Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.     

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.     

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.     

Immunomodulatory Mechanisms of Mesenchymal Stem Cells and Their Potential Clinical Applications

Mesenchymal stem cells (MSCs) are multipotent stem cells with the capacity of self-renewal, homing, and low immunogenicity. These distinct biological characteristics have already shown immense potential in regenerative medicine. MSCs also possess immunomodulatory properties that can maintain immune homeostasis when the immune response is over-activated or under-activated. The secretome of MSCs consists of cytokines, chemokines, signaling molecules, and growth factors, which effectively contribute to the regulation of immune and inflammatory responses. The immunomodulatory effects of MSCs can also be achieved through direct cell contact with microenvironmental factors and immune cells. Furthermore, preconditioned and engineered MSCs can specifically improve the immunomodulation effects in diverse clinical applications. These multifunctional properties of MSCs enable them to be used as a prospective therapeutic strategy to treat immune disorders, including autoimmune diseases and incurable inflammatory diseases. Here we review the recent exploration of immunomodulatory mechanisms of MSCs and briefly discuss the promotion of the genetically engineered MSCs. Additionally, we review the potential clinical applications of MSC-mediated immunomodulation in four types of immune diseases, including systemic lupus erythematosus, Crohn’s disease, graft-versus-host disease, and COVID-19.     

CD9 Upregulation-Decreased CCL21 Secretion in Mesenchymal Stem Cells Reduces Cancer Cell Migration

Tetraspanin CD9 is widely expressed on various cell types, such as cancer cells and mesenchymal stem cells (MSCs), and/or cell-released exosomes. It has been reported that exosomal CD9 plays an important role in intercellular communications involved in cancer cell migration and metastasis. However, reports on the effect of the CD9 of MSCs or MSC-derived exosomes on cancer cell migration are still lacking. In this study, using a transwell migration assay, we found that both dextran-coated iron oxide nanoparticles (dex-IO NPs) and ionomycin stimulated exosomal CD9 expression in human MSCs (hMSCs); however, hMSCs could not deliver them to melanoma cells to affect cell migration. Interestingly, a reduced migration of melanoma cell line was observed when the ionomycin-incubated hMSC-conditioned media but not dex-IO NP-labeled hMSC-conditioned media were in the bottom chamber. In addition, we found that dex-IO NPs decreased cellular CD9 expression in hMSCs but ionomycin increased this. Simultaneously, we found that ionomycin suppressed the expression and secretion of the chemokine CCL21 in hMSCs. The silencing of CD9 demonstrated an inhibitory role of cellular CD9 in CCL21 expression in hMSCs, suggesting that ionomycin could upregulate cellular CD9 to decrease CCL21 expression and secretion of hMSCs, which would reduce the migration of B16F10, A549 and U87MG cancer cell lines due to chemoattraction reduction of CCL21. The present study not only highlights the important role of bone marrow-derived hMSCs’ CD9-mediated CCL21 regulation in cancer bone metastasis but also suggests a new distinct pharmaceutical strategy for prevention or/and therapy of cancer metastasis.     

Tolerogenic IDO1+CD83− Langerhans Cells in Sentinel Lymph Nodes of Patients with Melanoma

Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin⁺ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1⁻CD83⁺ LCs; real immature IDO1⁻CD83⁻ LCs; tolerogenic mature IDO1⁺CD83⁺ LCs; tolerogenic immature IDO1⁺CD83⁻ LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma.     

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.     

Alteration of 3D Matrix Stiffness Regulates Viscoelasticity of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material against osteoporosis. Although interrogation of directing effect on lineage specification by physical cues has been proposed, how mechanical stimulation impacts intracellular viscoelasticity during osteogenesis remained enigmatic. Cyto-friendly 3D matrix was prepared with polyacrylamide and conjugated fibronectin. The hMSCs were injected with fluorescent beads and chemically-induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. Inverted epifluorescence microscope was exploited to capture the Brownian trajectory of hMSCs. Mean square displacement was calculated and transformed into intracellular viscoelasticity. Two different stiffness of microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. hMSCs possessed equivalent mechanical traits initially in the first week, while cells cultured in rigid matrix displayed significant elevation over elastic (G′) and viscous moduli (G″) on day 7 (p < 0.01) and 14 (p < 0.01). However, after two weeks, soft niches no longer stiffened hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course. Stiffness of matrix impacted the viscoelasticity of hMSCs. Detailed recognition of how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.     

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.     

Favorable Biological Performance Regarding the Interaction between Gold Nanoparticles and Mesenchymal Stem Cells

Gold nanoparticles (AuNPs) are well known to interact with cells, leading to different cell behaviors such as cell proliferation and differentiation capacity. Biocompatibility and biological functions enhanced by nanomedicine are the most concerning factors in clinical approaches. In the present research, AuNP solutions were prepared at concentrations of 1.25, 2.5, 5 and 10 ppm for biocompatibility investigations. Ultraviolet–visible spectroscopy was applied to identify the presence of AuNPs under the various concentrations. Dynamic Light Scattering assay was used for the characterization of the size of the AuNPs. The shape of the AuNPs was observed through a Scanning Electron Microscope. Afterward, the mesenchymal stem cells (MSCs) were treated with a differentiation concentration of AuNP solutions in order to measure the biocompatibility of the nanoparticles. Our results demonstrate that AuNPs at 1.25 and 2.5 ppm could significantly enhance MSC proliferation, decrease reactive oxygen species (ROS) generation and attenuate platelet/monocyte activation. Furthermore, the MSC morphology was observed in the presence of filopodia and lamellipodia while being incubated with 1.25 and 2.5 ppm AuNPs, indicating that the adhesion ability was enhanced by the nanoparticles. The expression of matrix metalloproteinase (MMP-2/9) in MSCs was found to be more highly expressed under 1.25 and 2.5 ppm AuNP treatment, relating to better cell migrating ability. Additionally, the cell apoptosis of MSCs investigated with Annexin-V/PI double staining assay and the Fluorescence Activated Cell Sorting (FACS) method demonstrated the lower population of apoptotic cells in 1.25 and 2.5 ppm AuNP treatments, as compared to high concentrations of AuNPs. Additionally, results from a Western blotting assay explored the possibility that the anti-apoptotic proteins Cyclin-D1 and Bcl-2 were remarkably expressed. Meanwhile, real-time PCR analysis demonstrated that the 1.25 and 2.5 ppm AuNP solutions induced a lower expression of inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6 and IL-8). According to the tests performed on an animal model, AuNP 1.25 and 2.5 ppm treatments exhibited the better biocompatibility performance, including anti-inflammation and endothelialization. In brief, 1.25 and 2.5 ppm of AuNP solution was verified to strengthen the biological functions of MSCs, and thus suggests that AuNPs become the biocompatibility nanomedicine for regeneration research.     

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.     

Interaction between Mesenchymal Stem Cells and B-Cells

Mesenchymal stem cells (MSCs) are multipotent; non-hematopoietic stem cells. Because of their immunoregulatory abilities; MSCs are widely used for different clinical applications. Compared with that of other immune cells; the investigation of how MSCs specifically regulate B-cells has been superficial and insufficient. In addition; the few experimental studies on this regulation are often contradictory. In this review; we summarize the various interactions between different types or states of MSCs and B-cells; address how different types of MSCs and B-cells affect this interaction and examine how other immune cells influence the regulation of B-cells by MSCs. Finally; we hypothesize why there are conflicting results on the interaction between MSCs and B-cells in the literature.     

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.     

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.     

Treatment of Chronic Kidney Disease with Extracellular Vesicles from Mesenchymal Stem Cells and CD133+ Expanded Cells: A Comparative Preclinical Analysis

Chronic kidney disease (CKD) is characterized by structural abnormalities and the progressive loss of kidney function. Extracellular vesicles (EVs) from human umbilical cord tissue (hUCT)-derived mesenchymal stem cells (MSCs) and expanded human umbilical cord blood (hUCB)-derived CD133⁺ cells (eCD133⁺) maintain the characteristics of the parent cells, providing a new form of cell-free treatment. We evaluated the effects of EVs from hUCT-derived MSCs and hUCB-derived CD133⁺ cells on rats with CDK induced by an adenine-enriched diet. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The animals were randomized and divided into the MSC-EV group, eEPC-EV group and control group. Infusions occurred on the seventh and 14th days after CKD induction. Evaluations of kidney function were carried out by biochemical and histological analyses. Intense labeling of the α-SMA protein was observed when comparing the control with MSC-EVs. In both groups treated with EVs, a significant increase in serum albumin was observed, and the increase in cystatin C was inhibited. The results indicated improvements in renal function in CKD, demonstrating the therapeutic potential of EVs derived from MSCs and eCD133⁺ cells and suggesting the possibility that in the future, more than one type of EV will be used concurrently.     

Effects of Normal Synovial Fluid and Interferon Gamma on Chondrogenic Capability and Immunomodulatory Potential Respectively on Equine Mesenchymal Stem Cells

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-β1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-β1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.     

Strategies to Potentiate Paracrine Therapeutic Efficacy of Mesenchymal Stem Cells in Inflammatory Diseases

Mesenchymal stem cells (MSCs) have been developed as cell therapeutics for various immune disorders using their immunoregulatory properties mainly exerted by their paracrine functions. However, variation among cells from different donors, as well as rapid clearance after transplantation have impaired the uniform efficacy of MSCs and limited their application. Recently, several strategies to overcome this limitation have been suggested and proven in pre-clinical settings. Therefore, in this review article, we will update the knowledge on bioengineering strategies to improve the immunomodulatory functions of MSCs, including genetic modification and physical engineering.     

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.