The iPSC secretome is beneficial for in vitro propagation of primary osteoarthritic chondrocytes cell lines.

BACKGROUND: One of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells. METHODS: We added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles. RESULTS: iPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time. CONCLUSION: Our preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

Conditioned medium of human mesenchymal stem cells affects stem cell senescence in osteoporosis.

Systemic transplantation of mesenchymal stem cells (MSCs) and conditioned medium derived from MSCs have been reported to recover bone loss in animal models of osteoporosis; however, the underlying mechanisms remain unclear. We recently reported that extracellular vesicles released from human mesenchymal stem cells (hMSCs) prevent senescence of stem cells in bisphosphonate-related osteonecrosis of the jaw model. In this study, we aimed to compare the effects of conditioned medium (hMSCs-CM) from early and late passage hMSCs on cellular senescence and to verify the benefits of CM from early passage hMSCs in mitigating the progression of osteoporosis through the prevention of cellular senescence. We investigated the distinct endocrine effects of early (P5) and late (P17) passage hMSCs in vitro, as well as the preventive benefits of early passage hMSCs-CM in osteoporosis model triggered by ovariectomy. Our results indicate that long-term cultured hMSCs contributed to the progression of inflammatory transcriptional programs in P5 hMSCs, ultimately impairing their functionality and enhancing senescence-related characteristics. Conversely, early passage hMSCs reversed these alterations. Moreover, early passage hMSCs-CM infused intravenously in a postmenopausal osteoporosis mouse model suppressed bone degeneration and prevented osteoporosis by reducing ovariectomy-induced senescence in bone marrow MSCs and reducing the expression of senescence-associated secretory phenotype-related cytokines. Our findings highlight the high translational value of early passage hMSCs-CM in antiaging intervention and osteoporosis prevention.

Platelet-rich plasma-derived exosomes enhance mesenchymal stem cell paracrine function and nerve regeneration potential.

BACKGROUND: Peripheral nerve injury (PNI) presents a significant clinical challenge, leading to enduring sensory-motor impairments. While mesenchymal stem cell (MSC)-based therapy holds promise for PNI treatment, enhancing its neurotrophic effects remains crucial. Platelet-rich plasma-derived exosomes (PRP-Exo), rich in bioactive molecules for intercellular communication, offer potential for modulating cellular biological activity. METHODS: PRP-Exo was isolated, and its impact on MSC viability was evaluated. The effects of PRP-Exo-treated MSCs (MSCPExo) on Schwann cells (SCs) from injured sciatic nerves and human umbilical vein endothelial cells (HUVECs) were assessed. Furthermore, the conditioned medium from MSCPExo (MSCPExo-CM) was analyzed using a cytokine array and validated through ELISA and Western blot. RESULTS: PRP-Exo enhanced MSC viability. Coculturing MSCPExo with SCs ameliorated apoptosis and promoted SC proliferation following PNI. Similarly, MSCPExo-CM exhibited pro-proliferative, migratory, and angiogenic effects. Cytokine array analysis identified 440 proteins in the MSCPExo secretome, with 155 showing upregulation and 6 showing downregulation, many demonstrating potent pro-regenerative properties. ELISA confirmed the enrichment of several angiotrophic and neurotrophic factors. Additionally, Western blot analysis revealed the activation of the PI3K/Akt signaling pathway in MSCPExo. CONCLUSION: Preconditioning MSCs with PRP-Exo enhanced the paracrine function, particularly augmenting neurotrophic and pro-angiogenic secretions, demonstrating an improved potential for neural repair.

Proinflammatory signaling mechanism of endocan in macrophages: Involvement of TLR2 mediated MAPK-NFkB pathways.

Endocan is an endothelial cell-specific proteoglycan that contributes to vascular dysfunction by impairing endothelial function and inducing vascular smooth muscle cell migration. However, its role in regulating macrophage inflammation, a key pathological feature of vascular dysfunction, is not well understood. In this study, we investigated the effect of endocan on macrophage inflammation to better understand its contribution to vascular dysfunction. We found that endocan upregulated pro-inflammatory cytokines including IL-1ß, IL-6 and TNF-α in RAW 264.7 cells and activated MAPK/NFkB signaling pathways. Inhibiting these pathways reduced endocan-induced cytokine levels, while inhibiting TLR2 compromised the MAPK/NFkB regulation. Additionally, LPS-induced HUVEC conditioned medium stimulated cytokine levels in RAW 264.7 cells, which were reduced by endocan siRNA treatment in HUVEC. These results suggest that endocan positively regulates pro-inflammation in macrophages through the TLR2-MAPK-NFkB axis, highlighting the potential of targeting endocan to reduce inflammation in vascular dysfunction.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

The proliferative effects of stem cells from apical papilla-conditioned medium on rat corneal endothelial cells.

The cornea, positioned at the forefront of the eye, refracts the light for focusing images on the retina. Damage to this transparent structure can lead to various visual disorders. The corneal endothelial cells (CECs) are crucial for transparency and homeostasis, but lack the ability to reproduce. Significant damage results in structure destruction and vision impairment. While extensive research has aimed at the restoring the corneal endothelial layer, including endothelial proliferation for functional monolayers remains challenging. Our previous studies confirmed the proliferative activity of stem cells from apical papilla-conditioned medium (SCAP-CM) on the retinal pigmented epithelium as a single cell layer. This study investigates how SCAP-CM influences the proliferation and migration of CECs. Our results introduced Matrigel, as a new matrix component for in vitro culture of CECs. Moreover, 60% of SCAP-CM was able to stimulate CEC proliferation as well as migrate to repair wound healing during 24 h. Confluent CECs also expressed specific markers, ATP1a1, ZO-1 and CD56, indicative of CEC characteristics, aligning with the recapitulation of differentiation when forming a homogenous monolayer at the same level of isolated CECs without in vitro culture. These findings suggested that SCAP-CM administration could be useful for future preclinical and clinical applications.

A novel study of brain microvascular endothelial cells induced by astrocyte conditioned medium for constructing blood brain barrier model in vitro: A promising tool for meningitis of teleost.

The blood-brain barrier (BBB) is mainly composed of specialized endothelial cells, which can resist harmful substances, transport nutrients, and maintain the stability of the brain environment. In this study, an endothelial cell line from tilapia (Oreochromis niloticus) named TVEC-01 was successfully established. During the earlier establishment phase of the cell line, the TVEC-01 cells were persistently exposed to an astrocyte-conditioned medium (ACM). TVEC-01 cells were identified as an endothelial cell line. TVEC-01 cells retained the multiple functions of endothelial cells and were capable of performing various experiments in vitro. Furthermore, TVEC-01 cells efficiently expressed BBB-related tight junctions and key efflux transporters. From the results of the qRT-PCR, we found that the TVEC-01 cell line did not gradually lose BBB characteristics after persistent and repetitive passages, which was different from the vast majority of immortalized endothelial cells. The results showed that ACM induced up-regulation of the expression levels of multiple BBB-related genes in TVEC-01 cells. We confirmed that Streptococcus agalactiae was capable of invading the TVEC-01 cells and initiating a series of immune responses, which provided a theoretical basis for S. agalactiae to break through the BBB of teleost through the transcellular traversal pathway. In summary, we have successfully constructed an endothelial cell line of teleost, named TVEC-01, which can be used in many experiments in vitro and even for constructing BBB in vitro. Moreover, it was confirmed that S. agalactiae broke through the BBB of teleost through the transcellular traversal pathway and caused meningitis.

Benefits of bone marrow mesenchymal stem cells compared to their conditioned medium in valproic acid-induced autism in rats.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, a limited range of activities, and deficiencies in social communications. Bone marrow mesenchymal stem cells (BM-MSCs), which secrete factors that stimulate surrounding microenvironment, and BM-MSCs conditioned medium (BM-MSCs-CM), which contains cell-secreted products, have been speculated to hold potential as a therapy for ASD. This study aimed to compare the therapeutic effects of BM-MSCs and BM-MSCs-CM on behavioral and microglial changes in an animal model of autism induced by valproic acid (VPA). METHODS AND RESULTS: Pregnant Wistar rats were administered by VPA at a dose of 600 mg/kg at 12.5 days post-conception. After birth, male pups were included in the study. At 6 weeks of age, one group of rats received intranasal administration of BM-MSCs, while another group received BM-MSCs-CM. The rats were allowed to recover for 2 weeks. Behavioral tests, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry were performed. Both BM-MSCs and BM-MSCs-CM administration significantly improved some behavioral deficits. Furthermore, these treatments notably reduced Iba-1 marker associated with microgliosis. Additionally, there was a significant reduction in the expression of pro-inflammatory cytokines IL-1ß and IL-6, and an increase in the levels of the anti-inflammatory cytokine IL-10 in rats administered by BM-MSCs and BM-MSCs-CM. CONCLUSIONS: Post-developmental administration of BM-MSCs and BM-MSCs-CM can ameliorate prenatal neurodevelopmental deficits, restore cognitive and social behaviors, and modulate microglial and inflammatory markers. Results indicated that the improvement rate was higher in the BM-MSCs group than BM-MSCs-CM group.

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

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

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

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

A modified system using macrophage-conditioned medium revealed that the indirect effects of anti-inflammatory food-derived compounds improve inflammation-induced suppression of UCP-1 mRNA expression in 10T1/2 adipocytes.

Recently, it has been suggested that brown and beige adipocytes may ameliorate obesity because these adipocytes express uncoupling protein-1 (UCP-1), which generates heat by consuming lipid. However, obesity-induced inflammation suppresses the expression of UCP-1. To improve such conditions, food components with anti-inflammatory properties are attracting attention. In this study, we developed a modified system to evaluate only the indirect effects of anti-inflammatory food-derived compounds by optimizing the conventional experimental system using conditioned medium. We validated this new system using 6-shogaol and 6-gingerol, which have been reported to show the anti-inflammatory effects and to increase the basal expression of UCP-1 mRNA. In addition, we found that the acetone extract of Sarcodon aspratus, an edible mushroom, showed anti-inflammatory effects and rescued the inflammation-induced suppression of UCP-1 mRNA expression. These findings indicate that the system with conditioned medium is valuable for evaluation of food-derived compounds with anti-inflammatory effects on the inflammation-induced thermogenic adipocyte dysfunction.

Serum-free spontaneously immortalized bovine oviduct epithelial cell conditioned medium promotes the early development of bovine in vitro fertilized embryos.

Embryonic transfer of bovine blastocysts produced using in vitro fertilization (IVF) is widely used, although the challenge of compromised conception rates remains. Using bovine oviduct epithelial cells (BOEC) to improve embryo culture conditions has attracted attention, particularly since the recent discovery of extracellular vesicles from BOEC. The selection of embryos for transfer has also been the subject of various studies, and a set of evaluation criteria to predict pregnancy success has been suggested, in which the embryos are judged by their kinetics and morphology at the early stages. In the present study, we established a spontaneously immortalized BOEC line (SI-BOEC) and examined the effects of conditioned medium on IVF embryos, focusing on the results of the recommended criteria. A modified KSOM (mKSOM) was used to prepare conditioned media. Presumptive zygotes were cultured in mKSOM (control), SI-BOEC-conditioned medium, mKSOM supplemented with sediment (pellet) collected after the ultracentrifugation of the conditioned medium (mKSOM/sediment), and the supernatant. A significantly higher percentage of embryos satisfied the recommended criteria when grown in the conditioned medium than in the mKSOM. A higher proportion of embryos developed into blastocysts after achieving the four criteria. A similar tendency was observed when grown in mKSOM/sediment compared to mKSOM; however, this was not observed in the supernatant. Vesicles with a size similar to that of exosomes were observed in the sediment. In conclusion, the culture medium conditioned by SI-BOEC promoted the production of bovine blastocysts that satisfied the four evaluation criteria recommended for embryo selection.

Photobiomodulation and conditioned medium of adipose-derived stem cells for enhancing wound healing in rats with diabetes: an investigation on the proliferation phase.

This investigation tried to evaluate the combined and solo effects of photobiomodulation (PBM) and conditioned medium derived from human adipose tissue-derived stem cells (h-ASC-CM) on the inflammatory and proliferative phases of an ischemic infected delayed healing wound model (IIDHWM) in rats with type I diabetes mellitus (TIDM). The present investigation consisted of four groups: group 1 served as the control, group 2 treated with h-ASC-CM, group 3 underwent PBM treatment, and group 4 received a combination of h-ASC-CM and PBM. Clinical and laboratory assessments were conducted on days 4 and 8. All treatment groups exhibited significantly higher wound strength than the group 1 (p = 0.000). Groups 4 and 3 demonstrated significantly greater wound strength than group 2 (p = 0.000). Additionally, all therapeutic groups showed reduced methicillin -resistant Staphylococcus aureus (MRSA) in comparison with group 1 (p = 0.000). While inflammatory reactions, including neutrophil and macrophage counts, were significantly lower in all therapeutic groups rather than group 1 on days 4 and 8 (p < 0.01), groups 4 and 3 exhibited superior results compared to group 2 (p < 0.01). Furthermore, proliferative activities, including fibroblast and new vessel counts, as well as the measurement of new epidermal and dermal layers, were significantly increased in all treatment groups on 4 and 8 days after the surgery (p < 0.001). At the same times, groups 4 and 3 displayed significantly higher proliferative activities compared to group 2 (p < 0.001). The treatment groups exhibited significantly higher mast cell counts and degranulation phenotypes in comparison with the group 1 on day 4 (p < 0.05). The treatment groups showed significantly lower mast cell counts and degranulation phenotypes than group 1 on day 8 (p < 0.05).The combined and individual application of h-ASC-CM and PBM remarkably could accelerate the proliferation phase of wound healing in the IIDHWM for TIDM in rats, as indicated by improved MRSA control, wound strength, and stereological evaluation. Furthermore, the combination of h-ASC-CM and PBM demonstrated better outcomes compared to the individual application of either h-ASC-CM or PBM alone.

Transcriptome wide functional analysis of HBx expressing human hepatocytes stimulated with endothelial cell cross-talk.

Identification of genes dysregulated during the hepatitis B virus (HBV)-host cell interaction adds to the understanding of underlying molecular mechanisms and aids in discovering effective therapies to improve prognosis in hepatitis B virus (HBV)-infected individuals. Through bioinformatics analyses of transcriptomics data, this study aimed to identify potential genes involved in the cross-talk of human hepatocytes expressing the HBV viral protein HBx with endothelial cells. Transient transfection of HBV viral gene X (HBx) was performed in THLE2 cells using pcDNA3 constructs. Through mRNA Sequencing (RNA Seq) analysis, differentially expressed genes (DEGs) were identified. THLE2 cells transfected with HBx (THLE2x) were further treated with conditioned medium from cultured human umbilical vein derived endothelial cells (HUVEC-CM). Gene Ontology (GO) enrichment analysis revealed that interferon and cytokine signaling pathways were primarily enriched for the downregulated DEGs in THLE2x cells treated with HUVEC-CM. One significant module was selected following protein-protein interaction (PPI) network generation, and thirteen hub genes were identified from the module. The prognostic values of the hub genes were evaluated using Kaplan-Meier (KM) plotter, and three genes (IRF7, IFIT1, and IFITM1) correlated with poor disease specific survival (DSS) in HCC patients with chronic hepatitis. A comparison of the DEGs identified in HUVEC-stimulated THLE2x cells with four publicly available HBV-related HCC microarray datasets revealed that PLAC8 was consistently downregulated in all four HCC datasets as well as in HUVEC-CM treated THLE2x cells. KM plots revealed that PLAC8 correlated with worse relapse free survival and progression free survival in HCC patients with hepatitis B virus infection. This study provided molecular insights which may help develop a deeper understanding of HBV-host stromal cell interaction and open avenues for future research.

Further Studies are Needed for Testing the Effects of Topical Conditioned Medium of Stem Cells in Facial Skin Nonsurgical Resurfacing Modalities for Antiaging.

We would like to respond to the commentary with further understanding of the issue of potential statistical power in the analysis of our original finding. This further analysis has been planned to be carried out using the data from the wrinkle outcome because it has been contributed by the largest sample size with a dramatic effect size among all outcomes. Sequential analysis in this letter has been down with alpha 0.05 (type I error) and power of 0.80 (1-type II error) based on the O'Brien Fleming method. In addition to the common settings abovementioned, we chose a small effect size (SMD = 0.2) for avoiding underestimation in the optimal information size calculation and power analysis. The analysis was conducted using R via RStudio. The figure of sequential analysis shows that the cumulative effect of topical CM of stem cells on wrinkle outcome reaches statistical significance (z score of the end of blue line > 2), which is consistent with our original finding. Nevertheless, the information size of the outcome is insufficient (n = 118), which is lower than the required sample size (n = 1419). The observed power of the effects of topical CM of stem cells on the wrinkle outcome is only about 0.64, which is lower than the pre-defined or expected power of 0.80. Based on the fraction of information, although the observed z score of 3.232 for the cumulative effect surpasses 2, it does not surpass the monitoring boundary of 6.795 at the fraction (8.3%).Level of Evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Potential Consequences of the Use of Adipose-Derived Stem Cells in the Treatment of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) ranks as the most prevalent of primary liver cancers and stands as the third leading cause of cancer-related deaths. Early-stage HCC can be effectively managed with available treatment modalities ranging from invasive techniques, such as liver resection and thermoablation, to systemic therapies primarily employing tyrosine kinase inhibitors. Unfortunately, these interventions take a significant toll on the body, either through physical trauma or the adverse effects of pharmacotherapy. Consequently, there is an understandable drive to develop novel HCC therapies. Adipose-derived stem cells (ADSCs) are a promising therapeutic tool. Their facile extraction process, coupled with the distinctive immunomodulatory capabilities of their secretome, make them an intriguing subject for investigation in both oncology and regenerative medicine. The factors they produce are both enzymes affecting the extracellular matrix (specifically, metalloproteinases and their inhibitors) as well as cytokines and growth factors affecting cell proliferation and invasiveness. So far, the interactions observed with various cancer cell types have not led to clear conclusions. The evidence shows both inhibitory and stimulatory effects on tumor growth. Notably, these effects appear to be dependent on the tumor type, prompting speculation regarding their potential inhibitory impact on HCC. This review briefly synthesizes findings from preclinical and clinical studies examining the effects of ADSCs on cancers, with a specific focus on HCC, and emphasizes the need for further research.

Proposal of Simplified Standardization of the Cell-Growth-Promoting Activity of Human Adipose Tissue Mesenchymal Stromal Cell Culture Supernatants.

The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.

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

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

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion.

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2'-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7highMyoDlowEdUpos) and activated satellite cells (Pax7highMyoDhighEdUpos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.

Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair.

The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCsBMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects.