Stem Cell-derived Conditioned Medium Research Articles

The effect of mesenchymal stem cell conditioned medium incorporated within chitosan nanostructure in clearance of common gastroenteritis bacteria in-vitro and in-vivo

Gastroenteritis infection is a major public health concern worldwide, especially in developing countries due to the high annual mortality rate. The antimicrobial and antibiofilm activity of human mesenchymal stem cell-derived conditioned medium (hMSCsCM) encapsulated in chitosan nanoparticles (ChNPs) was studied in vitro and in vivo against common gastroenteritis bacteria. The synthesized ChNPs were characterized using Zeta potential, scanning electron microscopy (SEM), and dynamic light scattering (DLS) techniques. HMSC-derived conditioned medium incorporated into chitosan NPs (hMSCsCM-ChNPs) composite was fabricated by chitosan nanoparticles loaded with BM-MSCs (positive for CD73 and CD44 markers). The antimicrobial and antibiofilm activity of composite was investigated against four common gastroenteritis bacteria (Campylobacter jejuni ATCC29428, Salmonella enteritidis ATCC13076, Shigella dysenteriae PTCC1188, and E. coli ATCC25922) in-vitro and in-vivo. Majority of ChNPs (96%) had an average particle size of 329 nm with zeta potential 7.08 mV. The SEM images confirmed the synthesis of spherical shape for ChNPs and a near-spherical shape for hMSCsCM-ChNPs. Entrapment efficiency of hMSCsCM-ChNPs was 75%. Kinetic profiling revealed that the release rate of mesenchymal stem cells was reduced following the pH reduction. The antibacterial activity of hMSCsCM-ChNPs was significantly greater than that of hMSCsCM and ChNPs at dilutions of 1:2 to 1:8 (P < 0.05) against four common gastroenteritis bacteria. The number of bacteria present decreased more significantly in the group of mice treated with the hMSCsCM-ChNPs composite than in the groups treated with hMSCsCM and ChNPs. The antibacterial activity of hMSCsCM against common gastroenteritis bacteria in an in vivo assay decreased from > 106 CFU/ml to approximately (102 to 10) after 72 h. Both in vitro and in vivo assays demonstrated the antimicrobial and antibiofilm activities of ChNPs at a concentration of 0.1% and hMSCsCM at a concentration of 1000 μg/ml to be inferior to that of hMSCsCM-ChNPs (1000 μg/ml + 0.1%) composite. These results indicated the existence of a synergistic effect between ChNPs and hMSCsCM. The designed composite exhibited notable antibiofilm and antibacterial activities, demonstrating optimal release in simulated intestinal lumen conditions. The utilization of this composite is proposed as a novel treatment approach to combat gastroenteritis bacteria in the context of more challenging infections.

Bioregenerative Applications of the Human Mesenchymal Stem Cell- Derived Secretome: Part-I

Mesenchymal stem cells hold many therapeutic benefits in treating diverse disease states, but autologous requirements, high costs, lack of standardization and other factors limit their widespread application. Additionally, researchers have discovered that many mesenchymal stem cell in-vivo benefits originate from their paracrine factors. Therefore, cell-free therapeutics, including mesenchymal stem cell-derived conditioned medium and extracellular vesicles have been suggested as alternative bioregenerative therapies. This literature review summarizes mesenchymal stem cell application, the benefits of cell-free strategies and the components of its secretome. This manuscript is a companion piece to “Part 2: Bioregenerative Applications of the Human Mesenchymal Stem Cell-Derived Secretome,” included in this issue, which contains the results of in-vivo studies applying the conditioned medium and extracellular vesicles to human and animal models, ongoing clinical trials, limitations to cell-free strategies and future directions for the wide-scale adoption of these therapies.

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.

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.

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.

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.

Protective effect of human umbilical cord mesenchymal stem cell derived conditioned medium in a mutant TDP-43 induced motoneuron-like cellular model of ALS

Amyotrophic lateral sclerosis (ALS) is a multi-factor neurodegenerative disease, characterized by the loss of motor neurons. TAR DNA-binding protein 43 (TDP-43) mutation, accumulation and aggregation, as well as oxidative stress are recognized as major pathological denominators and biochemical markers for ALS. Recently, human umbilical cord mesenchymal stem cell-derived conditioned medium (UC-CM) has been introduced to treat ALS patients. However, there is no research for the protective effect of UC-CM on the TDP-43 model of ALS. In this study, we evaluated the potential neuroprotective effect of UC-CM on a cellular ALS model expressing TDP-43mutant M337V, as well as its underlying mechanism. We found that 24 h UC-CM treatment could protect M337V expressing motor neurons by increasing cell viability and reducing LDH leakage. Furthermore, the aggregation of M337V, generation of ROS, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), protein carbonyl and 8-OHdG were also reduced by UC-CM, indicating that UC-CM protected cells by reducing oxidative damage. Moreover, UC-CM significantly increased the expression of nuclear Nrf2 and its downstream enzyme HO1. The Nrf2 translocation inhibitor ML385 could inhibit the effect of UC-CM on the cell viability and aggregate of M337V. Our results suggest that UC-CM protect cells against M337V expression by its strong antioxidative effect via Nrf-2/HO-1 axis activation.

Therapeutic Effect of Three-Dimensional Cultured Adipose-Derived Stem Cell-Conditioned Medium in Renal Ischemia-Reperfusion Injury.

We evaluated the effect of adipose-derived stem cell-derived conditioned medium (ADSC-CM) on the renal function of rats with renal ischemia-reperfusion injury (IRI)-induced acute kidney injury. Forty male Sprague-Dawley rats were randomly divided into four groups: sham, nephrectomy control, IRI control, ADSC-CM. The ADSC-CM was prepared using the three-dimensional spheroid culture system and injected into renal parenchyme. The renal function of the rats was evaluated 28 days before and 1, 2, 3, 4, 7, and 14 days after surgical procedures. The rats were sacrificed 14 days after surgical procedures, and kidney tissues were collected for histological examination. The renal parenchymal injection of ADSC-CM significantly reduced the serum blood urea nitrogen and creatinine levels compared with the IRI control group on days 1, 2, 3, and 4 after IRI. The renal parenchymal injection of ADSC-CM significantly increased the level of creatinine clearance compared with the IRI control group 1 day after IRI. Collagen content was significantly lower in the ADSC-CM group than in the IRI control group in the cortex and medulla. Apoptosis was significantly decreased, and proliferation was significantly increased in the ADSC-CM group compared to the IRI control group in the cortex and medulla. The expressions of anti-oxidative makers were higher in the ADSC-CM group than in the IRI control group in the cortex and medulla. The renal function was effectively rescued through the renal parenchymal injection of ADSC-CM prepared using a three-dimensional spheroid culture system.

Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs.

In this study, we assessed the effects of human deciduous dental pulp-derived mesenchymal stem cell-derived conditioned medium (SHED-CM) on the properties of various cell types. The effects of vascular endothelial growth factor (VEGF) in SHED-CM on the luminal architecture, proliferative ability, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) were determined. We also investigated the effects of SHED-CM on the proliferation of human-bone-marrow mesenchymal stem cells (hBMSCs) and mouse calvarial osteoblastic cells (MC3T3-E1) as well as the expression of ALP, OCN, and RUNX2. The protein levels of ALP were examined using Western blot analysis. VEGF blockade in SHED-CM suppressed the proliferative ability and angiogenic potential of HUVECs, indicating that VEGF in SHED-CM contributes to angiogenesis. The culturing of hBMSCs and MC3T3-E1 cells with SHED-CM accelerated cell growth and enhanced mRNA expression of bone differentiation markers. The addition of SHED-CM enhanced ALP protein expression in hBMSCs and MT3T3-E1 cells compared with that of the 0% FBS group. Furthermore, SHED-CM promoted the metabolism of HUVECs, MC3T3-E1 cells, and hBMSCs. These findings indicate the potential benefits of SHED-CM in bone tissue regeneration.

LPS preconditioning of MSC-CM improves protection against hypoxia/reoxygenation-induced damage in H9c2 cells partly via HMGB1/Bach1 signalling.

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function after myocardial infarction; however, this cardioprotective effect is moderate and transient. Lipopolysaccharide (LPS) pretreatment partially improves MSC-CM-mediated cardioprotective effects owing to the presence of paracrine factors. However, the mechanism underlying these improved effects remains unknown. To study the effect of LPS-pretreated MSC-CM on hypoxia/reoxygenation (H/R)-induced injury, MSCs were treated with or without LPS (400 ng/mL) for 48 h, and the supernatant was collected (MSC-CM). Subsequently, H9c2 cells were co-cultured with Nor-CM (CM derived from LPS-untreated MSCs) and LPS-CM (CM derived from LPS-pretreated MSCs) for 24 h and subjected to H/R. MSC-CM inhibited the progression of H/R-induced injury in H9c2 cells, and this protective effect was enhanced via LPS pretreatment as evidenced by the improved apoptosis assessment index (i.e. caspase-3 and B-cell lymphoma-2 [Bcl-2] expression) and decreased levels of lactic dehydrogenase (LDH) and cardiac troponin (cTn). In addition, the results of haematoxylin-eosin staining (H&E), transmission electron microscopy (TEM) and TdT-mediated dUTP nick-end labelling (TUNEL) validated that MSC-CM inhibited H/R-induced injury in H9c2 cardiomyocytes. LPS pretreatment downregulated the expression of high mobility group box-1 (HMGB1) and BTB and CNC homology-1 (Bach1) proteins in MSCs but upregulated the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF). HMGB1 knockdown (MSC/siHMGB1-CM) significantly decreased the expression of Bach1 and increased the expression of VEGF, HGF and IGF. Bach1 knockdown (MSC/siBach1-CM) did not alter the production of HMGB1 but increased the expression of VEGF and IGF. LPS pretreatment did not alter the expression of the paracrine factors VEGF and HGF in the MSC/siHMGB1 group but increased their expression in the MSC/siBach1 group. The myocyte anti-apoptotic effects of MSCs/siBach1-CM were similar to those of untreated MSCs, which were not enhanced by LPS. LPS-pretreated MSC-CM protects H9c2 cells against H/R-induced injury partly through the HMGB1/Bach1 signalling pathway.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Conditioned Medium Promotes Human Endometrial Cell Proliferation through Wnt/β-Catenin Signaling.

Purpose Mesenchymal stem cells (MSCs) and their derivant are among the promising treatments for intrauterine adhesion (IUA); they have been reported to repair the endometrial injury by proliferating endometrial cells. However, the signal pathways involved are not clear. This study investigated the role of human umbilical cord mesenchymal stem cell-derived conditioned medium (hUCMSC-CM) in relieving IUA to find out whether Wnt/β-catenin signaling was involved, and if so, to determine the possible ligands. Methods After endometrial epithelial cells (EECs) were treated with hUCMSC-CM, their proliferation and migration were measured by the CCK8 assay and the scratch assay. The activation of Wnt/β-catenin signaling was measured by Western blots, fluorescent staining, and T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase. A Wnt inhibitor (XAV393) was used to inhibit the proliferation effect of hUCMSC-CM in EECs. Wnt5a expression in hUCMSC was measured by Western blots and fluorescent staining, and Wnt5a in hUCMSC-CM was detected by enzyme-linked immunosorbent assay (ELISA), to further clarify the mechanism. Results As shown by the CCK8 assay, hUCMSC-CM promoted proliferation and migration of EECs. The expression of β-catenin, c-myc, and cyclin D1 increased in EECs after being treated with hUCMSC-CM. Moreover, hUCMSC-CM was found to promote β-catenin delivery into nuclei by Western blot and fluorescent staining; meanwhile, the inhibitor (XAV393) could restrain this process and inhibit the effect of hUCMSC-CM on EEC proliferation. Wnt5a was detected in hUCMSCs and hUCMSC-CM, which might be a potential therapeutic target. Conclusion This study demonstrated that hUCMSC-CM promoted human endometrial cell proliferation through Wnt/β-catenin signaling, and Wnt5a might be a potential activator. This would be one of the activating signal pathways in the MSC-related treatment of IUA.

Identification and characterization of stem cell secretome-based recombinant proteins for wound healing applications.

Stem cells have been introduced as a promising therapy for acute and chronic wounds, including burn injuries. The effects of stem cell-based wound therapies are believed to result from the secreted bioactive molecules produced by stem cells. Therefore, treatments using stem cell-derived conditioned medium (CM) (referred to as secretome) have been proposed as an alternative option for wound care. However, safety and regulatory concerns exist due to the uncharacterized biochemical content and variability across different batches of CM samples. This study presents an alternative treatment strategy to mitigate these concerns by using fully characterized recombinant proteins identified by the CM analysis to promote pro-regenerative healing. This study analyzed the secretome profile generated from human placental stem cell (hPSC) cultures and identified nine predominantly expressed proteins (ANG-1, FGF-7, Follistatin, HGF, IL-6, Insulin, TGFβ-1, uPAR, and VEGF) that are known to contribute to wound healing and angiogenesis. These proteins, referred to as s (CMFs), were used in combination to test the effects on human dermal fibroblasts (HDFs). Our results showed that CMF treatment increased the HDF growth and accelerated cell migration and wound closure, similar to stem cell and CM treatments. In addition, the CMF treatment promoted angiogenesis by enhancing new vessel formation. These findings suggest that the defined CMF identified by the CM proteomic analysis could be an effective therapeutic solution for wound healing applications. Our strategy eliminates the regulatory concerns present with stem cell-derived secretomes and could be developed as an off-the-shelf product for immediate wound care and accelerating healing.

Is human umbilical cord mesenchymal stem cell-derived conditioned medium effective against oxidative and inflammatory status in CCl4-induced acute liver injury?

IntroductionAcute liver injury (ALI) is diagnosed by detection of elevated liver enzymes within six months after liver damage. Mesenchymal stem cells (MSCs) have recently been considered a beneficial strategy for treating various diseases due to healing secretory factors. Therapeutic effects of human umbilical cord MSCs-derived conditioned medium (hMSC-CM) were evaluated on CCl4-induced ALI. Materials and methodsTwenty-four male Wistar rats were divided into groups including N (received saline), ALI (received CCl4), RPMI (received CCl4 and RPMI medium), and ALI-CM (received CCl4 and hMSC-CM) groups. The expression of TNF-α and TGFβ-1 genes was evaluated with qPCR. Hepatic levels of TNF-α and TGF-β were measured by ELISA. Total antioxidant capacity (TAC), total oxidant status (TOS), malondialdehyde (MDA), glutathione peroxidase (GPx) activity, and catalase (CAT) activity were also assayed. Hematoxylin-eosin (H&E), Masson's trichrome, reticulin, and Periodic Acid–Schiff (PAS) stainings were conducted to evaluate tissue lesions. ResultsCCl4 increased expression of TNF-α and TGF-1β at both mRNA and protein levels, while hMSC-CM decreased these parameters in the ALI-CM group. TAC levels significantly decreased in the ALI group, and CCl4 increased TOS and MDA levels compared with the N group. hMSC-CM treatment led to the return of these parameters to their baseline levels. GPx and CAT activity in the ALI group were significantly lower than in the N group and hMSC-CM reduced these parameters to the baseline in the ALI-CM group. hMSC-CM modulated CCl4-induced tissue lesions. ConclusionThe present study suggests hMSC-CM probably improves CCl4-induced ALI through its antioxidant and anti-inflammatory effects.

Lipopolysaccharide-pretreated mesenchymal stem cell-conditioned medium optimized with 10 kDa filter attenuates the injury of H9c2 cardiomyocytes in a model of hypoxia/reoxygenation.

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function, which is partly attributed to the released paracrine factors. Since such cardioprotection is moderate and transient, it is essential that MSC-CM's effective components are optimized to alleviate myocardial injury. To optimize MSC-CM, MSCs were treated with or without lipopolysaccharides (LPSs) for 48 h (serum-free), and the supernatant was collected. Then, LPS-CM (MSC stimulated by LPS) was further treated with LPS remover (LPS Re-CM) or was concentrated with a 10 kDa cutoff filter (10 kDa-CM). Enzyme-linked immunosorbent assay showed that all the pretreatments increased the levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin growth factor (IGF) except LPS Re-CM; 10 kDa-CM was superior to the other CMs. Cell Counting Kit-8 displayed that the viability of injured H9c2 cells was enhanced with the increase in the MSC-CM concentration. We also found that the 10 kDa-CM significantly alleviated H9c2 hypoxia/reoxygenation (H/R) injury, as evidenced by the increased Bcl-2/Bax ratio, and decreased the levels of lactate dehydrogenase and cardiac troponin. Transmission electron microscopy (TEM), TdT-mediated dUTP nick-end labelling (TUNEL), and hematoxylin and eosin staining (H&E) confirmed that 10 kDa-CM inhibited H/R-induced H9c2 morphological changes. Proteomic analysis identified 41 differentially expressed proteins in 10 kDa-CM, among which anti-inflammation, proangiogenesis, and antiapoptosis were related to cardiac protection. This study indicates that 10 kDa-CM protects H9c2 cardiomyocytes from H/R injury by preserving most of the protective factors, such as VEGF, HGF, and IGF, in MSC-CM.

Therapeutic Effect of Human Mesenchymal Stem Cell-Conditioned Medium on Erectile Dysfunction.

PurposeOwing to the safety and cost effectiveness of conditioned medium (CM), its therapeutic effects have attracted significant attention from many researchers. To date, numerous studies have been conducted on CM; however, little has been done with regard to erectile dysfunction (ED). In this research, the potential of human mesenchymal stem cell-derived CM (MSC-CM) for the treatment of ED was investigated.Materials and MethodsA high concentration of MSC-CM was prepared through 3D spheroid culturing with bone marrow-derived MSCs and cut-off filtering. The composition of CM was analyzed using biochemical assays, and the effect of the preparation process on the quality of CM was investigated. The therapeutic effects of MSC-CM were evaluated through animal studies using a cavernous nerve (CN) injury rat model.Results3D spheroid culturing afforded a 278-fold increase in the total protein content of CM, as compared to that from 2D cultures; the protein concentration increased by 19 times on increasing the centrifugation time for cut-off filtering. Biochemical assays indicated that the CM contains various xlink:types of angiogenic, neurotrophic, and anti-inflammatory factors. Histological assay results showed that MSC-CM has angio- and neuro-trophic effect in a CN injury rat model in vivo, and these therapeutic effects appear in a dose-dependent manner.ConclusionsThe experimental results confirmed the therapeutic effect of MSC-CM in healing damaged cavernosal tissue and restoring erectile function. These results successfully demonstrated that MSC-CM has significant potential for the treatment of ED.

Protective effects of low-molecular-weight components of adipose stem cell-derived conditioned medium on dry eye syndrome in mice

The present study demonstrated the protective effects of low-molecular-weight adipose-derived stem cell-conditioned medium (LADSC-CM) in a mouse model of dry eye syndrome. Mice subjected to desiccating stress and benzalkonium chloride had decreased tear secretion, impaired corneal epithelial tight junction with microvilli, and decreased conjunctival goblet cells. Topical application of adipose-derived stem cell-conditioned medium (ADSC-CM) stimulated lacrimal tear secretion, preserved tight junction and microvilli of the corneal epithelium, and increased the density of goblet cells and MUC16 expression in the conjunctiva. The low-molecular-weight fractions (< 10 kDa and < 3 kDa) of ADSC-CM (LADSC-CM) provided better protections than the > 10 kDa or > 3 kDa fractions of ADSC-CM. In the in vitro study, desiccation for 10 min or hyperosmolarity (490 osmols) for 24 h caused decreased viability of human corneal epithelial cells, which were reversed by LADSC-CM. The active ingredients in the LADSC-CM were lipophobic and stable after heating and lyophilization. Our study demonstrated that LADSC-CM had beneficial effects on experimental dry eye. It is worthy of further exploration for the active ingredient(s) and the mechanism.

Combination therapy of iPSC-derived conditioned medium with ceftriaxone alleviates bacteria-induced lung injury by targeting the NLRP3 inflammasome.

The lung is the first and most frequent organ to fail among sepsis patients. The mortality rate of sepsis-related acute lung injury (ALI) is high. Despite appropriate antimicrobial therapy, no treatment strategies are available for sepsis-induced ALI. Stem cell-mediated paracrine signaling is a potential treatment method for various diseases. This study aimed to examine the effects of induced pluripotent stem cell-derived conditioned medium (iPSC-CM) combined with antibiotics on ALI in a rat model of Escherichia coli-induced sepsis. Rats were administered either iPSC-CM or the vehicle (saline) with antibiotics (ceftriaxone). After 72 h, liquid biopsy, bronchoalveolar lavage fluid (BALF), and tissues were harvested for analysis. Survival rates were observed for up to 3 days. Furthermore, we examined the effects of iPSC-CM on cytokine production, metalloproteinase 9 (MMP-9) expression, and NLRP3-ASC interaction in RAW264.7 cells stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ). Combined treatment of iPSC-CM with antibiotics significantly improved survival in E. coli-infected rats (p = 0.0006). iPSC-CM ameliorated E. coli-induced infiltration of macrophages, reducing the number of cells in BALF, and suppressing interleukin (IL)-1β, MIP-2, IL-6, and MMP-9 messenger RNA in lung sections. iPSC-CM treatment attenuated NLRP3 expression and inhibited NLRP3 inflammasome activation by disrupting NLRP3-mediated ASC complex formation in LPS/IFN-γ-primed RAW264.7 cells. This study reveals the mechanisms underlying iPSC-CM-conferred anti-inflammatory activity in ALI through the attenuation of macrophage recruitment to the lung, thus inactivating NLRP3 inflammasomes in macrophages. iPSC-CM therapy may be a useful adjuvant treatment to reduce sepsis-related mortality by ameliorating ALI.

Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation.

This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory medium, suggesting proliferative capacity was maintained. In contrast, all of these genes were significantly downregulated in the other groups. Senescence marker β-galactosidase-positive cells significantly decreased in either MSC-CM and/or Y-27632 mixed media. Senescence-related genes downregulated LMNB1 and MAP2K6, and upregulated MMP2. Cell cycle checkpoint genes such as CDC25C, CDCA2, and CIP2A did not vary in the combinatory medium but were significantly downregulated in either ROCK inhibitor or MSC-CM alone. These results imply the synergistic effect of combinatory culture medium on corneal endothelial cell proliferation and high cell number. This study supports high potential for translation to the development of human corneal endothelial tissue regeneration.

Topical Application of Conditioned Medium from Hypoxically Cultured Amnion-Derived Mesenchymal Stem Cells Promotes Wound Healing in Diabetic Mice.

Mesenchymal stem cells or their conditioned medium improve chronic wound healing, and their effect is enhanced by hypoxia. Diabetic foot ulcers are chronic wounds characterized by abnormal and delayed healing, which frequently require amputation. The authors evaluated the effect of topical application of conditioned medium from hypoxically cultured amnion-derived mesenchymal stem cells on wound healing in diabetic mice. Amnion-derived mesenchymal stem cells were cultured under 21% oxygen to prepare normoxic conditioned medium and under 1% oxygen to prepare hypoxic conditioned medium. Hydrogels containing standard medium, normoxic conditioned medium, or hypoxic conditioned medium were topically applied to excisional wounds of mice with streptozotocin-induced diabetes. Ulcer tissues were harvested on day 9; immunohistochemical and quantitative polymerase chain reaction analyses were performed to analyze angiogenesis, inflammatory cell infiltration, and expression levels of inflammation-related genes. Hypoxic conditioned medium significantly enhanced wound closure, increased capillary density and epithelization, and reduced macrophage infiltration. It also tended to reduce the infiltration of neutrophils and enhance the infiltration of regulatory T cells; it showed a tendency to downregulate the expression of the inflammation-related genes interleukin-1β, interleukin-6, chemokine ligand 1, and chemokine ligand 2. Normoxic conditioned medium exhibited similar effects, although they were of lesser magnitude than those of hypoxic conditioned medium. Hydrogels containing hypoxically cultured, amnion-derived mesenchymal stem cell conditioned medium accelerated wound healing in diabetic mice by enhancing angiogenesis, accelerating epithelization, and suppressing inflammation. Therefore, topical application of amnion mesenchymal stem cell-derived hypoxic conditioned medium could be a novel treatment for diabetic foot ulcers.

Effects of the prevention of medication-related osteonecrosis of the jaw by local administration of a dental pulp stem cell-conditioned medium to the rat tooth extraction socket.

The onset of osteonecrosis of the jaw, which is a side effect of bisphosphonates, often develops after tooth extraction; measures for its prevention have not yet been established. While treatment with systemic administration of bone marrow stem cell-derived conditioned medium for medication-related osteonecrosis of the jaw (MRONJ) has been reported, its preventive effects have not been clarified yet, and the high degree of invasiveness of bone marrow fluid collection remains an issue. Therefore, we created a rat model of MRONJ using BP zoledronic acid, used a dental pulp stem cell-conditioned medium (DPSC-CM), which can be collected relatively easily, and locally applied it to the tooth extraction socket with atelocollagen and gelatin sponges. The preventive effect on the onset of MRONJ was subsequently examined. The results demonstrated that the bone exposure width of the extraction socket was reduced, and the mucosal covering was promoted in the atelocollagen + DPSC-CM group as compared with the other groups. Furthermore, histological results indicated a decrease in the number of empty bone lacunae, whereas immunohistochemical staining revealed the presence of many vascular endothelial growth factor (VEGF)-positive cells. Moreover, the results of the investigation of the sustained release of atelocollagen using VEGF indicated the release of VEGF over time. Our results suggest that local administration of DPSC-CM using atelocollagen may be a useful method for the prevention of MRONJ triggered by tooth extraction.