Concentrated Conditioned Medium Research Articles

Bone marrow stromal cell-conditioned medium regenerates injured sciatic nerve by increasing expression of MPZ and NGF and decreasing apoptosis.

Despite the many benefits of mesenchymal stem cell (MSC) transplantation for tissue regeneration, there are some limitations to using them, including the high costs, applying invasive procedures, the possibility of transplant rejection, and cell malignancy. This study aimed to investigate the effect of secretions of bone marrow stromal cells (BMSCs) with the cell-free strategy on damaged sciatic nerve with an emphasis on the role of apoptosis and the expression of myelin protein zero (MPZ) and nerve growth factor (NGF) proteins. BMSCs were cultured and a 25-fold concentrated conditioned medium (CM) from the cells was provided. After creating a crush injury in the left sciatic nerve of male rats, BMSCs or CM were injected into the injured site of the nerve. Four weeks later, the expression of MPZ, NGF, Bax, and Bcl-2 proteins in the sciatic nerve and histological parameters of the sciatic nerve and gastrocnemius muscle were assessed. The results demonstrated that injection of CM decreased apoptosis and increased expression of MPZ and NGF proteins, improving remyelination and regeneration of the sciatic nerve almost as much as the transplantation of the BMSCs themselves compared to the control group. The results suggest that BMSC secretions may improve remyelination and regeneration of damaged sciatic nerve by increasing the expression of MPZ and NGF and decreasing apoptosis.

Local transplantation of mesenchymal stem cells improves encephalo-myo-synangiosis-mediated collateral neovascularization in chronic brain ischemia

BackgroundTo explore whether local transplantation of mesenchymal stem cells (MSCs) in temporal muscle can promote collateral angiogenesis and to analyze its main mechanisms of promoting angiogenesis.MethodsBilateral carotid artery stenosis (BCAS) treated mice were administrated with encephalo-myo-synangiosis (EMS), and bone marrow mesenchymal stem cells (BMSCs) were transplanted into the temporal muscle near the cerebral cortex. On the 30th day after EMS, the Morris water maze, immunofluorescence, laser speckle imaging, and light sheet microscopy were performed to evaluate angiogenesis; In addition, rats with bilateral common carotid artery occlusion were also followed by EMS surgery, and BMSCs from GFP reporter rats were transplanted into the temporal muscle to observe the survival time of BMSCs. Then, the concentrated BMSC-derived conditioned medium (BMSC-CM) was used to stimulate HUVECs and BMECs for ki-67 immunocytochemistry, CCK-8, transwell and chick chorioallantoic membrane assays. Finally, the cortical tissue near the temporal muscle was extracted after EMS, and proteome profiler (angiogenesis array) as well as RT-qPCR of mRNA or miRNA was performed.ResultsThe results of the Morris water maze 30 days after BMSC transplantation in BCAS mice during the EMS operation, showed that the cognitive impairment in the BCAS + EMS + BMSC group was alleviated (P < 0.05). The results of immunofluorescence, laser speckle imaging, and light sheet microscopy showed that the number of blood vessels, blood flow and astrocytes increased in the BCAS + EMS + BMSC group (P < 0.05). The BMSCs of GFP reporter rats were applied to EMS and showed that the transplanted BMSCs could survive for up to 14 days. Then, the results of ki-67 immunocytochemistry, CCK-8 and transwell assays showed that the concentrated BMSC-CM could promote the proliferation and migration of HUVECs and BMECs (P < 0.05). Finally, the results of proteome profiler (angiogenesis array) in the cerebral cortex showed that the several pro-angiogenesis factors (such as MMP-3, MMP-9, IGFBP-2 or IGFBP-3) were notably highly expressed in MSC transplantation group compared to others.ConclusionsLocal MSCs transplantation together with EMS surgery can promote angiogenesis and cognitive behavior in chronic brain ischemia mice. Our study illustrated that MSC local transplantation can be the potential therapeutical option for improving EMS treatment efficiency which might be translated into clinical application.

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation.

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5mg of peptide for 10min with 10µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.

Trimetazidine Preconditioning Potentiates the Effect of Mesenchymal Stem Cells Secretome on the Preservation of Rat Pancreatic Islet Survival and Function In Vitro.

Islet transplantation offers improved glycemic control in individuals with type 1 diabetes mellitus. However, in vitro islet culture is associated with islet apoptosis and eventually will lose their functionality prior to transplantation. In this study, we examined the effects of mesenchymal stem cells (MSCs) secretome preconditioned with diazoxide (DZ) and trimetazidine (TMZ) on rat islet cells during pre-transplant culture. With and without preconditioned hAD-MSCs' concentrated conditioned media (CCM) were added to the culture medium containing rat islets every 12h for 24 and 48h, after testing for selected cytokine concentrations (interleukin (IL)-4, IL-6, IL-13). Insulin content, glucose-stimulated insulin secretion, islet cell apoptosis, and mRNA expression of pro-apoptotic (BAX, BAK-1, and PUMA) and anti-apoptotic factors (BCL-2,BCL-xL, and XIAP) in rat islets were assessed after 24 and 48h of culture. The protein level of IL-6 and IL-4 was significantly higher in TMZ-MSC-CM compared to MSC-non-CM. In rat isolated islets, normalized secreted insulin in the presence of 16.7mM glucose was significantly higher in treated islet groups compared to control islets at both 24 and 48h cultivation. Also, the percentage of apoptotic islet cells TMZ-MSC-CCM-treated islets was significantly lower compared to MSC-CM and MSC-CCM-treated islets in both 24 and 48h cultivation. Consistent with the number of apoptotic cells, after 24h culture, the expression of BCL-2 and BCL-xL genes in the control islets was lower than all treatment islet groups and in 48h was lower than only TMZ-MSC-CM-treated islets. Also, the expression of the XIAP gene in control islets was significantly lower compared to the TMZ-MSC-CCM-treated islets at both at 24 and 48h. In addition, mRNA level of the BAX gene in TMZ-MSC-CCM-treated islets was significantly lower compared to other groups at 48h. Our findings revealed that TMZ proved to be more effective than DZ and could enhance the potential of hAD-MSCs-CM to improve the function and viability of islets prior to transplantation.

The Effect of Mast Cells Stimulated by Toll-Like Receptors on Keratinocytes

Abstract The mast cells, as located in the skin’s dermal layer, are important innate immune cells that mediate inflammation as a defense response against outer pathogens. Mast cells are activated by innate immune receptors such as Toll-like receptors receptrs (TLRs) and interact with a variety of cells including keratinocytes. This study confirmed the effect of TLRs-stimulated mast cells on the migration of keratinocytes residing in the epidermis. The HaCaT cells, a human keratinocyte cell line, was treated with the concentrated conditioned medium (CM) of the HMC-1, a human mast cell line. After that, the migratory capacity via scratch and transwell assays. In addition, we tried to confirm whether TLRs activations affect these effects of mast cells on keratinocyte migration. Thus the same experiments were carried out using the CM of HMC-1 obtained under various stimulation by TLRs agonist. As a result, HMG-1 CM promoted the migratory ability of HaCaT cells in scratch assay and transwell assay. Those effects were further increased when HMC-1 was exposed to various TLRs stimulation. This work was supported by a grant of the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (NRF-2021R1A2C1012551).

Mesenchymal stem cell-derived secretomes-enriched alginate/ extracellular matrix hydrogel patch accelerates skin wound healing

BackgroundThe secretomes of mesenchymal stem cells (MSCs) have great therapeutic potential and thereby their efficient delivery into the target site is of particular interest. Here, we propose a new strategy of hMSCs-derived secretomes delivery for advanced wound healing upon harnessing the working principle of extracellular matrix (ECM)-growth factors interaction in vivo.MethodsWe prepared an alginate hydrogel based wound patch, where it contains both human MSC-derived secretomes and ECM. The ECM was obtained from the decellularization of in vitro cultured human lung fibroblasts. The alginate solution was blended with ECM suspension, crosslinked, air-dried, then rehydrated with the secretomes contained in the concentrated conditioned media (CCM) as a highly saturated form of conditioned media (CM). We tested four different groups, with or without the ECM to investigate not only the role of ECM but the therapeutic effect of secretomes.ResultsThe secretomes reserved many, diverse bioactive factors, such as VEGF, HGF, IGFBPs, IL-6, and IL-8. Alginate/ECM/CCM (AEC) patch could hold significantly larger amount of secretomes and release them longer than the other groups. Our AEC patch was the most effective in stimulating not only cell migration and proliferation but the collagen synthesis of dermal fibroblasts in vitro. Moreover, the AEC patch-treated full-thickness skin wounds disclosed significantly better wound healing indications: cell recruitment, neovascularization, epidermis thickness, keratinocyte migration, and mature collagen deposition, as assessed via histology (H&E, Herovici staining) and immunofluorescence, respectively. In particular, our AEC patch enabled a phenotype shift of myofibroblast into fibroblast over time and led to mature blood vessel formation at 14 day.ConclusionsWe believe that ECM certainly contributed to generate a secretomes-enriched milieu via ECM-secretomes interactions and thereby such secretomes could be delivered more efficiently, exerting significant therapeutic impact either individually or collectively during wound healing process.Graphical abstract

Bone marrow stem cells secretome accelerates simulated birth trauma-induced stress urinary incontinence recovery in rats.

Stress urinary incontinence (SUI) is defined as involuntary urine leakage during physical activities that increase the intra-abdominal pressure on the bladder. We studied bone marrow stem cell (BMSC) secretome-induced activation of anterior vaginal wall (AVW) fibroblasts and its ability to accelerate SUI recovery following vaginal distention (VD) in a rat model of birth trauma using BMSC-conditioned medium (BMSC-CM) and concentrated conditioned medium (CCM). BMSC-CM enhanced the proliferation, migration, and collagen synthesizing abilities of fibroblasts. Differentially expressed genes in BMSC-CM-induced fibroblasts were mainly enriched for cell adhesion, extracellular fibril organization and angiogenesis. Treatment with the JAK2 inhibitor AG490 reversed BMSC-CM-induced activation of the JAK2/STAT4 pathway. Periurethral injection with BMSC-CCM markedly enhanced the abdominal leak point pressure (LPP) in rats after VD. Histological analysis revealed increased numbers of fibroblasts, improved collagen fibers arrangement and elevated collagens content in the AVW of rats receiving BMSC-CCM. These findings suggest the BMSC secretome activates AVW fibroblasts and contributes to the functional and anatomic recovery of simulated birth trauma-induced SUI in rats.

Combination of lyophilized adipose-derived stem cell concentrated conditioned medium and polysaccharide hydrogel in the inhibition of hypertrophic scarring

BackgroundMesenchymal stem cell-based acellular therapies have been widely exploited in managing hypertrophic scars. However, low maintenance dose and transitory therapeutic effects during topical medication remain a thorny issue. Herein, this study aimed to optimize the curative effect of adipose-derived stem cell conditioned medium (ADSC-CM) in the prevention of hypertrophic scarring.MethodsIn the present study, ADSC-CM was concentrated via the freeze-drying procedure. The efficacy of different dose groups (CM, CM5, CM10) was conducted on the proliferation, apoptosis, and α-smooth muscle actin (α-SMA) expression of human keloid fibroblasts (HKFs) in vitro. Incorporation of adipose-derived stem cell concentrated conditioned medium (ADSCC-CM) into polysaccharide hydrogel was investigated in rabbit ear, in vivo. Haematoxylin-eosin (H&E) and Masson’s trichrome staining were performed for the evaluation of scar hyperplasia.ResultsWe noted that ADSCC-CM could downregulate the α-SMA expression of HKFs in a dose-dependent manner. In the rabbit ear model, the scar hyperplasia in the medium-dose group (CM5) and high-dose group (CM10) was inhibited with reduced scar elevation index (SEI) under 4 months of observation. It is noteworthy that the union of CM5 and polysaccharide hydrogel (CM5+H) yielded the best preventive effect on scar hyperplasia. Briefly, melanin, height, vascularity, and pliability in the CM5+H group were better than those of the control group. Collagen was evenly distributed, and skin appendages could be regenerated.ConclusionsAltogether, ADSCC-CM can downregulate the expression of α-SMA due to its anti-fibrosis effect and promote the rearrangement of collagen fibres, which is integral to scar precaution. The in situ cross bonding of ADSCC-CM and polysaccharide hydrogel could remarkably enhance the therapeutic outcomes in inhibiting scar proliferation. Hence, the alliance of ADSCC-CM and hydrogel may become a potential alternative in hypertrophic scar prophylaxis.

Ultrafiltration and Injection of Islet Regenerative Stimuli Secreted by Pancreatic Mesenchymal Stromal Cells.

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and β cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.

Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts

Conditioned medium (CM) and extracellular vesicles (EV) from Adipose-derived Stem/stromal cells (ASC) and Dermal fibroblasts (DF) represent promising tools for therapeutic applications. Which one should be preferred is still under debate and no direct comparison of their proteome has been reported yet. Here, we apply quantitative proteomics to explore the protein composition of CM and EV from the two cell types. Data are available via ProteomeXchange (identifier PXD020219). We identified 1977 proteins by LC-MS/MS proteomic analysis. Unsupervised clustering analysis and PCA recognized CM and EV as separate groups. We identified 68 and 201 CM and EV specific factors. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation factors. The analysis of ASC and DF secretomes revealed the presence of cell type-specific proteins. ASC-CM and -EV carried factors involved in ECM organization and immunological regulation, respectively. Conversely, DF-CM and –EV were enriched in epithelium development associated factors and -EV in Wnt signaling factors. In conclusion, this analysis provides evidence of a different protein composition between CM and EV and of the presence of cell type-specific bioactive mediators suggesting their specific future use as advanced therapy medicinal products. SignificanceThe use of cell secretome presents several advantages over cell therapy such as the lower risks associated to the administration step and the avoidance of any potential risk of malignant transformation. The main secretome preparations consist in concentrated conditioned medium (CM) and extracellular vesicles (EV). Both of them showed well-documented therapeutic potentials. However, it is still not clear in which case it should be better to use one preparation over the other and an exhaustive comparison between their proteome has not been performed yet. The choice of the cell source is another relevant aspect that still needs to be addressed. In order to shed light on these questions we explored the protein composition of CM and EV obtained from Adipose-derived Stem/stromal Cells (ASC) and Dermal Fibroblasts (DF), by a comprehensive quantitative proteomics approach. The analysis showed a clear distinction between CM and EV proteome. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation-related factors. Furthermore, the analysis of ASC and DF secretomes revealed specific biological processes for the different cell products. ASC secretome presented factors involved in ECM organization (hyaluronan and glycosaminoglycan metabolism) and immunological regulation (e.g. macrophage and IkB/NFkB signaling regulation), respectively. On the other hand, DF-CM and –EV were both enriched in epithelium development associated factors, whilst DF-CM in proteins involved in cellular processes regulation and -EV in Wnt signaling factors. In conclusion, our study shed a light on the different protein composition of CM and EV of two promising cell types, spanning from basic processes involved in secretion to specific pathways supporting their therapeutic potential and their possible future use as advanced therapy medicinal products.

Potential of Extracellular Vesicle-Associated TSG-6 from Adipose Mesenchymal Stromal Cells in Traumatic Brain Injury.

Multipotent mesenchymal stromal cells (MSC) represent a promising strategy for a variety of medical applications. Although only a limited number of MSC engraft and survive after in vivo cellular infusion, MSC have shown beneficial effects on immunomodulation and tissue repair. This indicates that the contribution of MSC exists in paracrine signaling, rather than a cell-contact effect of MSC. In this review, we focus on current knowledge about tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and mechanisms based on extracellular vesicles (EV) that govern long-lasting immunosuppressive and regenerative activity of MSC. In this context, in particular, we discuss the very robust set of findings by Jha and colleagues, and the opportunity to potentially extend their research focus on EV isolated in concentrated conditioned media (CCM) from adipose tissue derived MSC (ASC). Particularly, the authors showed that ASC-CCM mitigated visual deficits after mild traumatic brain injury in mice. TSG-6 knockdown ASC were, then, used to generate TSG-6-depleted CCM that were not able to replicate the alleviation of abnormalities in injured animals. In light of the presented results, we envision that the infusion of much distilled ASC-CCM could enhance the alleviation of visual abnormalities. In terms of EV research, the advantages of using size-exclusion chromatography are also highlighted because of the enrichment of purer and well-defined EV preparations. Taken together, this could further delineate and boost the benefit of using MSC-based regenerative therapies in the context of forthcoming clinical research testing in diseases that disrupt immune system homeostasis.

Abstract PR15: Acting at a distance: Medulloblastoma-secreted ligands disrupt normal neural stem cell function

Abstract Long-term cognitive impairments are common in pediatric brain cancer survivors. While these impairments are thought to arise following radiation treatment, recent reports suggest a link to tumor-specific mechanisms. We therefore hypothesized that pediatric brain tumors, more specifically medulloblastoma (MB), can directly affect neural stem and precursor cell function in the forebrain stem cell niche—the subventricular zone (V-SVZ) —by secreting bioactive factors. Mice harboring subcutaneous flank MB tumors had fewer proliferating neural precursor cells in the V-SVZ than controls as well as decreased olfactory bulb neurogenesis and white matter oligodendrogenesis. To assess the effects of the MB secretome in the brain, concentrated conditioned media from MB cell lines (MB-CM) was injected intracerebroventricular (ICV) into mouse pups. ICV injection of MB-CM decreased neural precursor cell proliferation as well as decreased numbers of V-SVZ neurospheres in culture. MB-CM from multiple cell lines decreased V-SVZ neurosphere number and promoted astrocyte differentiation in culture. To identify the ligands contributing to the phenotype, an interaction model was developed extracting ligands from MB microarray data, networking them to receptors on NSCs. Of the predicted ligands, IL6-family cytokine expression and secretion was validated in MB cells. When added in culture, recombinant IL6, IL11, and CT1 decreased neurosphere number and ICV injection of IL11 into mouse pups decreased V-SVZ neural precursor cell proliferation. Overall, this work demonstrates that medulloblastoma secretes bioactive compounds that perturb neural stem cell function and the circuity involved in normal cognitive function. This abstract is also being presented as Poster B64. Citation Format: Alexander Gont, Jaclin V. Simonetta, Jenna Park, Alice R. Shan, Michael J. Borrett, Michael D. Taylor, Freda D. Miller, David R. Kaplan. Acting at a distance: Medulloblastoma-secreted ligands disrupt normal neural stem cell function [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR15.

Characterisation of extracellular vesicles surface markers and co-expression studies with single particle interferometric imaging platform

Background & Aim Extracellular vesicle (EVs)-based therapeutics have been anticipated as an alternative to cell therapies based on the premise that EVs shed by stem cells exert similar therapeutic effects. A major challenge in EV research workflow is the lack of a platform that offers characterisation at the single EV level. In light of this, we tested and validated the single particle interferometric imaging using conditioned media in comparison against purified EVs obtained using ultracentrifugation and size exclusion chromatography. We performed multiplexed measurement of typical EV surface markers (e.g. tetraspanins) and simultaneously measured the size of antigen positive EVs. Methods, Results & Conclusion Human amniotic epithelial cells (hAECs) were cultured in serum free media for 96 hours. We prepared both unpurified (conditioned media, concentrated conditioned media) and purified EV samples (ultracentrifugation and size exclusion chromatography). Single particle interferometric imaging measurement was performed using the ExoView platform (Nanoview Biosciences). CD9, CD63 and CD81-positive EVs were immunocaptured on a tetraspanin microarray chip and imaged as single particles. Co-expression of tetraspanin proteins were then assessed by labeling the captured EVs with a cocktail of fluorescence antibodies conjugated to CD81-Alexa555, CD63-Alexa647 and CD9-Alexa488. The chips were then imaged with ExoView R100 reader and analysed using ExoViewer software with sizing thresholds set to 50-200nm diameter. The label free measurement used in this study captured particles based on their expression of classical EV markers (CD9/CD63/CD81) and provided specific EV particle characterisation down to a resolution of 50nm. Further labelling the captured EVs with fluorescence antibodies proved to increase sensitivity and include particles

SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs (miRNAs) from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular miRNAs in concentrated conditioned medium (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEVmiRNA profiles – but not cCMmiRNAs – were impacted by lipedema. Seven sEVmiRNAs (miR–16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR–144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy individuals, whereas only one cCMmiRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

TSG-6 in conditioned media from adipose mesenchymal stem cells protects against visual deficits in mild traumatic brain injury model through neurovascular modulation

BackgroundRetinal inflammation affecting the neurovascular unit may play a role in the development of visual deficits following mild traumatic brain injury (mTBI). We have shown that concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) can limit retinal damage from blast injury and improve visual function. In this study, we addressed the hypothesis that TNFα-stimulated gene-6 (TSG-6), an anti-inflammatory protein released by mesenchymal cells, mediates the observed therapeutic potential of ASCs via neurovascular modulation.MethodsAbout 12-week-old C57Bl/6 mice were subjected to 50-psi air pulse on the left side of the head overlying the forebrain resulting in an mTBI. Age-matched sham blast mice served as control. About 1 μl of ASC-CCM (siControl-ASC-CCM) or TSG-6 knockdown ASC-CCM (siTSG-6-ASC-CCM) was delivered intravitreally into both eyes. One month following injection, the ocular function was assessed followed by molecular and immunohistological analysis. In vitro, mouse microglial cells were used to evaluate the anti-inflammatory effect of ASC-CCM. Efficacy of ASC-CCM in normalizing retinal vascular permeability was assessed using trans-endothelial resistance (TER) and VE-cadherin expression in the presence of TNFα (1 ng/ml).ResultsWe show that intravitreal injection of ASC-CCM (siControl-ASC-CCM) but not the TSG-6 knockdown ASC-CCM (siTSG-6-ASC-CCM) mitigates the loss of visual acuity and contrast sensitivity, retinal expression of genes associated with microglial and endothelial activation, and retinal GFAP immunoreactivity at 4 weeks after blast injury. In vitro, siControl-ASC-CCM but not the siTSG-6-ASC-CCM not only suppressed microglial activation and STAT3 phosphorylation but also protected against TNFα-induced endothelial permeability as measured by transendothelial electrical resistance and decreased STAT3 phosphorylation.ConclusionsOur findings suggest that ASCs respond to an inflammatory milieu by secreting higher levels of TSG-6 that mediates the resolution of the inflammatory cascade on multiple cell types and correlates with the therapeutic potency of the ASC-CCM. These results expand our understanding of innate mesenchymal cell function and confirm the importance of considering methods to increase the production of key analytes such as TSG-6 if mesenchymal stem cell secretome-derived biologics are to be developed as a treatment solution against the traumatic effects of blast injuries and other neurovascular inflammatory conditions of the retina.

Concentrated Conditioned Medium-Loaded Silk Nanofiber Hydrogels with Sustained Release of Bioactive Factors To Improve Skin Regeneration

An injectable hydrogel is a powerful carrier for therapeutic bioactive molecules. Here, an injected concentrated conditioned medium (CCM)-silk nanofiber composite hydrogel was developed to achieve the sustained release of multiple proteins and better wound healing. All the encapsulated proteins showed slow delivery for more than 9 days in vitro. Bioactive molecules such as transforming growth factor-beta1 (TGF-β1), insulin-like growth factor binding protein-1 (IGFBP-1), and platelet-derived growth factor-AB (PDGF-AB) were successfully released from CCM-loaded hydrogels, and they induced the proliferation and migration of fibroblasts and endothelial cells in a dose- and time-dependent manner. The differentiation of fibroblasts into myofibroblasts was also inhibited, implying less scar formation in vivo. Skin wound regeneration results indicated that the CCM-loaded hydrogel enhanced neovascularization, accelerated wound closure, and promoted hair follicle regeneration. The injectable multiple protein delivery system shows a promising application in skin wound repair.

Bovine fetal mesenchymal stem cells exert antiproliferative effect against mastitis causing pathogen Staphylococcus aureus

Staphylococcus aureus is the most commonly isolated pathogen from clinical bovine mastitis samples and a difficult pathogen to combat. Mesenchymal stem cells (MSC) are multipotent progenitor cells equipped with a variety of factors that inhibit bacterial growth. The aim of the present study was to evaluate the in vitro antibacterial potential against S. aureus of conditioned medium (CM) from MSC derived from fetal bovine bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC, AT-MSC and fetal fibroblasts (FB) cultures were activated by infection with S. aureus. Bacterial growth was evaluated in presence of CM, concentrated CM (CCM), activated CM (ACM) and concentrated ACM (CACM) from BM-MSC, AT-MSC and FB. Gene expression of β-defensin 4A (bBD-4A), NK-lysine 1 (NK1), cathelicidin 2 (CATHL2), hepcidin (HEP) and indoleamine 2,3 dioxygenase (IDO) and protein expression of bBD-4A were determined in activated and non-activated cells. The majority of BM-MSC and AT-MSC expressed CD73, Oct4 and Nanog, and were negative for CD34. Growth of S. aureus decreased when it was exposed to CM from BM-MSC, AT-MSC and FB. Moreover, growth of S. aureus in CCM, ACM and CACM was lower compared to controls of CM from BM-MSC and AT-MSC. Activated AT-MSC increased mRNA levels of bBD4A and NK1, and protein levels of bBD4A in CM. Thus, CM from fetal bovine BM-MSC and AT-MSC has the capacity to reduce in average ~30% of S. aureus relative growth under in vitro conditions. The in vitro antibacterial effect of fetal bovine MSC may be mediated by bBD4A and NK1 activity.

Differentiation of eye field neuroectoderm from human adipose-derived stem cells by using small-molecules and hADSC-conditioned medium

Recently, stem-cell therapy as a promising therapeutic alternative is considered to treat retinal degenerative diseases. Here, we used small molecules and concentrated conditioned medium selectively enriched with Amicon filter units from human adipose-derived stem cells (hADSC-CM) containing various neurotrophic factors to induce hADSCs toward eye field neuroectoderm (EFN). For induction of stem cells, hADSC-CM and small molecules CKI-7, SB431542 and LDN193189 as inhibitors of Wnt, Nodal and BMP4 signaling pathways were used, respectively. We found the highest expression of β-TUB III as a neural marker in the group in which small molecules and conditioned medium were applied simultaneously. Moreover, EFN markers SIX3, PAX6 and RAX had higher expression in the presence of a conditioned medium. However, the superior expression of ENF marker OTX2 was seen in the small molecules group. Our results indicated that neurotrophic factors present in hADSCs-CM could induce hADSCs into EFN cells. Therefore, a more thorough study of these factors and their effects in hADSC-CM might pave the way for cellular and non-cellular therapy in retinal degenerative diseases.

Abstract 3559: Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation

Abstract Background: Despite aggressive treatment, including surgical resection, radiation and chemotherapy, over 90% of glioblastoma (GB) patients experience tumor recurrence. This may be due to high migratory potential, abnormal angiogenesis, molecular heterogeneity and a strong immunosuppressive environment. An earlier report from our laboratory demonstrated that GB cells express high levels of carbohydrate-binding galectin protein: galectin-1 (Gal-1). However, the role of galectin-1 in GB remains elusive. The aim of this study was to evaluate whether galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB in in vitro GB models. Methods: To investigate the molecular mechanisms of Gal-1 on tumor progression, we used RT-PCR and western blots techniques. Gal-1 was stably over-expressed in the U118 and U87MG cell line. Gal-1 (+/-) sub-clones were assayed for attachment, proliferation, migration, and tube formation. Results: Gal-1 protein expression was assessed in human GB cells. We found increased Gal-1 protein expression in human GB cells and tissue. To investigate the effects of Gal-1 silencing on angiogenesis in vitro, we collected conditioned media from Gal-1-knockdown U118 and U87MG cells then applied this to human umbilical vein endothelial cells (HUVEC) culture to analyze endothelial cell proliferation, migration, and tube formation. Proliferation rate, migrating capability, and tube formation decreased significantly when HUVECs were cultured with concentrated conditioned media from Gal-1-knockdown cells. This data suggests that Gal-1 in GB cells may promote tumor angiogenesis both in vitro and co-culture. Results showed that silencing of Gal-1 in U118 and U87MG cells significantly repressed CXCR4 expression at the mRNA level and protein levels. To further explore the role of CXCR4 in GB cells, CXCR4 knockdown resulted in significant inhibition of cell migration and clonogenic ability in vitro. Thus, Gal-1 might upregulate CXCR4 expression to promote tumor progression. A previous laboratory study indicated that NF-κB promotes cancer cell motility by upregulating CXCR4 expression. Luciferase reporter assay was used to determine the involvement of NF-κB in Gal-1-induced CXCR4 expression in U118 and U87MG cells. NF-κB luciferase activity was significantly decreased in Gal-1-knockdown cells. Moreover, decreased CXCR4 promoter activity and protein expression in Gal-1-knockdown cells were observed. This data indicates that Gal-1 might modulate CXCR4 expression via NF-κB activation. Conclusion: Our in vitro cell culture results suggest that upregulation of galectin-1 and CXCR4 might at least in part explain the aggressive infiltrative biology of GB and the Gal-1-CXCR4 axis may serve as a therapeutic target. Further studies in different animal models are warranted. Citation Format: David Cachia, Rachel Malley, William A. Vandergrift, Sunil Patel, Gabriel A. Rabinovich, Arabinda Das. Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3559.

Conditioned medium from umbilical cord mesenchymal stem cells improves nasal mucosa damage by radiation

To explore therapeutic effects of conditioned medium from human umbilical cord mesenchymal stem cells (hUC-MSCs) on nasal mucosa radiation damage both in vivo and in vitro. The mucus cilia clearance time (7 and 30days), degree of mucosal edema (7, 30, 90 and 180days), cilia coverage (180days) of concentrated conditioned medium group improved compared with radiotherapy control group. The proliferation and migration abilities of irradiated and non-irradiated nasal epithelial cells significantly increased after culture in bronchial epithelial cell growth medium (BEGM) containing 10% conditioned medium of hUC-MSCs compared to cells cultured in BEGM alone. Soluble factors secreted by hUC-MSCs may promote nasal epithelial cell proliferation and migration. Intranasal administration of hUC-MSC conditioned medium effectively repairs nasal mucosa radiation damage.