation with lipopolysaccharide (LPS). Furthermore, these compounds blocked inflammasome activation when administered just before ATP-induced NLRP3 activation, as evidenced by the abrogation of IL-1ß release in mouse macrophages and human peripheral blood monocytes. The addition of CAP, SA, or ENL during the differentiation of bone marrow-derived macrophages was also sufficient to inhibit LPS-induced IL-6 and TNFα production. Finally, CAP, END, and ENL treatment during differentiation of 3T3-L1 adipocytes induced an adiponectin-high phenotype accompanied by increases in thermogenic gene expression, and conditioned media from these adipocytes inhibited LPS-induced production of IL-1ß, IL-6, and TNFα from J774 macrophages. These polarizing effects were partially mediated by the elevated adiponectin and decreased syndecan-4 in the adipocyte-conditioned media. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.NEW & NOTEWORTHY The utility of food-based products to prevent or alleviate chronic conditions such as obesity and its associated comorbidities is an attractive approach. Capsaicin, schisandrin A, enterodiol, and enterolactone, phytochemicals present in traditional medicinal food, decreased proinflammatory cytokine production from macrophages that, in turn, reduced obesity-associated adipocyte dysfunction. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.

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.

Vascular smooth muscle cells in response to cholesterol crystals modulates inflammatory cytokines release and promotes neutrophil extracellular trap formation.

BACKGROUND: The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response. METHODS: Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy. RESULTS: VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K-AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation. CONCLUSION: We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

Conditioned medium of induced pluripotent stem cell derived neuromesodermal progenitors enhances cell migration in vitro.

BACKGROUND: Identification of novel cell-based therapy sources has been of great interest in recent years to provide alternative and available therapy options in clinics. Conditioned medium (CM) can be a valuable supply for growth factors, cytokines and chemokines as a source of stem cell secretome. Exploring the role of new CM sources for tissue regeneration might be a promising approach for therapeutic purposes. METHODS AND RESULTS: In the current study, neuromesodermal progenitors (NMPs) derived from induced pluripotent stem cells (iPSCs) were used to collect CM. Fibroblast derived iPSCs were successfully differentiated into NMPs and NMPs were characterized by double positive T/Bra and Sox2 staining. CM was collected from NMPs, and the content was characterized by membrane analysis. In vitro wound healing assay was used as a model system to observe potential activity of CM on cell migration. Fibroblasts, keratinocytes and endothelial cells were used to evaluate the effect of NMP-derived CM (NMP-CM) on cell migration in vitro. Several important proteins related to wound healing such as ANGPT 1, ANGPT 2, MCP-1, PDGF-AA, SDF-1α, TIMP-1 and TIMP-2 were increased in NMP-CM. NMP-CM increased cell proliferation and migration in vitro. CONCLUSIONS: In vitro data obtained from three distinct cell types suggest a promising role of NMP-CM on cell migration. NMP-CM can be used for wound management in the further future after detailed in vitro and in vivo research.

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.

Cytoprotective role of human dental pulp stem cell-conditioned medium in chemotherapy-induced alopecia.

BACKGROUND: Chemotherapy-induced alopecia (CIA) is a distressing adverse effect of chemotherapy, with an estimated incidence of 65% and limited treatment options. Cyclophosphamide (CYP) is a common alopecia-inducing chemotherapy agent. Human dental pulp stem cells (DPSCs) secrete several paracrine factors that up-regulate hair growth. Conditioned medium (CM) collected from DPSCs (DPSC-CM) promotes hair growth; culturing mesenchymal stem cells under hypoxic conditions can enhance this effect. METHODS: The effect of DPSC-CM cultured under normoxic (N-) and hypoxic (H-) conditions against CYP-mediated cytotoxicity in keratinocytes was examined using cell viability assay, lactate dehydrogenase (LDH) cytotoxicity assay, and apoptosis detection. The damage-response pathway was determined in a well-established CIA mouse model by analyzing macroscopic effects, histology, and apoptosis. Reverse transcription-quantitative PCR and Caspase-3/7 activity assay were used to investigate the impact of DPSC-CM on the molecular damage-response pathways in CYP-treated mice. The effect of post-CIA DPSC-CM application on post-CIA hair regrowth was analyzed by macroscopic effects and microstructure observation of the hair surface. Furthermore, to investigate the safety of DPSC-CM as a viable treatment option, the effect of DPSC-CM on carcinoma cell lines was examined by cell viability assay and a subcutaneous tumor model. RESULTS: In the cell viability assay, DPSC-CM was observed to increase the number of keratinocytes over varying CYP concentrations. Furthermore, it reduced the LDH activity level and suppressed apoptosis in CYP-treated keratinocytes. DPSC-CM exhibited the cytoprotective role in vivo via the dystrophic anagen damage-response pathway. While both N-CM and H-CM downregulated the Caspase-3/7 activity level, H-CM downregulated Caspase-3 mRNA expression. The proportion of post-CIA H-CM-treated mice with > 90% normal hair was nearly twice that of vehicle- or N-CM-treated mice between days 50 and 59 post-depilation, suggesting that post-CIA H-CM application may accelerate hair regrowth and improve hair quality. Furthermore, DPSC-CM suppressed proliferation in vitro in certain carcinoma cell lines and did not promote the squamous cell carcinoma (SCC-VII) tumor growth rate in mice. CONCLUSIONS: The potentiality of DPSC-CM and H-CM as a promising cytoprotective agent and hair regrowth stimulant, respectively, for CIA needs in-depth exploration.

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.

Translating proteome and transcriptome dynamics of periodontal ligament stem cell-derived secretome/conditioned medium in an in vitro model of periodontitis.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) have been proposed as therapeutic candidates in periodontal diseases and periodontium defects. Paracrine factors of PDLSCs, namely, secretome, can contribute to tissue regeneration comparable to direct stem cell application. This study explored restoration effects of PDLSC-derived secretome/conditioned medium (PDLSC-CM) on PDLSCs themselves in an inflammatory microenvironment and identified its action mechanisms using proteomics and transcriptomic profiling. METHODS: PDLSC-CM was prepared from cells under healthy culture conditions. Mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were then performed to analyze the PDLSC-CM proteome. Osteogenic differentiation of PDLSCs under inflammatory conditions or in the presence of PDLSC-CM was then characterized in assays of alkaline phosphatase activity, intracellular calcium levels, protein expression of osteogenic markers, and matrix mineralization. Furthermore, the transcriptomic profile was assessed to identify significantly enriched signaling pathways and associated molecular networks by RNA sequencing. RESULTS: LC-MS/MS proteomics identified a total of 203 proteins and distinguished 187 significant protein changes in PDLSC-CM compared to control-CM. LPS-treated PDLSCs significantly attenuated osteogenic differentiation. When PDLSCs were treated with PDLSC-CM alone, their osteogenic activity was significantly upregulated compared to the control group. Moreover, the LPS-impaired osteogenesis of PDLSCs was reconstituted by PDLSC-CM treatment. RNA sequencing revealed 252, 1,326, and 776 differentially expressed genes in the control vs. LPS, control vs. PDLSC-CM, and LPS vs. LPS + PDLSC-CM groups, respectively. CONCLUSION: This study suggest that PDLSC-CM restores the osteogenic potential of PDLSCs in an inflammatory environment through secretory functions representing potential repair and regenerative mechanisms.

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.

Multifaceted impact of adipose conditioned media: Obesity-driven promotion of prostate cancer and cancer stem cell dynamics.

Obesity is an established risk factor for the development and progression of prostate cancer (PC). This study used adipose conditioned media (ACM) from differentiated adipocytes to assess its effect on PC development and aggressiveness. Due to limited research on ACM's impact on isolated PC stem cells (PCSCs), we also examined CD44+ PCSCs. ACM notably boosted interleukin-1ß (IL-1ß), IL-6, and IL-8 production in normal prostate epithelial cells and LNCaP cells. It also increased IL-6 and IL-8 production in PC3 and CD44+ LNCaP cells, and IL-1ß and IL-6 production in CD44+ PC3 cells. This indicates that ACM induces the production of inflammatory cytokines in both cancer and prostate epithelial cells. Furthermore, ACM promoted proliferation in androgen receptor (AR)-negative PC3 cells, CD44+ PC3 PCSCs, and nonmalignant RWPE cells, without affecting AR-positive LNCaP cells. In addition, ACM-enhanced invasion and migration potential in both PC3 and CD44+ PC3 cells. Western blot analysis indicated the involvement of NF-κB and AKT pathways in ACM-induced proliferation in PC3 cells and NF-κB in PCSCs. In ACM-treated PC3 cells, E-cadherin was downregulated, while N-cadherin, Snail, vimentin, fibronectin, and Twist were upregulated, suggesting ACM-induced invasion via classical epithelial-to-mesenchymal transition (EMT) pathways. In response to ACM, PCSCs exhibited increased expression of E-cadherin, Snail, and vimentin, which are partial EMT markers promoting stemness and resistance to apoptosis. In addition, increased expressions of Nanog, Oct3/4, survivin, and Bcl-2 were observed. Although the molecules we studied have diverse effects on cellular regulation, our data emphasize obesity's multifaceted role in promoting and aggressing PC, notably affecting PCSC populations.