Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.     

Osteogenic Efficacy of Human Trophoblasts-Derived Conditioned Medium on Mesenchymal Stem Cells

Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We found that TB-CM promoted the osteogenic differentiation of MSCs in a dose-dependent manner. Furthermore, it inhibited adipogenesis of MSCs. We also found that the primary trophoblast-derived conditioned medium (PTB-CM) significantly enhanced the proliferation and osteogenic differentiation of human MSCs. Our study demonstrated the regulatory mechanisms underlying the TB-CM-induced osteogenesis in MSCs. An upregulation of genes associated with cytokines/chemokines was observed. The treatment of MSCs with TB-CM stimulated osteogenesis by activating several biological processes, such as mitogen-activated protein kinase (MAPK) and bone morphogenetic protein 2 (BMP2) signaling. This study demonstrated the proliferative and osteogenic efficacies of the trophoblast-derived secretomes, suggesting their potential for use in clinical interventions for bone regeneration and treatment.     

Exosomes Derived from Mesenchymal Stem Cells

The functional mechanisms of mesenchymal stem cells (MSCs) have become a research focus in recent years. Accumulating evidence supports the notion that MSCs act in a paracrine manner. Therefore, the biological factors in conditioned medium, including exosomes and soluble factors, derived from MSC cultures are being explored extensively. The results from most investigations show that MSC-conditioned medium or its components mediate some biological functions of MSCs. Several studies have reported that MSC-derived exosomes have functions similar to those of MSCs, such as repairing tissue damage, suppressing inflammatory responses, and modulating the immune system. However, the mechanisms are still not fully understood and the results remain controversial. Compared with cells, exosomes are more stable and reservable, have no risk of aneuploidy, a lower possibility of immune rejection following in vivo allogeneic administration, and may provide an alternative therapy for various diseases. In this review, we summarize the properties and biological functions of MSC-derived exosomes and discuss the related mechanisms.     

Mesenchymal Stem Cells: Therapeutic Mechanisms for Stroke

Due to aging of the world’s population, stroke has become increasingly prevalent, leading to a rise in socioeconomic burden. In the recent past, stroke research and treatment have become key scientific issues that need urgent solutions, with a sharp focus on stem cell transplantation, which is known to treat neurodegenerative diseases related to traumatic brain injuries, such as stroke. Indeed, stem cell therapy has brought hope to many stroke patients, both in animal and clinical trials. Mesenchymal stem cells (MSCs) are most commonly utilized in biological medical research, due to their pluripotency and universality. MSCs are often obtained from adipose tissue and bone marrow, and transplanted via intravenous injection. Therefore, this review will discuss the therapeutic mechanisms of MSCs and extracellular vehicles (EVs) secreted by MSCs for stroke, such as in attenuating inflammation through immunomodulation, releasing trophic factors to promote therapeutic effects, inducing angiogenesis, promoting neurogenesis, reducing the infarct volume, and replacing damaged cells.     

Hydrogel Encapsulation of Mesenchymal Stem Cells and Their Derived Exosomes for Tissue Engineering

Tissue engineering has been an inveterate area in the field of regenerative medicine for several decades. However, there remains limitations to engineer and regenerate tissues. Targeted therapies using cell-encapsulated hydrogels, such as mesenchymal stem cells (MSCs), are capable of reducing inflammation and increasing the regenerative potential in several tissues. In addition, the use of MSC-derived nano-scale secretions (i.e., exosomes) has been promising. Exosomes originate from the multivesicular division of cells and have high therapeutic potential, yet neither self-replicate nor cause auto-immune reactions to the host. To maintain their biological activity and allow a controlled release, these paracrine factors can be encapsulated in biomaterials. Among the different types of biomaterials in which exosome infusion is exploited, hydrogels have proven to be the most user-friendly, economical, and accessible material. In this paper, we highlight the importance of MSCs and MSC-derived exosomes in tissue engineering and the different biomaterial strategies used in fabricating exosome-based biomaterials, to facilitate hard and soft tissue engineering.     

Mesenchymal Stem Cells and Induced Pluripotent Stem Cells as Therapies for Multiple Sclerosis

Multiple sclerosis (MS) is a chronic, autoimmune, inflammatory demyelinating disorder of the central nervous system that leads to permanent neurological deficits. Current MS treatment regimens are insufficient to treat the irreversible neurological disabilities. Tremendous progress in the experimental and clinical applications of cell-based therapies has recognized stem cells as potential candidates for regenerative therapy for many neurodegenerative disorders including MS. Mesenchymal stem cells (MSC) and induced pluripotent stem cell (iPSCs) derived precursor cells can modulate the autoimmune response in the central nervous system (CNS) and promote endogenous remyelination and repair process in animal models. This review highlights studies involving the immunomodulatory and regenerative effects of mesenchymal stem cells and iPSCs derived cells in animal models, and their translation into immunomodulatory and neuroregenerative treatment strategies for MS.     

Hematopoietic–Mesenchymal Signals Regulate the Properties of Mesenchymal Stem Cells

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic–mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.     

The Effects of Synthetic Oligopeptide Derived from Enamel Matrix Derivative on Cell Proliferation and Osteoblastic Differentiation of Human Mesenchymal Stem Cells

Enamel matrix derivative (EMD) is widely used in periodontal tissue regeneration therapy. However, because the bioactivity of EMD varies from batch to batch, and the use of a synthetic peptide could avoid use from an animal source, a completely synthetic peptide (SP) containing the active component of EMD would be useful. In this study an oligopeptide synthesized derived from EMD was evaluated for whether it contributes to periodontal tissue regeneration. We investigated the effects of the SP on cell proliferation and osteoblast differentiation of human mesenchymal stem cells (MSCs), which are involved in tissue regeneration. MSCs were treated with SP (0 to 1000 ng/mL), to determine the optimal concentration. We examined the effects of SP on cell proliferation and osteoblastic differentiation indicators such as alkaline phosphatase activity, the production of procollagen type 1 C-peptide and osteocalcin, and on mineralization. Additionally, we investigated the role of extracellular signal-related kinases (ERK) in cell proliferation and osteoblastic differentiation induced by SP. Our results suggest that SP promotes these processes in human MSCs, and that ERK inhibitors suppress these effects. In conclusion, SP promotes cell proliferation and osteoblastic differentiation of human MSCs, probably through the ERK pathway.     

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30–200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.     

MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3′ untranslated region (3′-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/β-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.     

Human Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Neural Differentiation of Neural Progenitor Cells

Mesenchymal stem cells (MSCs) have been adopted in various preclinical and clinical studies because of their multipotency and low immunogenicity. However, numerous obstacles relating to safety issues remain. Therefore, MSC-derived extracellular vesicles (EVs) have been recently employed. EVs are nano-sized endoplasmic reticulum particles generated and released in cells that have similar biological functions to their origin cells. EVs act as cargo for bioactive molecules such as proteins and genetic materials and facilitate tissue regeneration. EVs obtained from adipose-derived MSC (ADMSC) also have neuroprotective and neurogenesis effects. On the basis of the versatile effects of EVs, we aimed to enhance the neural differentiation ability of ADMSC-derived EVs by elucidating the neurogenic-differentiation process. ADMSC-derived EVs isolated from neurogenesis conditioned media (differentiated EVs, dEVs) increased neurogenic ability by altering innate microRNA expression and cytokine composition. Consequently, dEVs promoted neuronal differentiation of neural progenitor cells in vitro, suggesting that dEVs are a prospective candidate for EV-based neurological disorder regeneration therapy.     

Multilineage Potential Research of Bovine Amniotic Fluid Mesenchymal Stem Cells

The use of amnion and amniotic fluid (AF) are abundant sources of mesenchymal stem cells (MSCs) that can be harvested at low cost and do not pose ethical conflicts. In human and veterinary research, stem cells derived from these tissues are promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. This work aimed to obtain and characterize bovine amniotic fluid mesenchymal stem cells (AFMSC). The bovine AF from the amniotic cavity of pregnant gilts in the early stages of gestation (3- and 4-m-old bovine embryos) was collected. AFMSCs exhibit a fibroblastic-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that AFMSCs were positive for β-integrin, CD44, CD73 and CD166, but negative for CD34, CD45. Meanwhile, AFMSCs expressed ES cell markers, such as Oct4, and when appropriately induced, are capable of differentiating into ectodermal and mesodermal lineages. This study reinforces the emerging importance of these cells as ideal tools in veterinary medicine; future studies aimed at a deeper evaluation of their immunological properties will allow a better understanding of their role in cellular therapy.     

Engineering of Self-Assembled Fibronectin Matrix Protein and Its Effects on Mesenchymal Stem Cells

Fibronectin (FN) contributes to cell adhesion, proliferation, and differentiation in various cell types. To enhance the activity of fibronectin at the sites of focal adhesion, we engineered a novel recombinant fibronectin (FNIII10) fragment connected to the peptide amphiphile sequence (PA), LLLLLLCCCGGDS. In this study, the effects of FNIII10-PA on rat mesenchymal stem cells (rMSCs) were compared with those of FNIII10. FNIII10-PA showed the prominent protein adhesion activity. In addition, FNIII10-PA showed a significantly higher effect on adhesion, proliferation, and differentiation of rMSCs than FNIII10. Taken together, the FNIII10-containing self-assembled sequence enhanced rMSCs adhesion, proliferation, and differentiation.     

Potential Spermatogenesis Recovery with Bone Marrow Mesenchymal Stem Cells in an Azoospermic Rat Model

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.     

Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.     

Human Adipose-Derived Mesenchymal Stem Cells Are Resistant to HBV Infection during Differentiation into Hepatocytes in Vitro

The therapeutic methods for chronic hepatitis B are limited. The shortage of organ donors and hepatitis B virus (HBV) reinfection obstruct the clinical application of orthotopic liver transplantation (OLT). In the present study, adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated from chronic hepatitis B patients and characterized for morphology, growth potency, surface phenotype and the differentiation potential. The results showed that both MSCs had adipogenic, osteogenic and neuron differentiation potential, and nearly all MSCs expressed CD105, CD44 and CD29. Compared with AD-MSCs, BM-MSCs of chronic hepatitis B patients proliferated defectively. In addition, the ability of AD-MSCs to differentiate into hepatocyte was evaluated and the susceptibility to HBV infection were assessed. AD-MSCs could differentiate into functional hepatocyte-like cells. These cells express the hepatic-specific markers and have glycogen production and albumin secretion function. AD-MSCs and hepatic differentiation AD-MSCs were not susceptible to infection by HBV in vitro. Compared with BM-MSCs, AD-MSCs may be alternative stem cells for chronic hepatitis B patients.     

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.     

Human Mesenchymal Stem Cells Modulate Inflammatory Cytokines after Spinal Cord Injury in Rat

Transplantation of mesenchymal stem cells (MSC) improves functional recovery in experimental models of spinal cord injury (SCI); however, the mechanisms underlying this effect are not completely understood. We investigated the effect of intrathecal implantation of human MSC on functional recovery, astrogliosis and levels of inflammatory cytokines in rats using balloon-induced spinal cord compression lesions. Transplanted cells did not survive at the lesion site of the spinal cord; however, functional recovery was enhanced in the MSC-treated group as was confirmed by the Basso, Beattie, and Bresnahan (BBB) and the flat beam test. Morphometric analysis showed a significantly higher amount of remaining white matter in the cranial part of the lesioned spinal cords. Immunohistochemical analysis of the lesions indicated the rearrangement of the glial scar in MSC-treated animals. Real-time PCR analysis revealed an increased expression of Irf5, Mrc1, Fgf2, Gap43 and Gfap. Transplantation of MSCs into a lesioned spinal cord reduced TNFα, IL-4, IL-1β, IL-2, IL-6 and IL-12 and increased the levels of MIP-1α and RANTES when compared to saline-treated controls. Intrathecal implantation of MSCs reduces the inflammatory reaction and apoptosis, improves functional recovery and modulates glial scar formation after SCI, regardless of cell survival. Therefore, repeated applications may prolong the beneficial effects induced by MSC application.