Cell-derived Conditioned Medium Research Articles

Human amniotic mesenchymal stem cells-derived conditioned medium and exosomes alleviate oxidative stress-induced retinal degeneration by activating PI3K/Akt/FoxO3 pathway

Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly, which is primarily attributed to oxidative stress-induced damage to the retinal pigment epithelium (RPE). Human amniotic mesenchymal stem cells (hAMSC) were considered to be one of the most promising stem cells for clinical application due to their low immunogenicity, tissue repair ability, pluripotent potential and potent paracrine effects. The conditional medium (hAMSC-CM) and exosomes (hAMSC-exo) derived from hAMSC, as mediators of intercellular communication, play an important role in the treatment of retinal diseases, but their effect and mechanism on oxidative stress-induced retinal degeneration are not explored. Here, we reported that hAMSC-CM alleviated H2O2-induced ARPE-19 cell death through inhibiting mitochondrial-mediated apoptosis pathway in vitro. The overproduction of reactive oxygen species (ROS), alteration in mitochondrial morphology, loss of mitochondrial membrane potential and elevation of Bax/Bcl2 ratio in ARPE-19 cells under oxidative stress were efficiently reversed by hAMSC-CM. Moreover, it was found that hAMSC-CM protected cells against oxidative injury via PI3K/Akt/FoxO3 signaling. Intriguingly, exosome inhibitor GW4869 alleviated the inhibitory effect of hAMSC-CM on H2O2-induced decrease in cell viability of ARPE-19 cells. We further demonstrated that hAMSC-exo exerted the similar protective effect on ARPE-19 cells against oxidative damage as hAMSC-CM. Additionally, both hAMSC-CM and hAMSC-exo ameliorated sodium iodate-induced deterioration of RPE and retinal damage in vivo. These results first indicate that hAMSC-CM and hAMSC-exo protect RPE cells from oxidative damage by regulating PI3K/Akt/FoxO3 pathway, suggesting hAMSC-CM and hAMSC-exo will be a promising cell-free therapy for the treatment of AMD in the future.

Treatment of Equine Tarsus Long Medial Collateral Ligament Desmitis with Allogenic Synovial Membrane Mesenchymal Stem/Stromal Cells Enhanced by Umbilical Cord Mesenchymal Stem/Stromal Cell-Derived Conditioned Medium: Proof of Concept

Horses are high-performance athletes prone to sportive injuries such as tendonitis and desmitis. The formation of fibrous tissue in tendon repair remains a challenge to overcome. This impels regenerative medicine to develop innovative therapies that enhance regeneration, retrieving original tissue properties. Multipotent Mesenchymal Stem/Stromal Cells (MSCs) have been successfully used to develop therapeutic products, as they secrete a variety of bioactive molecules that play a pivotal role in tissue regeneration. These factors are released in culture media for producing a conditioned medium (CM). The aforementioned assumptions led to the formulation of equine synovial membrane MSCs (eSM-MSCs)—the cellular pool that naturally regenerates joint tissue—combined with a medium enriched in immunomodulatory factors (among other bioactive factors) produced by umbilical cord stroma-derived MSCs (eUC-MSCs) that naturally contribute to suppressing the immune rejection in the maternal–fetal barrier. A description of an equine sport horse diagnosed with acute tarsocrural desmitis and treated with this formulation is presented. Ultrasonographic ligament recovery occurred in a reduced time frame, reducing stoppage time and allowing for the horse’s return to unrestricted competition after the completion of a physical rehabilitation program. This study focused on the description of the therapeutic formulation and potential in an equine desmitis treatment using the cells themselves and their secretomes.

Preemptive administration of mesenchymal stem cells-derived conditioned medium can attenuate the development of neuropathic pain in rats via downregulation of proinflammatory cytokines

Neuropathic pain (NP) is a chronic condition characterized by persistent pain following nerve injury. It is a challenging clinical problem to manage due to limited treatment options. Mesenchymal stem cells (MSCs)-derived conditioned medium (CM) is a cell-free product that contains the secretome of MSCs and has been shown to have therapeutic potential in various inflammatory and degenerative disorders. Several animal studies have examined the antinociceptive effects of MSCs-CM on established neuropathic pain, but none have investigated the early prevention of neuropathic pain using MSCs-CM. Therefore, in this study, we tested whether preemptive administration of MSCs-CM could attenuate the development of NP in rats. To this end, NP was induced in Wistar rats using a chronic constriction injury (CCI) model (day 0), and then the animals were divided into four groups: Sham, CCI, CCI-Dulbecco's Modified Eagle Medium (DMEM), and CCI-CM. The CCI-CM group received 1 ml intraperitoneal administration of MSCs-CM on days − 1, 1, and 2, while the Sham, CCI, and CCI-DMEM groups received vehicle only (normal saline or DMEM). Mechanical withdrawal threshold and thermal withdrawal latency were assessed to evaluate pain sensitivities. In addition, the expression levels of proinflammatory cytokines (TNF-α and IL-1β) in the spinal cord tissues were measured using quantitative real-time PCR (qRT-PCR). The results demonstrated that preemptive treatment with MSCs-CM can significantly attenuate the development of NP, as evidenced by improved mechanical withdrawal threshold and thermal withdrawal latency in the CCI-CM group compared to the CCI and CCI-DMEM groups. Furthermore, the relative gene expression of proinflammatory cytokines TNF-α and IL-1β were significantly decreased in the spinal cord tissues of the CCI-CM group compared to the control groups. These findings suggest that preemptive administration of MSCs-CM can attenuate the development of NP in rats, partly due to the downregulation of proinflammatory cytokines.

MMP-2-mediated Scube2 degradation promotes blood-brain barrier disruption by blocking the interaction between astrocytes and endothelial cells via inhibiting Sonic hedgehog pathway during early cerebral ischemia.

We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an invitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an invivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.

Curcumin Disrupts a Positive Feedback Loop between ADMSCs and Cancer Cells in the Breast Tumor Microenvironment via the CXCL12/CXCR4 Axis.

Adipose tissue has a significant impact on breast cancer initiation and progression owing to its substantial proportion in the breast. Adipose-derived mesenchymal stem cells (ADMSCs) are major players in the breast tumor microenvironment (TME) as they interact with cancer cells. The intricate interaction between ADMSCs and cancer cells not only drives the differentiation of ADMSCs into cancer-associated fibroblasts (CAFs) but also the metastasis of cancer cells, which is attributed to the CXCL12/CXCR4 axis. We investigated the effects of curcumin, a flavonoid known for CXCL12/CXCR4 axis inhibition, on breast TME by analyzing whether it can disrupt the ADMSC-cancer positive loop. Using MCF7 breast cancer cell-derived conditioned medium (MCF7-CM), we induced ADMSC transformation and verified that curcumin diminished the phenotypic change, inhibiting CAF marker expression. Additionally, curcumin suppressed the CXCL12/CXCR4 axis and its downstream signaling both in ADMSCs and MCF7 cells. The CM from ADMSCs, whose ADMSC-to-CAF transformation was repressed by the curcumin treatment, inhibited the positive feedback loop between ADMSCs and MCF7 as well as epithelial-mesenchymal transition in MCF7. Our study showed that curcumin is a potent anti-cancer agent that can remodel the breast TME, thereby restricting the ADMSC-cancer positive feedback loop associated with the CXCL12/CXCR4 axis.

Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro.

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro.

Antiangiogenic Action of JZL184 on Endothelial Cells via Inhibition of VEGF Expression in Hypoxic Lung Cancer Cells.

JZL184, an inhibitor of monoacylglycerol lipase (MAGL) and thus of the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG), mediates various anticancer effects in preclinical studies. However, studies on the effect of this or other MAGL inhibitors under hypoxia, an important factor in tumor biology and response to cancer therapy, have not yet been performed in cancer cells. In the present study, the impact of the conditioned media (CM) of A549 and H358 lung cancer cells incubated with JZL184 under hypoxic conditions on the angiogenic properties of human umbilical vein endothelial cells (HUVECs) was investigated. Treatment of HUVECs with CM derived from cancer cells cultured for 48 h under hypoxic conditions was associated with a substantial increase in migration and tube formation compared with unconditioned medium, which was inhibited when cancer cells were incubated with JZL184. In this process, JZL184 led to a significant increase in 2-AG levels in both cell lines. Analysis of a panel of proangiogenic factors revealed inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) expression by JZL184. Antiangiogenic and VEGF-lowering effects were also demonstrated for the MAGL inhibitor MJN110. Receptor antagonist experiments suggest partial involvement of the cannabinoid receptors CB1 and CB2 in the antiangiogenic and VEGF-lowering effects induced by JZL184. The functional importance of VEGF for angiogenesis in the selected system is supported by observations showing inhibition of VEGF receptor 2 (VEGFR2) phosphorylation in HUVECs by CM from hypoxic cancer cells treated with JZL184 or when hypoxic cancer cell-derived CM was spiked with a neutralizing VEGF antibody. On the other hand, JZL184 did not exert a direct effect on VEGFR2 activation induced by recombinant VEGF, so there seems to be no downstream effect on already released VEGF. In conclusion, these results reveal a novel mechanism of antiangiogenic action of JZL184 under conditions of hypoxic tumor-endothelial communication.

Effect of Periodontal Ligament Stem Cells-Derived Conditioned Medium on Gene Expression and Differentiation of Tumor Necrosis Factor-α-Challenged Osteoblasts.

Tumor necrosis factor-α (TNF-α) causes bone resorption in periodontitis. It induces the production of receptor activator of NF-κB ligand (RANKL) from osteoblasts, leading to the disturbance of bone homeostasis through RANKL, RANK, and osteoprotegerin (OPG) axis. This study aimed to explore the effect of periodontal ligament stem cells-derived conditioned medium (PDLSCs-CM) on gene expression related to bone homeostasis and the differentiation of TNF-α-challenged osteoblasts. Human osteoblasts were cultured with 50 ng/mL of TNF-α and 0, 1, 10, and 100 µg/ mL of PDLSCs-CM. Osteoblasts cultured without TNF-α and PDLSCs-CM were served as control. Gene expression of RANKL, OPG, and interleukin-1β (IL-1β) was evaluated by reverse transcription quantitative polymerase chain reaction at 48 hours. The early-stage and late-stage differentiation of TNF-α-challenged osteoblasts without or with PDLSCs-CM was explored by alkaline phosphatase (ALP) activity and alizarin red staining, respectively, at day 1, 3, 6, 9, and 12. Mann-Whitney U test was used to analyze the differences in gene expression of TNF-α-challenged osteoblasts at 24 and 48 hours, and Kruskal-Wallis test was used to analyze the effect of PDLSCs-CM on gene expression and ALP activity among all experimental groups using SPSS software version 21.0. Statistical significance was considered with p-value less than 0.05. Expression of RANKL, OPG and IL-1β was significantly upregulated in TNF-α-challenged osteoblasts compared to the untreated control. The PDLSCs-CM at 1 and 10 μg/mL downregulated gene expression of TNF-α-challenged osteoblasts compared to the group without PDLSCs-CM, but the difference did not reach statistical significance. The ALP activity was decreased in TNF-α-challenged osteoblasts. The addition of PDLSCs-CM did not alter ALP activity of TNF-α-challenged osteoblasts. Alizarin red staining was comparable in the TNF-α-challenged osteoblasts cultured without or with PDLSCs-CM. The PDLSCs-CM did not alter gene expression involved in bone homeostasis and differentiation of TNF-α-challenged osteoblasts.

SALL4 promotes angiogenesis in gastric cancer by regulating VEGF expression and targeting SALL4/VEGF pathway inhibits cancer progression

BackgroundSpalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown.MethodsAnalyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression.ResultsSALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM.ConclusionThese findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway.

Serum granulosa cell-derived TNF-α promotes inflammation and apoptosis of renal tubular cells and PCOS-related kidney injury through NF-κB signaling.

Polycystic ovary syndrome (PCOS) is a disorder with endocrinal and metabolic problems in reproductive aged women. Evidence shows that PCOS is in a high prone trend to develop kidney diseases. In this study, we investigated the mediators responsible for PCOS-related kidney injury. We found that tumor necrosis factor (TNF-α) levels were significantly increased in serum and primary cultured granulosa cells (GCs) from PCOS patients. Serum TNF-α levels were positively correlated with serum testosterone and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, suggesting its positive role in the severity of PCOS. Serum TNF-α levels were also positively correlated with the levels of urinary KapU, LamU,α1-MU and β2-MU, the markers for renal tubular cell-derived proteinuria. We established a PCOS mouse model by resection of the right kidney, followed by daily administration of dihydrotestosterone (DHT, 27.5 μg, i.p.) from D7 for 90 days. We found that TNF-α levels were significantly increased in the ovary and serum of the mice, accompanied by increased renal tubular cell apoptosis, inflammation and fibrosis in kidneys. Furthermore, the receptor of TNF-α, tumor necrosis factor receptor 1 (TNFR1), was significantly upregulated in renal tubular cells. We treated human ovarian granulosa-like tumor cells (KGN) with DHT (1 μg/ml) in vitro, the conditioned medium derived from the granulosa cellculture greatly accelerated apoptotic injury in human proximal tubular epithelial cells (HKC-8), which was blocked after knockdown of TNF-α in KGN cells. Furthermore, knockdown of TNFR1 in renal tubular epithelial cells greatly ameliorated cell injury induced by granulosa cell-derived conditioned medium. These results suggest that serum TNF-α plays a key role in mediating inflammation and apoptosis in renal tubular cells associated with PCOS-related kidney injury.

E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways.

E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.

A novel therapeutic approach for endometriosis using adipose-derived stem cell-derived conditioned medium- A new hope for endometriotic patients in improving fertility.

Endometriosis is defined as the growth of endometrial glands and stromal cells in a heterotopic location with immune dysregulation. It usually leads to chronic pelvic pain and subfertility. Although various treatments are available, the recurrence rate remains high. Adipose tissue is an abundant source of multipotent mesenchymal adipose-derived stem cells (ADSCs). ADSCs display effects on not only tissue regeneration, but also immune regulation. Thus, the current study aims to test the effects of ADSCs on the growth of endometriosis. ADSCs isolated from lipoaspiration-generated adipose tissue and their conditioned medium (ADSC-CM) were subjected to quality validation, including karyotyping as well as growth promotion and sterility tests for microbial contamination under Good Tissue Practice and Good Manufacturing Practice regulations. An autologous endometriosis mouse model was established by suturing endometrial tissue to peritoneal wall followed by treating with DMEM/F12 medium, ADSC-CM, ADSCs or ADSC-CM+ADSCs for 28 days. The area of endometriotic cysts and the degree of pelvic adhesion were measured. ICAM-1, VEGF and caspase 3 expression was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Moreover, the mice were allowed to mate and deliver. The pregnancy outcomes were recorded. The ADSC-CM was subjected to proteomics analysis with further data mining with Ingenuity Pathway Analysis (IPA). Both ADSC-CM and ADSCs passed quality validation. ADSC-CM reduced the area of endometriotic cysts. The inhibition by ADSC-CM was obliterated by adding ADSCs. The presence of ADSCs with or without ADSC-CM increased the peritoneal adhesion. ADSC-CM inhibited ICAM-1 and VEGF mRNA and protein expression, whereas the addition of ADSCs not only did not inhibit by itself, but also blocked the inhibition by ADSC-CM. The resorption rate was reduced by ADSC-CM. The number of live birth/dam and the survival rate of pup at 1 week-old were both increased by ADSC-CM in mice with endometriosis. IPA demonstrated that PTX3 was potentially critical for the inhibition of endometriosis by ADSC-CM due to its anti-inflammatory and antiangiogenic properties as well as its importance in implantation. ADSC-CM inhibited endometriosis development and improved pregnancy outcomes in mice. Potential translation to clinical treatment for human endometriosis is expected.

Mitoprotective effect of mesenchymal stem cells-derived conditioned medium in myocardial reperfusion injury of aged rats: role of SIRT-1/PGC-1α/NRF-2 network.

The aged myocardium experiences various forms of stress that cause reduction of its tolerance to injury induced by ischemia/reperfusion (I/R). Developing effective cardioprotective modalities to prevent the amplification of I/R injury during aging is under focus of investigation. Mesenchymal stem cells (MSCs) have the ability to regenerate infarcted myocardium mostly by producing multiple secretory factors. This study aimed to explore the mechanisms of mitoprotection by MSCs-conditioned medium (CM) in myocardial I/R injury of aged rats. Male Wistar rats (n = 72, 400-450g, 22-24months old) were randomized into groups with/without I/R and/or MSCs-CM treatment. To establish myocardial I/R injury, the method of LAD occlusion and re-opening was employed. MSCs-CM was administered intramyocardially (150μl) at the onset of reperfusion in recipient group. After 24h reperfusion, myocardial infarct size, LDH level, mitochondrial functional endpoints, expression of mitochondrial biogenesis-associated genes, and the levels of pro-inflammatory cytokines were evaluated. After 28days reperfusion, echocardiographic assessment of cardiac function was performed. MSCs-CM treatment improved myocardial function and decreased infarct size and LDH level in aged I/R rats (P < .05 to P < .001). It also decreased mitochondrial ROS formation, enhanced mitochondrial membrane potential and ATP content, upregulated mitochondrial biogenesis-related genes including SIRT-1, PGC-1α, and NRF-2, and lessened TNF-α, IL-1β, and IL-6 levels (P < .05 to P < .01). MSCs-CM treatment attenuated myocardial I/R injury in aged rats, in part by improving mitochondrial function and biogenesis and restraining inflammatory reaction. the upregulation of SIRT-1/PGC-1α/NRF-2 profiles is a possible target for the mitoprotective effects of MSCs-CM following I/R injury during aging.

Mesenchymal-Stem Cell-Derived Conditioned Media Versus Exosomes in the Treatment of Rat Model of Polycystic Ovary: An Attempt to Understand the Underlying Mechanisms (Biochemical and Histological Study).

Polycystic ovary syndrome (PCOS) is one of the most common endocrine and reproductive disorders throughout female reproductive age. Cell free therapy [conditioned media (CM) & exosomes (EXO)] is a promising approach in regenerative medicine. This study aimed to compare between the therapeutic effects of stem cell-derived CM and exosomes on induced animal model of polycystic ovary. Polycystic ovary (PCO) was induced in female rats (3-4 weeks old, weighing 70-80 g) by letrozole with a dose of 1 mg/kg/day dissolved in carboxymethylcellulose 1% orally once daily for 5 weeks. Animals were divided into four groups: control group, PCO group, EXO-treated group, and CM-treated group. Serum levels of testosterone hormone, leutinizing hormone, follicle stimulatimg hormone, and insulin hormone were estimated. Immunohistochemistry using anti-P53, anti-AMP-dependent protein kinase antibodies were done. Six rats/group were used for matting with adult male rats for testing fertility. The results showed that CM had significant superior therapeutic effects on exosomes in restoring the normal histological architecture of the ovary and fertility. In summary, cell free treatment is a safe approach for tissue regeneration. Stem cell-derived CM was more effective than exosomes in restoring normal histological structure of the ovaries and fertility in animal models of polycystic ovary.

Abstract 367: The heparan sulfate mimetic PG545 inhibits tumor growth in both male and female mouse model of pancreatic ductal adenocarcinoma (PDAC) with sex difference in the effect on cachexia

Abstract BACKGROUND: Cachexia occurs in up to 80% of PDAC patients and is characterized by weight loss mainly due to muscle and fat wasting. Cachexia leads to functional impairment, reduced anticancer therapy tolerance and increased mortality. PG545, a heparan sulfate (HS) mimetic, shows anticancer activity; however, it is unknown whether sex affects the anticancer activity and what effect PG545 has on cancer associated muscle and fat wasting. Given that many cytokines induced in patients with cancer cachexia can bind HS, the aim of this study was to assess PG545 effects on PDAC cachexia with emphasis on sex difference. METHODS: PDAC cells were injected into the pancreas of 10-week C57BL/6 male and female mice. The orthotopic tumor-bearing mice were treated with PG545 (10 mg/kg; i.p.). Body weight and body composition were monitored, and tumor and organs were collected. Skeletal muscles were cryosectioned for evaluation of cross-sectional area (CSA). C2C12 myotubes, the in vitro model of muscle wasting, were treated with PDAC cell-derived conditioned medium (CM) without or with PG545 and analyzed for the changes in size. PDAC cell proliferation was assessed by MTT assay. RESULTS: PG545 treatment markedly reduced tumor mass in both males and females compared to the vehicle controls. PG545 also reduced body weight but the difference gradually dissipated over time despite repeated dosing. However, male mice recovered from the negative effects more slowly than female mice. Similar sex difference was observed in the changes in the PG545-induced lean and fat mass loss. Of note, fat mass in PG545-treated females at the end of experiment was higher than in the vehicle controls. Furthermore, males had more reductions in muscle and fat weights than females in response to PG545. Grip strength in males was weakened by PG545 but returned to the level like that in the vehicle controls at the end, while PG545 did not weaken grip strength in females at the same time points. In addition, males, but not females, had smaller muscle fiber CSA after PG545 treatment versus their vehicle controls. In in vitro assay, PG545 inhibited PDAC cell proliferation, indicating a direct effect on cancer cells. Lastly, opposed to the in vivo effect, PG545 partly prevented myotube wasting induced by PDAC CM. CONCLUSION: PG545 has strong anti-PDAC activity in both male and female mice, at least partly through inhibition of cancer cell proliferation. Its initial negative effects on cachexia are gradually reversed with male tumor-bearing mice reversing more slowly than females. Because of the protective effect on C2C12 myotubes treated with PDAC CM, PG545 might induce some counteracting factors in vivo in the initial treatment and identification of these factors may offer molecular targets for combination therapy to counteract the negative effects while take advantage of the strong anti-cancer potential. Citation Format: Xiaoling Zhong, Keith Dredge, John M. Chirgwin, Michael G. House, Harikrishna Nakshatri, Teresa A. Zimmers. The heparan sulfate mimetic PG545 inhibits tumor growth in both male and female mouse model of pancreatic ductal adenocarcinoma (PDAC) with sex difference in the effect on cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 367.

Mesenchymal stem cell-derived conditioned medium and Methysergide give rise to crosstalk inhibition of 5-HT2A and 5-HT7 receptors in neuroblastoma cells

Objective(s): We aimed to investigate the effects of mesenchymal stem cell secretome and methysergide combination on 5-hydroxytryptamine 2A, (5-HT2AR), 5-hydroxytryptamine 7 (5-HT7R), adenosine 2A (A2AR) receptors and CD73 on neuroblastoma cell line and how they affect biological characteristics. Methysergide was used as a serotonin antagonist on the neuroblastoma cells. Materials and methodsHuman dental pulp-derived stem cells (hDPSCs) used to obtain conditioned medium (CM). Methysergide drug was prepared in CM and applied to neuroblastoma cells. Analysis of 5-HT7R, 5-HT2AR, A2AR and CD73 expressions was performed by western blot and immunofluorescence staining. Total apoptosis, mitochondrial membrane depolarization, Ki-67 proliferation test, viability analysis, DNA damage and cell cycle analysis were performed in accordance with the product procedure by using biological activity test kits. ResultsOur results showed that neuroblastoma cancer cells are normally on the Gs signaling axis via the serotonin 7 receptor and the adenosine 2A receptor. CM and Methysergide inhibited the 5-HT7 and A2A receptor levels in neuroblastoma cells. We found that CM and methysergide formed crosstalk inhibition between 5-HT2AR, 5-HT7R, A2AR and CD73. CM and Methysergide increased the total apoptosis in neuroblastoma cells and induced the mitochondrial membrane depolarization. CM and Methysergide induced the DNA damage and arrested in G0/G1 phase of cell cycle of the neuroblastoma cells. ConclusionThese findings suggest that the combination of CM and methysergite may exert a therapeutic effect on neuroblastoma cancer cells, and future in vivo studies may be important in area of neuroblastoma research to support the findings.

Paracrine effects of stem cell conditioned medium on production of oxygen reactive species in blood neutrophils in acetaminophen-induced liver failure

The current study examined the effects of mesenchymal stem cells-derived conditioned medium on the severity of systemic inflammatory response induced by the administration of acetaminophen, as well as its long-term effects on hepatic tissues. A conditioned medium fraction ˂30 кDа showed maximum protective effect. Proteins of this fraction reduce the degree of systemic inflammatory response and liver tissue fibrosis long after the toxin administration. Liver tissues from experimental animals were examined, and a correlation between the reduction of granulocytic infiltration scoring and the degree hepatic parenchymal necrosis was demonstrated (confirmed by the cytolytic enzyme level). The ˂30 кDа fraction increased the spontaneous but decreased phobrol-12-myristate-13-acetate-induced intracellular production of reactive oxygen species. Results from this study show that the level of macrophage migration inhibitory factor and expression of thioredoxin significantly increase as compared to controls during increased spontaneous production of reactive oxygen species in neutrophils.

Andrographolide-treated bone marrow mesenchymal stem cells-derived conditioned medium protects cardiomyocytes from injury by metabolic remodeling.

Bone marrow mesenchymal stem cells (BMSCs) transplantation therapy providing a great hope for the recovery of myocardial ischemic hypoxic injury. However, the microenvironment after myocardial injury is not conducive to the survival of BMSCs, which limits the therapeutic application of BMSCs. Our previous study has confirmed that the survival of BMSCs cells in the glucose and serum deprivation under hypoxia (GSDH) is increased after Andrographolide (AG) pretreatment, but whether this treatment could improve the effect of BMSCs in repairing of myocardial injury has not been verified. We first treated H9C2 with GSDH to simulate the microenvironment of myocardial injury in vitro, then we pretreated rat primary BMSCs with AG, and collected conditioned medium derived from BMSCs (BMSCs-CM) and conditioned medium derived from AG-pretreated BMSCs (AG-BMSCs-CM) after GSDH treatment. And they were used to treat H9C2 cells under GSDH to further detect oxidative stress and metabolic changes. The results showed that AG-BMSCs-CM could be more advantageous for cardiomyocyte injury repair than BMSCs-CM, as indicated by the decrease of apoptosis rate and oxidative stress. The changes of mitochondria and lipid droplets results suggested that AG-BMSCs-CM can regulate metabolic remodeling of H9C2 cells to repair cell injury, and that AMPK was activated during this process. This study demonstrates, for the first time, the protective effect of AG-BMSCs-CM on GSDH-induced myocardial cell injury, providing a potential therapeutic strategy for clinical application.

Characterization of cellular senescence in radiation ulcers and therapeutic effects of mesenchymal stem cell-derived conditioned medium

Radiation ulcers are a common and severe injury after uncontrolled exposure to ionizing radiation. The most important feature of radiation ulcers is progressive ulceration, which results in the expansion of radiation injury to the nonirradiated area and refractory wounds. Current theories cannot explain the progression of radiation ulcers. Cellular senescence refers to as irreversible growth arrest that occurs after exposure to stress, which contributes to tissue dysfunction by inducing paracrine senescence, stem cell dysfunction and chronic inflammation. However, it is not yet clear how cellular senescence facilitates the continuous progression of radiation ulcers. Here, we aim to investigate the role of cellular senescence in promoting progressive radiation ulcers and indicate a potential therapeutic strategy for radiation ulcers. Radiation ulcer animal models were established by local exposure to 40Gy X-ray radiation and continuously evaluated for >260days. The roles of cellular senescence in the progression of radiation ulcers were assessed using pathological analysis, molecular detection and RNA sequencing. Then, the therapeutic effects of conditioned medium from human umbilical cord mesenchymal stem cells (uMSC-CM) were investigated in radiation ulcer models. Radiation ulcer animal models with features of clinical patients were established to investigate the primary mechanisms responsible for the progression of radiation ulcers. We have characterized cellular senescence as being closely associated with the progression of radiation ulcers and found that exogenous transplantation of senescent cells significantly aggravated them. Mechanistic studies and RNA sequencing suggested that radiation-induced senescent cell secretions were responsible for facilitating paracrine senescence and promoting the progression of radiation ulcers. Finally, we found that uMSC-CM was effective in mitigating the progression of radiation ulcers by inhibiting cellular senescence. Our findings not only characterize the roles of cellular senescence in the progression of radiation ulcers but also indicate the therapeutic potential of senescent cells in their treatment.

Therapeutic Approach for Hair Growth and Regeneration Using Bioactive Formulation Containing Mesenchymal Stromal Cell-Derived Conditioned Medium

Therapeutic Approach for Hair Growth and Regeneration Using Bioactive Formulation Containing Mesenchymal Stromal Cell-Derived Conditioned Medium