Regenerative Potential of Mesenchymal Stem Cells’ (MSCs) Secretome for Liver Fibrosis Therapies

Chronic liver injuries lead to liver fibrosis and then to end-stage liver cirrhosis. Liver transplantation is often needed as a course of treatment for patients in critical conditions, but limitations associated with transplantation prompted the continuous search for alternative therapeutic strategies. Cell therapy with stem cells has emerged as an attractive option in order to stimulate tissue regeneration and liver repair. Transplanted mesenchymal stem cells (MSCs) could trans-differentiate into hepatocyte-like cells and, moreover, show anti-fibrotic and immunomodulatory effects. However, cell transplantation may lead to some uncontrolled side effects, risks associated with tumorigenesis, and cell rejection. MSCs’ secretome includes a large number of soluble factors and extracellular vesicles (EVs), through which they exert their therapeutic role. This could represent a cell-free strategy, which is safer and more effective than MSC transplantation. In this review, we focus on cell therapies based on MSCs and how the MSCs’ secretome impacts the mechanisms associated with liver diseases. Moreover, we discuss the important therapeutic role of EVs and how their properties could be further used in liver regeneration.     

Applications of Mesenchymal Stem Cells in Skin Regeneration and Rejuvenation

Mesenchymal stem cells (MSCs) are multipotent stem cells derived from adult stem cells. Primary MSCs can be obtained from diverse sources, including bone marrow, adipose tissue, and umbilical cord blood. Recently, MSCs have been recognized as therapeutic agents for skin regeneration and rejuvenation. The skin can be damaged by wounds, caused by cutting or breaking of the tissue, and burns. Moreover, skin aging is a process that occurs naturally but can be worsened by environmental pollution, exposure to ultraviolet radiation, alcohol consumption, tobacco use, and undernourishment. MSCs have healing capacities that can be applied in damaged and aged skin. In skin regeneration, MSCs increase cell proliferation and neovascularization, and decrease inflammation in skin injury lesions. In skin rejuvenation, MSCs lead to production of collagen and elastic fibers, inhibition of metalloproteinase activation, and promote protection from ultraviolet radiation-induced senescence. In this review, we focus on how MSCs and MSC-derived molecules improve diseased and aged skin. Additionally, we emphasize that induced pluripotent stem cell (iPSC)-derived MSCs are potentially advanced MSCs, which are suitable for cell therapy.     

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds—In Vitro Study

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.     

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.     

Cathepsin B Regulates Mice Granulosa Cells’ Apoptosis and Proliferation In Vitro

Cathepsin B (CTSB), a lysosomal cysteine protease’s high expression and activity, has been reported to cause poor-quality embryos in porcine and bovine. Nevertheless, CTSB functions in mice granulosa cells remain to explore. To discuss the CTSB functional role in follicular dynamics, we studied apoptosis, proliferation, cell cycle progression, and related signaling pathways in primary mouse granulosa cells transfected with small interference RNA specific to CTSB (siCTSB) for 48 h. Further, mRNA and protein expression of cell proliferation regulators (Myc and cyclin D2), apoptosis regulators (caspase 3, caspase 8, TNF-α, and Bcl2), steroidogenesis-related genes (FSHR and CYP11A1), and autophagy markers (LC3-I and ATG5) were investigated. In addition, the effect of CTSB on steroidogenesis and autophagy was also examined. Flow cytometry analysis assay displayed that silencing of CTSB decreased the early and total apoptosis rate by downregulating TNF-α, caspase 8, and caspase 3, and upregulating Bcl2. By regulating Myc and cyclin D2 expression and activating the p-Akt and p-ERK pathways, CTSB knockdown increased GC proliferation and number. A significant decline in estradiol and progesterone concentrations was observed parallel to a significant decrease in autophagy-related markers LC3-I and ATG5 compared to the control group. Herein, we demonstrated that CTSB serves as a proapoptotic agent and plays a critical role in folliculogenesis in female mice by mediating apoptosis, autophagy, proliferation, and steroidogenesis. Hence, CTSB could be a potential prognostic agent for female infertility.     

Stem Cells-Derived Extracellular Vesicles: Potential Therapeutics for Wound Healing in Chronic Inflammatory Skin Diseases

Endosome-derived small extracellular vesicles (EVs), often referred to as exosomes, are produced by almost all, if not all, cell types, and are critical for intercellular communication. They are composed of a lipid bilayer associated with membrane proteins and contain a payload of lipids, proteins and regulatory RNAs that depends on the parental cell physiological condition. By transferring their “cargo”, exosomes can modulate the phenotype of neighboring and distant cells. Stem cells (SC) were widely studied for therapeutic applications regarding their regenerative/reparative potential as well as their immunomodulatory properties. Whether from autologous or allogeneic source, SC beneficial effects in terms of repair and regeneration are largely attributed to their paracrine signaling notably through secreted EVs. Subsequently, SC-derived EVs have been investigated for the treatment of various diseases, including inflammatory skin disorders, and are today fast-track cell-free tools for regenerative/reparative strategies. Yet, their clinical application is still facing considerable challenges, including production and isolation procedures, and optimal cell source. Within the emerging concept of “allogeneic-driven benefit” for SC-based therapies, the use of EVs from allogeneic sources becomes the pragmatic choice although a universal allogeneic cell source is still needed. As a unique temporary organ that ensures the mutual coexistence of two allogeneic organisms, mother and fetus, the human placenta offers a persuasive allogeneic stem cell source for development of therapeutic EVs. Advancing cell-free therapeutics nurtures great hope and provides new perspectives for the development of safe and effective treatment in regenerative/reparative medicine and beyond. We will outline the current state of the art in regard of EVs, summarize their therapeutic potential in the context of skin inflammatory disorders, and discuss their translational advantages and hurdles.     

Menstrual Blood-Derived Endometrial Stem Cells’ Impact for the Treatment Perspective of Female Infertility

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues—the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins’ expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.     

Therapeutic Potential of Human Stem Cell Implantation in Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive debilitating neurodegenerative disease and the most common form of dementia in the older population. At present, there is no definitive effective treatment for AD. Therefore, researchers are now looking at stem cell therapy as a possible treatment for AD, but whether stem cells are safe and effective in humans is still not clear. In this narrative review, we discuss both preclinical studies and clinical trials on the therapeutic potential of human stem cells in AD. Preclinical studies have successfully differentiated stem cells into neurons in vitro, indicating the potential viability of stem cell therapy in neurodegenerative diseases. Preclinical studies have also shown that stem cell therapy is safe and effective in improving cognitive performance in animal models, as demonstrated in the Morris water maze test and novel object recognition test. Although few clinical trials have been completed and many trials are still in phase I and II, the initial results confirm the outcomes of the preclinical studies. However, limitations like rejection, tumorigenicity, and ethical issues are still barriers to the advancement of stem cell therapy. In conclusion, the use of stem cells in the treatment of AD shows promise in terms of effectiveness and safety.     

Effect of Multiple Sclerosis Cerebrospinal Fluid and Oligodendroglia Cell Line Environment on Human Wharton’s Jelly Mesenchymal Stem Cells Secretome

Multiple sclerosis (MS) is a neurological disorder of autoimmune aetiology. Experimental therapies with the use of mesenchymal stem cells (MSCs) have emerged as a response to the unmet need for new treatment options. The unique immunomodulatory features of stem cells obtained from Wharton’s jelly (WJ-MSCs) make them an interesting research and therapeutic model. Most WJ-MSCs transplants for multiple sclerosis use intrathecal administration. We studied the effect of cerebrospinal fluid (CSF) obtained from MS patients on the secretory activity of WJ-MSCs and broaden this observation with WJ-MSCs interactions with human oligodendroglia cell line (OLs). Analysis of the WJ-MSCs secretory activity with use of Bio-Plex Pro™ Human Cytokine confirmed significant and diverse immunomodulatory potential. Our data reveal rich WJ-MSCs secretome with markedly increased levels of IL-6, IL-8, IP-10 and MCP-1 synthesis and a favourable profile of growth factors. The addition of MS CSF to the WJ-MSCs culture caused depletion of most proteins measured, only IL-12, RANTES and GM-CSF levels were increased. Most cytokines and chemokines decreased their concentrations in WJ-MSCs co-cultured with OLs, only eotaxin and RANTES levels were slightly increased. These results emphasize the spectrum of the immunomodulatory properties of WJ-MSCs and show how those effects can be modulated depending on the transplantation milieu.     

Circ_0011129 Encapsulated by the Small Extracellular Vesicles Derived from Human Stem Cells Ameliorate Skin Photoaging

Photoaging is not only the main cause of skin aging caused by exogenous factors, it is also related to a variety of skin diseases and even malignant tumors. Excessive and repeated exposure to ultraviolet radiation, especially UVA induces oxidative stress, DNA damage, inflammation, and collagen and elastin degeneration, ultimately leads to skin photoaging, manifested by skin redness, coarse wrinkles, and pigmentation even skin cancer. There has been a large demand of effective prevention and medications but approaches in the current management of photoaging are very limited. In the previous study, we found that a non-coding circular RNA circ_0011129 acts as a miR-6732-5p adsorption sponge to inhibit the reduction of type I collagen and the denaturation and accumulation of elastin in UVA-induced HDF cells photoaging model. However, in vivo instability and efficient delivery to the target cell of circRNA is a major challenge for its clinical application. Therefore, improving its stability and delivery efficiency are desired. In this study, we proposed a strategy of delivering circ_0011129 with small extracellular vesicles (sEVs) from human adipose-derived stem cells (hADSCs) to intervene in the photoaging process. The results showed that sEVs from hADSCs in 3D bioreactor culture (3D-sEVs) can prevent photoaging. Consequently, by overexpressing circ_0011129 in hADSCs, we successfully loaded it into 3D-sEVs (3D-circ-sEVs) and its protective effect was better. Our studies provide a novel approach to preventing skin photoaging, which has important clinical significance and application value for the development of non-coding RNA drugs to treat skin photoaging. We first screened out hADSCs-derived sEVs with excellent anti-oxidant effects. We then compared the sEVs collected from traditional 2D culture with 3D bioreactor culture. By miRNA-seq and GEO data analysis, we found that miRNAs in 3D-sEVs were enriched in cell activities related to apoptosis, cellular senescence, and inflammation. Subsequently, we prepared circ_0011129-loaded 3D-sEVs (3D-circ-sEVs) by overexpressing it in hADSCs for the treatment of photoaging in vitro. We proved that 3D-circ-sEVs can interfere with the process of cell photoaging and protect cells from UVA radiation damage, as well as in a H2O2-induced oxidative stress model.     

Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy

Adipose-derived mesenchymal stromal cells (ADSC) are a promising source for cellular therapy of chronic wounds. However, the limited life span during in vitro expansion impedes their extensive use in clinical applications and basic research. We hypothesize that by introduction of an ectopic expression of telomerase into ADSC, the cells’ lifespans could be significantly extended. To test this hypothesis, we aimed at engineering an immortalized human ADSC line using a lentiviral transduction with human telomerase (hTERT). ADSC were transduced with a third-generation lentiviral system and a hTERT codifying plasmid (pLV-hTERT-IRES-hygro). A population characterized by increased hTERT expression, extensive proliferative potential and remarkable (potent) multilineage differentiation capacity was selected. The properties for wound healing of this immortalized ADSC line were assessed after 17 passages. Their secretome induced the proliferation and migration of keratinocytes, dermal fibroblasts, and endothelial cells similarly to untransduced ADSC. Moreover, they sustained the complete re-epithelialization of a full thickness wound performed on a skin organotypic model. In summary, the engineered immortalized ADSC maintain the beneficial properties of parent cells and could represent a valuable and suitable tool for wound healing in particular, and for skin regenerative therapy in general.     

Alleviation of Severe Skin Insults Following High-Dose Irradiation with Isolated Human Fetal Placental Stromal Cells

Skin exposure to high-dose irradiation, as commonly practiced in radiotherapy, affects the different skin layers, causing dry and wet desquamation, hyperkeratosis fibrosis, hard to heal wounds and alopecia and damaged hair follicles. Fetal tissue mesenchymal stromal cells (f-hPSC) were isolated from excised human fetal placental tissue, based on their direct migration from the tissue samples to the tissue dish. The current study follows earlier reports on for the mitigation of acute radiation syndrome following whole body high-dose exposure with remotely injected f-hPSC. Both the head only and a back skin flap of mice were irradiated with 16 &18 Gy, respectively, by 6MeV clinical linear accelerator electron beam. In both locations, the irradiated skin areas developed early and late radiation induced skin damages, including cutaneous fibrosis, lesions, scaring and severe hair follicle loss and reduced hair pigmentation. Injection of 2 × 10⁶ f-hPSC, 3 and 8 weeks following 16 Gy head irradiation, and 1 and 4 weeks following the 18 Gy back skin only irradiation, resulted in significantly faster healing of radiation induced damages, with reduction of wet desquamation as measured by surface moisture level and minor recovery of the skin viscoelasticity. Detailed histological morphometry showed a clear alleviation of radiation induced hyperkeratosis in f-hPSC treated mice, with significant regain of hair follicles density. Following 16 Gy head irradiation, the hair follicles density in the scalp skin was reduced significantly by almost a half relative to the controls. A nearly full recovery of hair density was found in the f-hPSC treated mice. In the 18 Gy irradiated back skin, the hair follicles density dropped in a late stage by ~70% relative to naïve controls. In irradiated f-hPSC treated mice, it was reduced by only ~30% and was significantly higher than the non-treated group. Our results suggest that local injections of xenogeneic f-hPSC could serve as a simple, safe and highly effective non-autologous pro-regenerative treatment for high-dose radiation induced skin insults. We expect that such treatment could also be applied for other irradiated organs.     

Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.     

Adipose-Derived Stem Cells in the Treatment of Perianal Fistulas in Crohn’s Disease: Rationale,Clinical Results and Perspectives

Between 20 to 25% of Crohn’s disease (CD) patients suffer from perianal fistulas, a marker of disease severity. Seton drainage combined with anti-TNFα can result in closure of the fistula in 70 to 75% of patients. For the remaining 25% of patients there is room for in situ injection of autologous or allogenic mesenchymal stem cells such as adipose-derived stem/stromal cells (ADSCs). ADSCs exert their effects on tissues and effector cells through paracrine phenomena, including the secretome and extracellular vesicles. They display anti-inflammatory, anti-apoptotic, pro-angiogenic, proliferative, and immunomodulatory properties, and a homing within the damaged tissue. They also have immuno-evasive properties allowing a clinical allogeneic approach. Numerous clinical trials have been conducted that demonstrate a complete cure rate of anoperineal fistulas in CD ranging from 46 to 90% of cases after in situ injection of autologous or allogenic ADSCs. A pivotal phase III-controlled trial using allogenic ADSCs (Alofisel^®) demonstrated that prolonged clinical and radiological remission can be obtained in nearly 60% of cases with a good safety profile. Future studies should be conducted for a better knowledge of the local effect of ADSCs as well as for a standardization in terms of the number of injections and associated procedures.     

Injectable Gel Form of a Decellularized Bladder Induces Adipose-Derived Stem Cell Differentiation into Smooth Muscle Cells In Vitro

Biologic scaffolds composed of extracellular matrix components have been proposed to repair and reconstruct a variety of tissues in clinical and pre-clinical studies. Injectable gels can fill and conform any three-dimensional shape and can be delivered to sites of interest by minimally invasive techniques. In this study, a biological gel was produced from a decellularized porcine urinary bladder by enzymatic digestion with pepsin. The enzymatic digestion was confirmed by visual inspection after dissolution in phosphate-buffered saline solution and Fourier-transform infrared spectroscopy. The rheological and biological properties of the gel were characterized and compared to those of the Matrigel^TM chosen as a reference material. The storage modulus G’ reached 19.4 ± 3.7 Pa for the 30 mg/mL digested decellularized bladder gels after ca. 3 h at 37 °C. The results show that the gel formed of the porcine urinary bladder favored the spontaneous differentiation of human and rabbit adipose-derived stem cells in vitro into smooth muscle cells to the detriment of cell proliferation. The results support the potential of the developed injectable gel for tissue engineering applications to reconstruct for instance the detrusor muscle part of the human urinary bladder.     

New Perspectives to Improve Mesenchymal Stem Cell Therapies for Drug-Induced Liver Injury

Drug-induced liver injury (DILI) is one of the leading causes of acute liver injury. Many factors may contribute to the susceptibility of patients to this condition, making DILI a global medical problem that has an impact on public health and the pharmaceutical industry. The use of mesenchymal stem cells (MSCs) has been at the forefront of regenerative medicine therapies for many years, including MSCs for the treatment of liver diseases. However, there is currently a huge gap between these experimental approaches and their application in clinical practice. In this concise review, we focus on the pathophysiology of DILI and highlight new experimental approaches conceived to improve cell-based therapy by the in vitro preconditioning of MSCs and/or the use of cell-free products as treatment for this liver condition. Finally, we discuss the advantages of new approaches, but also the current challenges that must be addressed in order to develop safer and more effective procedures that will allow cell-based therapies to reach clinical practice, enhancing the quality of life and prolonging the survival time of patients with DILI.     

Extracellular Vesicles of Adipose-Derived Stem Cells Promote the Healing of Traumatized Achilles Tendons

Healing of ruptured tendons remains a clinical challenge because of its slow progress and relatively weak mechanical force at an early stage. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have therapeutic potential for tissue regeneration. In this study, we isolated EVs from adipose-derived stem cells (ADSCs) and evaluated their ability to promote tendon regeneration. Our results indicated that ADSC-EVs significantly enhanced the proliferation and migration of tenocytes in vitro. To further study the roles of ADSC-EVs in tendon regeneration, ADSC-EVs were used in Achilles tendon repair in rabbits. The mechanical strength, histology, and protein expression in the injured tendon tissues significantly improved 4 weeks after ADSC-EV treatment. Decorin and biglycan were significantly upregulated in comparison to the untreated controls. In summary, ADSC-EVs stimulated the proliferation and migration of tenocytes and improved the mechanical strength of repaired tendons, suggesting that ADSC-EV treatment is a potential highly potent therapeutic strategy for tendon injuries.     

Unrestricted Somatic Stem Cells Loaded in Nanofibrous Scaffolds as Potential Candidate for Skin Regeneration

The authors have demonstrated the ability of cord blood–derived unrestricted somatic stem cells (USSCs) and gelatin-modified PHBV scaffold to promote skin regeneration. The scaffolds were designed by electrospinning method and evaluated by different analyses. Results of physical and mechanical analyses also were shown a good resilience as a skin graft. In animal model; all groups excluding the control group exhibited the most pronounced effect on wound closure, with the statistically significant improvement in wound healing being seen at post-operative day 21. Thus, the grafting of nanofibrous scaffold loaded with USSC showed better results during the healing process of skin defects in rat models.