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Mesenchymal stem/stromal cell (MSC)-based therapies have been evaluated in over 1500 human clinical trials for a diverse array of disease indication, but outcomes remain unpredictable due to knowledge gaps in the quality attributes that confer therapeutic potency onto cells and their mode of action in vivo. Based on accumulated evidence from pre-clinical models, MSCs exert therapeutic effects by repressing inflammatory and immune-mediated response via paracrine action following reprogramming by the host injury microenvironment, and by polarization of tissue resident macrophages following phagocytosis to an alternatively activated (M2) state. An important tenet of this existing paradigm is that well-established stem/progenitor functions of MSCs are independent of paracrine function and dispensable for their anti-inflammatory and immune suppressive functions. Herein, we review evidence that stem/progenitor and paracrine functions of MSCs are mechanistically linked and organized hierarchically and describe how this link may be exploited to develop metrics that predict MSC potency across a spectrum of activities and regenerative medicine applications.

Recent research indicated that extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) are a promising alternative to MSCs for immunomodulatory therapy. However, the contents of MSC-EVs would change as their parent MSCs change, hence the therapeutic efficacy of MSC-derived EVs (MSC-EVs) would largely depend on donors, tissue sources and culture conditions of MSCs. To overcome limitations of tissue-derived MSCs, we previously used MSCs derived from human induced pluripotent stem cells (iMSCs) to produce EVs and demonstrated their therapeutic potential in a mouse model of secondary Sjo¨gren's Syndrome. Here, we further found that EVs from early-passage iMSCs had better immunomodulatory potency than EVs from late-passage iMSCs in TLR4-stimulated splenocytes and in a mouse model of primary Sjogren's syndrome. Comparative molecular profiling using proteomics and microRNA sequencing revealed distinctive molecular profiles of iMSC-EVs with or without immunomodulation capacity. Amongst them, manipulation of TGF-β1, miR-21 and miR-125b levels in iMSC-EVs significantly affected their immunosuppressive effects. These findings would help improve our understanding of the molecular mechanism underlying iMSC-EV-mediated immunomodulation and further provide strategies to improve regulatory function of EVs for the treatment of immune-mediated diseases.

Corneal transplantation is a routine procedure for patients with corneal blindness. Despite the streamlining of surgical techniques and deeper understanding of the cellular and molecular pathways mediating rejection, corticosteroids are still the main immunosuppressive regimen in corneal transplantation, and the 15-year survival of corneal transplants remains as low as 50%, which is poorer than that for most solid organ transplants. Recently, mesenchymal stromal cells (MSCs) with unique regenerative and immune-modulating properties have emerged as a promising cell therapy to promote transplant tolerance, minimize the use of immunosuppressants, and prevent chronic rejection. Here, we review the literature on preclinical studies of MSCs for corneal transplantation and summarize the key findings from clinical trials with MSCs in solid organ transplantation. Finally, we highlight current issues and challenges regarding MSC therapies and suggest strategies for safe and effective MSC-based therapies in clinical transplantation.

Accumulating evidence indicates that mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) exhibit immunomodulatory effects by delivering therapeutic RNAs and proteins; however, the molecular mechanism underlying the EV-mediated immunomodulation is not fully understood. In this study, we found that EVs from early-passage MSCs had better immunomodulatory potency than did EVs from late-passage MSCs in T cell receptor (TCR)- or Toll-like receptor 4 (TLR4)-stimulated splenocytes and in mice with ocular Sjögren's syndrome. Moreover, MSC-EVs were more effective when produced from 3D culture of the cells than from the conventional 2D culture. Comparative molecular profiling using proteomics and microRNA sequencing revealed the enriched factors in MSC-EVs that were functionally effective in immunomodulation. Among them, manipulation of transforming growth factor β1 (TGF-β1), pentraxin 3 (PTX3), let-7b-5p, or miR-21-5p levels in MSCs significantly affected the immunosuppressive effects of their EVs. Furthermore, there was a strong correlation between the expression levels of TGF-β1, PTX3, let-7b-5p, or miR-21-5p in MSC-EVs and their suppressive function. Therefore, our comparative strategy identified TGF-β1, PTX3, let-7b-5p, or miR-21-5p as key molecules mediating the therapeutic effects of MSC-EVs in autoimmune disease. These findings would help understand the molecular mechanism underlying EV-mediated immunomodulation and provide functional biomarkers of EVs for the development of robust EV-based therapies.

Extracellular vesicles (EVs) from allogeneic-tissue-derived mesenchymal stem cells (MSCs) are promising to improve Sjögren’s syndrome (SS) treatment, but their application is hindered by high variations in and limited expandability of tissue MSCs. We derived standardized and scalable MSCs from iPS cells (iMSCs) and reported that EVs from young but not aging iMSCs (iEVs) inhibited sialadenitis onset in SS mouse models. Here, we aim to determine cellular mechanisms and optimization approaches of SS-inhibitory effects of iEVs. In NOD.B10.H2b mice at the pre-disease stage of SS, we examined the biodistribution and recipient cells of iEVs with imaging, flow cytometry, and qRT-PCR. Intravenously infused iEVs accumulated in the spleen but not salivary glands or cervical lymph nodes and were mainly taken up by macrophages. In the spleen, young but not aging iEVs increased M2 macrophages, decreased Th17 cells, and changed expression of related immunomodulatory molecules. Loading miR-125b inhibitors into aging iEVs significantly improved their effects on repressing sialadenitis onset and regulating immunomodulatory splenocytes. These data indicated that young but not aging iEVs suppress SS onset by regulating immunomodulatory splenocytes, and inhibiting miR-125b in aging iEVs restores such effects, which is promising to maximize production of effective iEVs from highly expanded iMSCs for future clinical application.

Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3‐fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5‐minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost‐effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential., A scalable system for the generation and rapid harvest of immunomodulatory induced pluripotent stem cell‐derived mesenchymal stem cells..

No effective treatment for Sjögren’s syndrome (SS), a chronic autoimmune disease affecting mainly salivary and lacrimal glands, is available now. Systemic infusion of allogeneic mesenchymal stem cells (MSCs) isolated from tissues such as bone marrow (BM) alleviated SS in mouse models and a small clinical trial, but further research and application of this MSC therapy were hindered by limited expandability, significant donor variations, and safety concerns of tissue-derived MSCs. To circumvent these issues, we derived MSCs from human iPSCs using an optimized protocol that can be easily scaled up to produce a huge amount of standardized MSCs. Our iPSC-MSCs inhibited the onset of lymphocyte infiltration into salivary glands in the NOD mouse model of SS in the same way as BM-MSCs. Extracellular vesicles (EVs) carry bioactive molecules in the same way as their originating cells and are more stable and considered much safer than cells for therapies. We found that EVs derived from BM-MSCs and iPSC-MSCs suppressed activation of immune cells and expression of proinflammation factors essential for SS progression in vitro and that infusion of iPSC-MSC EVs at the predisease stage decreased the lymphocyte infiltration in salivary glands and serum autoantibody levels in the same way as infusion of BM-MSCs and iPSC-MSCs. These data suggested that iPSC-MSC EVs have the potential to prevent the progression of SS before the onset of sialadenitis.

The cornea is a transparent tissue devoid of blood and lymphatic vessels. However, various inflammatory conditions can cause hemangiogenesis and lymphangiogenesis in the cornea, compromising transparency and visual acuity. Mesenchymal stem/stromal cells (MSCs) have therapeutic potentials in a variety of diseases because of anti-inflammatory properties. Herein, we investigated the effects of MSCs on corneal angiogenesis using a model of suture-induced inflammatory corneal neovascularization. Data demonstrated that an intravenous administration of MSCs suppressed corneal inflammation and neovascularization, inhibiting both hemangiogenesis and lymphangiogenesis. MSCs reduced the levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, Tek, MRC1, and MRC2 in the cornea, which are expressed by pro-angiogenic macrophages. Moreover, the number of CD11b+ monocytes/macrophages in the cornea, spleen, peripheral blood, and draining lymph nodes was decreased by MSCs. Depletion of circulating CD11b+ monocytes by blocking antibodies replicated the effects of MSCs. Importantly, knockdown of tumor necrosis factor alpha (TNF-α)-stimulated gene/protein 6 (TSG-6) in MSCs abrogated the effects of MSCs in inhibiting corneal hemangiogenesis and lymphangiogenesis and monocyte/macrophage infiltration. Together, the results suggest that MSCs inhibit inflammatory neovascularization in the cornea by suppressing pro-angiogenic monocyte/macrophage recruitment in a TSG-6-dependent manner.

Mesenchymal stromal cells, commonly referred to as MSCs, have emerged as a promising cell-based therapy for a range of autoimmune diseases thanks to several therapeutic advantages. Key among these are: 1) the ability to modulate innate and adaptive immune responses and to promote tissue regeneration, 2) the ease of their isolation from readily accessible tissues and expansion at scale in culture, 3) their low immunogenicity enabling use as an allogeneic "off-the-shelf" product, and 4) MSC therapy's safety and feasibility in humans, as demonstrated in more than one thousand clinical trials. Evidence from preclinical studies and early clinical trials indicate the therapeutic potential of MSCs and their derivatives for efficacy in ocular autoimmune diseases such as autoimmune uveoretinitis and Sjögren's syndrome-related dry eye disease. In this review, we provide an overview of the current understanding of the therapeutic mechanisms of MSCs, and summarize the results from preclinical and clinical studies that have used MSCs or their derivatives for the treatment of ocular autoimmune diseases. We also discuss the challenges to the successful clinical application of MSC therapy, and suggest strategies for overcoming them.

Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes., Graphical Abstract

Background aims Previously, we showed that human mesenchymal stromal cells (hMSCs) were activated to express tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) upon TNF-α stimulation, induced cell death in triple-negative breast cancer (TNBC) MDA-MB-231 cells (MDA), and RNA released from apoptotic MDA further increased TRAIL expression in hMSCs. This feed-forward stimulation increased apoptosis in MDA cells. Here, we tested whether TRAIL-expressing hMSCs, in combination with a sub-toxic-dose of a chemotherapy drug doxorubicin, would overcome TRAIL resistance and create synergistic effects on targeting metastatic TNBC. Methods To optimize conditions for the combination treatment, we (i) selected an optimal condition to activate hMSCs for TRAIL expression, (ii) selected an optimal dose of doxorubicin treatment, (iii) examined underlying mechanisms in vitro and (iv) tested the efficacy of the optimized conditions in a xenograft mouse model of human breast cancer lung metastasis. Results The results showed that DNA fragments from apoptotic MDA triggered hMSCs to increase further TRAIL expression in an absent in melanoma 2 (AIM2)–dependent manner, and thus higher TRAIL-expressing hMSCs stimulated with synthetic DNA, poly(deoxyadenylic-deoxythymidylic) acid [poly(dA:dT)], more effectively suppressed tumor progression in vivo . Furthermore, activated hMSCs increased apoptosis in MDA cells when combined with a sub-toxic dose of doxorubicin, which was mediated by up-regulating TRAIL and Fas-related pathways. When we combined the optimized conditions, pre-activated hMSCs with poly (dA:dT) synergistically reduced tumor burden even with minimal doxorubicin treatment in a xenograft mouse model of human breast cancer lung metastasis. Conclusions These results suggest that the treatment of hMSCs with a sub-toxic dose of doxorubicin can overcome TRAIL resistance and be a potential novel therapy for TNBC metastasis treatment.

Much of what we know about immunology suggests that little is to be gained from experiments in which human cells are administered to immunocompetent mice. Multiple reports have demonstrated that this common assumption does not hold for experiments with human mesenchymal stem/stromal cells (hMSCs). The data demonstrate that hMSCs can suppress immune responses to a variety of stimuli in immunocompetent mice by a range of different mechanisms that are similar to those employed by mouse MSCs. Therefore, further experiments with hMSCs in mice will make it possible to generate preclinical data that will improve both the efficacy and safety of the clinical trials with the cells that are now in progress.

One attractive strategy to treat cancers is to deliver an exogenous enzyme that will convert a non-toxic compound to a highly toxic derivative. The strategy was tested with viral vectors but was disappointing because the efficiency of transduction into tumor cells was too low. Recent reports demonstrated that the limitation can be addressed by using tissue-derived mesenchymal stromal cells (MSCs) to deliver enzyme/prodrug systems that kill adjacent cancer cells through bystander effects. Here we addressed the limitation that tissue-derived MSCs vary in their properties and are difficult to generate in the large numbers needed for clinical applications. We prepared a Feeder Stock of MSCs from induced pluripotent stem cells (iPSs) that provided an extensively expandable source of standardized cells. We then transduced the iPS-derived MSCs to express cytosine deaminase and injected them locally into a mouse xenogeneic model of human breast cancer. After administration of the prodrug (5-fluorocytosine), the transduced iPS-MSCs both limited growth of preformed tumors and decreased lung metastases.

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

Genetic and immunological screening for type 1 diabetes has led to the possibility of preventing disease in susceptible individuals. Here, we show that human mesenchymal stem/stromal cells (hMSCs) and tumor necrosis factor-α–stimulated gene 6 (TSG-6), a protein produced by hMSCs in response to signals from injured tissues, delayed the onset of spontaneous autoimmune diabetes in NOD mice by inhibiting insulitis and augmenting regulatory T cells (Tregs) within the pancreas. Importantly, hMSCs with a knockdown of tsg-6 were ineffective at delaying insulitis and the onset of diabetes in mice. TSG-6 inhibited the activation of both T cells and antigen-presenting cells (APCs) in a CD44-dependent manner. Moreover, multiple treatments of TSG-6 rendered APCs more tolerogenic, capable of enhancing Treg generation and delaying diabetes in an adoptive transfer model. Therefore, these results could provide the basis for a novel therapy for the prevention of type 1 diabetes.

The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell–cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics.

Stanniocalcin-1 (STC-1) is a multifunctional glycoprotein with antioxidant and anti-inflammatory properties. Ischemic myocardial necrosis generates "danger" signals that perpetuate detrimental inflammatory reactions often involving monocyte recruitment and their subsequent differentiation into proinflammatory macrophages. Therefore, we evaluated the effects of recombinant STC-1 (rSTC-1) on monocyte phenotype and in a mouse model of myocardial infarction. Using an established protocol to differentiate human monocytes into macrophages, we demonstrated that rSTC-1 did not alter morphology of the differentiated cells, toll-like receptor (TLR) 4 expression, or expression of the myeloid cell marker CD11b. However, rSTC-1 treatment before differentiation attenuated the rise in the expression of CD14, a TLR4 coreceptor and pathogen sensor that propagates innate immune responses, and suppressed levels of inflammatory cytokines produced by the differentiated cells in response to the CD14-TLR4 ligand lipopolysaccharide. Moreover, rSTC-1 treatment reduced CD14 expression in monocytes stimulated with endogenous danger signals. Interestingly, the effects of rSTC-1 on CD14 expression were not reproduced by a superoxide dismutase mimetic. In mice with induced myocardial infarcts, intravenous administration of rSTC-1 decreased CD14 expression in the heart as well as levels of tumor necrosis factor alpha, C-X-C motif ligand 2, interleukin 1 beta, and myeloperoxidase. It also suppressed the formation of scar tissue while enhancing cardiac function. The data suggests that one of the beneficial effects of STC-1 might be attributed to suppression of CD14 on recruited monocytes and macrophages that limits their inflammatory response. STC-1 may be a promising therapy to protect the heart and other tissues from ischemic injury.

Mesenchymal stem/progenitor cells (MSCs) improve functional outcome in a number of disease models through suppression of inflammation. However, their effects on neuroinflammation are unknown. In this study, we show that MSCs suppress endotoxin-induced glial activation in organotypic hippocampal slice cultures (OHSCs). Lipopolysaccharide-stimulated OHSCs activated MSCs to increase the expression of cyclo-oxygenase-2 and produce prostaglandin E2. MSC-derived prostaglandin E2, then suppressed pro-inflammatory cytokine production by the OHSCs. Together, the results suggest the potential anti-inflammatory mechanism of MSCs in models of disease and support earlier observations that MSCs may offer a therapy for neuroinflammation produced by trauma or disease.

Previous reports suggested that culture as 3D aggregates or as spheroids can increase the therapeutic potential of the adult stem/progenitor cells referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). Here we used a hanging drop protocol to prepare human MSCs (hMSCs) as spheroids that maximally expressed TNFα stimulated gene/protein 6 (TSG-6), the antiinflammatory protein that was expressed at high levels by hMSCs trapped in the lung after i.v. infusion and that largely explained the beneficial effects of hMSCs in mice with myocardial infarcts. The properties of spheroid hMSCs were found to depend critically on the culture conditions. Under optimal conditions for expression of TSG-6, the hMSCs also expressed high levels of stanniocalcin-1, a protein with both antiinflammatory and antiapoptotic properties. In addition, they expressed high levels of three anticancer proteins: IL-24, TNFα-related apoptosis inducing ligand, and CD82. The spheroid hMSCs were more effective than hMSCs from adherent monolayer cultures in suppressing inflammatory responses in a coculture system with LPS-activated macrophages and in a mouse model for peritonitis. In addition, the spheroid hMSCs were about one-fourth the volume of hMSCs from adherent cultures. Apparently as a result, larger numbers of the cells trafficked through the lung after i.v. infusion and were recovered in spleen, liver, kidney, and heart. The data suggest that spheroid hMSCs may be more effective than hMSCs from adherent cultures in therapies for diseases characterized by sterile tissue injury and unresolved inflammation and for some cancers that are sensitive to antiinflammatory agents.

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

We screened for surface proteins expressed only by the early progenitor cells present in low-passage, low-density cultures of the adult stem/progenitor cells from bone marrow referred to as mesenchymal stem cells or multipotent stromal cells (MSCs). Six proteins were identified that were selectively expressed in the early progenitors: podocalyxin-like protein (PODXL), α6-integrin (CD49f), α4-integrin (CD49d), c-Met, CXCR4, and CX3CR1. All were previously shown to be involved in cell trafficking or tumor progression. Antibodies to CD49f and PODXL, a sialomucin in the CD34 family, were the most robust for FACScan assays. PODXLhi/CD49fhi MSCs were more clonogenic and differentiated more efficiently than PODXLlo/CD49flo cells. Inhibition of expression of PODXL with RNA interference caused aggregation of the cells. Furthermore, PODXLhi/CD49fhi MSCs were less prone to produce lethal pulmonary emboli, and larger numbers were recovered in heart and kidney after intravenous infusion into mice with myocardial infarcts.

We tested the hypothesis that multipotent stromal cells from human bone marrow (hMSCs) can provide a potential therapy for human diabetes mellitus. Severe but nonlethal hyperglycemia was produced in NOD/ scid mice with daily low doses of streptozotocin on days 1–4, and hMSCs were delivered via intracardiac infusion on days 10 and 17. The hMSCs lowered blood glucose levels in the diabetic mice on day 32 relative to untreated controls (18.34 mM ± 1.12 SE vs. 27.78 mM ± 2.45 SE, P = 0.0019). ELISAs demonstrated that blood levels of mouse insulin were higher in the hMSC-treated as compared with untreated diabetic mice, but human insulin was not detected. PCR assays detected human Alu sequences in DNA in pancreas and kidney on day 17 or 32 but not in other tissues, except heart, into which the cells were infused. In the hMSC-treated diabetic mice, there was an increase in pancreatic islets and β cells producing mouse insulin. Rare islets contained human cells that colabeled for human insulin or PDX-1. Most of the β cells in the islets were mouse cells that expressed mouse insulin. In kidneys of hMSC-treated diabetic mice, human cells were found in the glomeruli. There was a decrease in mesangial thickening and a decrease in macrophage infiltration. A few of the human cells appeared to differentiate into glomerular endothelial cells. Therefore, the results raised the possibility that hMSCs may be useful in enhancing insulin secretion and perhaps improving the renal lesions that develop in patients with diabetes mellitus.

Controversies have arisen as to whether adult stem cells or progenitor cells from bone marrow can engraft into nonhematopoietic tissues in vivo. To resolve some of the controversies, we developed a highly sensitive polymerase chain reaction-based single nucleotide polymorphism (PCR-SNP) assay for competitive engraftment of mixtures of stem/progenitor cells. We used the assay to follow engraftment in immunodeficient mice of subpopulations of the stem/progenitor cells from human bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). The engraftment into adult mice without induced tissue injury was low and variable, but there was preferential engraftment of a subpopulation of rapidly self-renewing MSCs (RS-MSCs) compared with a subpopulation of slowly renewing MSCs (SR-MSCs). After intravenous infusion, there was a tendency for the cells to engraft into the hippocampal region that was previously designated a “vascular niche.” Migration assays suggested that preferential engraftment of RS-MSCs was in part explained by their expression of CXCR4 and CX3R1, the receptors for SDF-1 and fractalkine.

The peripheral benzodiazepine receptor (PBR) has been known to have many functions such as a role in cell proliferation, cell differentiation, steroidogenesis, calcium flow, cellular respiration, cellular immunity, malignancy, and apoptosis. However, the presence of PBR has not been examined in mesenchymal stem cells. In this study, we demonstrated the expression of PBR in human bone marrow stromal cells (hBMSCs) and human adipose stromal cells (hATSCs) by RT-PCR and immunocytochemistry. To determine the roles of PBR in cellular functions of human mesenchymal stem cells (hMSCs), effects of diazepam, PK11195, and Ro5-4864 were examined. Adipose differentiation of hMSCs was decreased by high concentration of PBR ligands (50 microM), whereas it was increased by low concentrations of PBR ligands (

The aquaporins constitute a family of homologous intrinsic membrane proteins that function as highly selective water channels and are highly expressed in tissues where rapid water movement across the cell membrane is required. Molecular mechanism of water transport through the plasma membrane of skeletal muscle is still not clear. This study was designed to identify aquaporin subtypes and their expression regulation in C2C12 cells, a mouse myoblastic cell line. RT-PCR, immunohistochemistry and Western blot analysis revealed that C2C12 cells express AQP5. AQP5 expression was increased by induction of C2C12 differentiation. Exposure of C2C12 cells to hypertonic solutions induced an increase in AQP5 expression and p38 kinase activation. However, a p38 kinase inhibitor failed to inhibit hyperosmolar induction of AQP5 expression in C2C12 cells. These data indicate that skeletal muscle cells express AQP5 protein and its expression is regulated by differentiation and hypertonic stress.

Human mesenchymal stem/progenitor cells (hMSCs) from bone marrow and other tissues are currently being administered to large numbers of patients even though there are no biomarkers that accurately predict their efficacy in vivo. Using a mouse model of chemical injury of the cornea, we found that bone-marrow–derived hMSCs isolated from different donors varied widely in their efficacy in modulating sterile inflammation. Importantly, RT-PCR assays of hMSCs for the inflammation-modulating protein TSG-6 expressed by the TNFα-stimulated gene 6 (TSG-6 or TNFAIP6) predicted their efficacy in sterile inflammation models for corneal injury, sterile peritonitis, and bleomycin-induced lung injury. In contrast, the levels of TSG-6 mRNA were negatively correlated with their potential for osteogenic differentiation in vitro and poorly correlated with other criteria for evaluating hMSCs. Also, a survey of a small cohort suggested that hMSCs from female donors compared with male donors more effectively suppressed sterile inflammation, expressed higher levels of TSG-6, and had slightly less osteogenic potential.

Embryonic stem (ES) cells, which have pluripotency in differentiation, have been considered as an excellent source for cell replacement therapy of damaged tissues. Although neurotrophic factors induce neural differentiation in neural precursor cells, their actions in ES cells are not clear. This study was designated to examine the effect of brain-derived neurotrophic factor (BDNF) on neural differentiation in mouse ES cells. Undifferentiated mouse ES cells were maintained at suspension culture in the presence of leukemia inhibitory factor (LIF), and neural precursor cells were isolated from the subventricular zone (SVZ) of the 2–3 day old mice brain. Oct-4 was expressed in undifferentiated ES cells and neural precursor cells, of which expression was markedly decreased by induction of differentiation. RT-PCR analysis showed that neural differentiation of ES cells induced expression of various neural markers, and that BDNF increased their expression. Western blot analysis demonstrated that BDNF induced expression of synaptophysin, a neuronal marker, and inhibited expression of glial fibrillary acidic protein, a glial marker. Undifferentiated ES cells expressed TrkA, TrkB and TrkC, of which expressions were increased during differentiation. Neural differentiation increased their expression, and BDNF further increased them. The treatment of BDNF during differentiation decreased expression of Notch1. These results indicated that BDNF induces neural differentiation and inhibits glial differentiation in ES cells as well as neuronal precursor cells.

cis-platinum(II) (cis-diammine dichloroplatinum; cisplatin) is a potent antitumor compound that is widely used for the treatment of many malignancies. An important side-effect of cisplatin is nephrotoxicity, which results from injury to renal tubular epithelial cells and can be manifested as either acute renal failure or a chronic syndrome characterized by renal electrolyte wasting. Recently, apoptosis has been recognized as an important mechanism of cell death mediating the antitumor effect of cisplatin. This study was undertaken to examine the mechanisms of cell death induced by cisplatin in M-1 cells, which were derived from the outer cortical collecting duct cells of SV40 transgenic mice. Treatment of M-1 cells with high concentrations of cisplatin (0.5 and 1 mM) for 2 hr led to necrotic cell death, whereas a 24-hr treatment with 5-20 microM cisplatin led to apoptosis. Antioxidants protected against cisplatin-induced necrosis, but not apoptosis, indicating that reactive oxygen species play a role in mediating necrosis but not apoptosis induced by cisplatin and that the mechanism of cell death induced by cisplatin is concentration dependent. The low concentrations of cisplatin, which induced apoptosis in M-1 cells, did not affect the expression levels of Bcl-2-related proteins and did not activate c-Jun NH2-terminal kinase (SAPK/JNK). Cisplatin induced the translocation of endogenous Bax from the cytosolic to the membrane fractions and, subsequently, the release of cytochrome c. Overexpression of Bcl-2 blocked cisplatin-induced apoptosis and Bax translocation. These observations suggest that the subcellular redistribution of Bax is a critical event in the apoptosis induced by cisplatin.

Our recent study showed that human mesenchymal stem/stromal cells (hMSCs) are activated to express tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) by exposure to TNF-α and these activated hMSCs effectively induce apoptosis in triple-negative breast cancer MDA-MB-231 (MDA) cells in vitro and in vivo. Here, we further demonstrated that activated hMSCs not only induced apoptosis of MDA cells but also reduced metastatic features in MDA cells. These activated hMSC-exposed MDA cells showed reduced tumorigenicity and suppressed formation of lung metastasis when implanted in the mammary fat pad. Surprisingly, the activated hMSC-exposed MDA cells increased TRAIL expression, resulting in apoptosis in MDA cells. Interestingly, upregulation of TRAIL in MDA cells was mediated by interferon-beta (IFN-β) secreted from activated hMSCs. Furthermore, IFN-β in activated hMSCs was induced by RNA and DNA released from apoptotic MDA cells in absent in melanoma 2 (AIM2) and IFN induced with helicase C domain 1 (IFIH1)-dependent manners. These observations were only seen in the TRAIL-sensitive breast cancer cell lines but not in the TRAIL-resistant breast cancer cell lines. Consistent with these results, Kaplan–Meier survival analysis also showed that lack of innate sensors detecting DNA or RNA is strongly associated with poor survival in estrogen receptor-negative breast cancer patients. In addition, cancer-associated fibroblasts (CAF) isolated from a breast cancer patient were also able to express TRAIL and IFN-β upon DNA and RNA stimulation. Therefore, our results suggest that the crosstalk between TRAIL-sensitive cancer cells and stromal cells creates a tumor-suppressive microenvironment and further provide a novel therapeutic approach to target stromal cells within cancer microenvironment for TRAIL sensitive cancer treatment.

Mesenchymal stem/progenitor cells (MSCs) were reported to enhance the survival of cellular and organ transplants. However, their mode of action was not established. We here used a mouse model of corneal allotransplantation and demonstrated that peri-transplant intravenous (i.v.) infusion of human MSCs (hMSCs) decreased the early surgically induced inflammation and reduced the activation of antigen-presenting cells (APCs) in the cornea and draining lymph nodes (DLNs). Subsequently, immune rejection was decreased, and allograft survival was prolonged. Quantitative assays for human GAPDH revealed that

Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).

Sterile inflammation underlies many diseases of the cornea including serious chemical burns and the common dry eye syndrome. In search for therapeutic targets for corneal inflammation, we defined the kinetics of neutrophil infiltration in a model of sterile injury to the cornea and identified molecular and cellular mechanisms triggering inflammatory responses. Neutrophil infiltration occurred in two phases: a small initial phase (Phase I) that began within 15 min after injury, and a larger second phase (Phase II) that peaked at 24–48 h. Temporal analysis suggested that the neuropeptide secretoneurin initiated Phase I without involvement of resident macrophages. Phase II was initiated by the small heat shock protein HSPB4 that was released from injured keratocytes and that activated resident macrophages via the TLR2/NF-κB pathway. The Phase II inflammation was responsible for vision-threatening opacity and was markedly suppressed by different means of inhibition of the HSPB4/TLR2/NF-κB axis: in mice lacking HSPB4 or TLR2, by antibodies to HSPB4 or by TNF-α stimulated gene/protein 6 that CD44-dependently inhibits the TLR2/NF-κB pathway. Therefore, our data identified the HSPB4/TLR2/NF-κB axis in macrophages as an effective target for therapy of corneal inflammation.

Previous reports demonstrated that the deleterious effects of chemical injury to the cornea were ameliorated by local or systemic administration of adult stem/progenitor cells from bone marrow referred to as mesenchymal stem or stromal cells (MSCs). However, the mechanisms for the beneficial effects of MSCs on the injured cornea were not clarified. Herein, we demonstrated that human MSCs (hMSCs) were effective in reducing corneal opacity and inflammation without engraftment after either intraperitoneal (i.p.) or intravenous (i.v.) administration following chemical injury to the rat cornea. A quantitative assay for human mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) demonstrated that less than 10 hMSCs were present in the corneas of rats 1-day and 3 days after i.v. or i.p. administration of 1 × 107 hMSCs. In vitro experiments using a transwell coculture system demonstrated that chemical injury to corneal epithelial cells activated hMSCs to secrete the multipotent anti-inflammatory protein TNF-α stimulated gene/protein 6 (TSG-6). In vivo, the effects of i.v. injection of hMSCs were largely abrogated by knockdown of TSG-6. Also, the effects of hMSCs were essentially duplicated by either i.v. or topical administration of TSG-6. Therefore, the results demonstrated that systemically administered hMSCs reduce inflammatory damage to the cornea without engraftment and primarily by secretion of the anti-inflammatory protein TSG-6 in response to injury signals from the cornea.

Human mesenchymal stem/progenitor cells (hMSCs) repair tissues and modulate immune systems but the mechanisms are not fully understood. We demonstrated that hMSCs are activated by inflammatory signals to secrete the anti-inflammatory protein, TNF-α–stimulated gene 6 protein (TSG-6) and thereby create a negative feedback loop that reduces inflammation in zymosan-induced peritonitis. The results demonstrate for the first time that TSG-6 interacts through the CD44 receptor on resident macrophages to decrease zymosan/TLR2-mediated nuclear translocation of the NF-κB. The negative feedback loop created by MSCs through TSG-6 attenuates the inflammatory cascade that is initiated by resident macrophages and then amplified by mesothelial cells and probably other cells of the peritoneum. Because inflammation underlies many pathologic processes, including immune responses, the results may explain the beneficial effects of MSCs and TSG-6 in several disease models.

Systemic administration of MSCs resulted in remarkable functional improvements in injured tissues without either long-term engraftment or differentiation in many clinical and experimental situations. Emerging evidence suggest that most of the beneficial effects of MSCs could be explained by secretion of soluble factors that have multiple effects including modulation of inflammatory and immune reactions, protection from cell death, and stimulation of endogenous progenitor cells. In this review, we focus on the therapeutic factors that account for the beneficial effects of MSCs in animal models of human diseases.

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α–stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.

Improved assays for human DNA and mRNA were used to examine the paradox that intravenously infused human multipotent stromal cells (hMSCs) enhance tissue repair with limited engraftment. After 106 ...

Recent data have implicated nuclear factor-ĸB (NF-ĸB) and Bcl-2 in the regulation of apoptotic and necrotic cell death in various cells. However, mechanisms of their effects on cell death of renal epithelial cells are not clear. First, we investigated the effect of specific inhibition of NF-ĸB and overexpression of Bcl-2 on necrotic cell death induced by hydrogen peroxide or cisplatin in renal collecting duct cells. M-1 cells, which were derived from outer cortical collecting duct, were stably transfected with the non-phosphorylatable mutant of inhibitory-ĸBα (I-ĸBα) and Bcl-2. Overexpression of I-ĸBα and Bcl-2 did not affect cisplatin-induced necrotic cell death, but overexpression of I-ĸBα significantly decreased H2O2-induced cell death. Regarding apoptotic cell death induced by cisplatin, serum deprivation and contact inhibition was increased by overexpression of I-ĸBα, whereas overexpression of bcl-2 inhibited the apoptotic cell death. I-ĸBα overexpression increased Bax expression and decreased cIAP-1 and -2 expression compared to vector-transfected cells, but did not alter SAPK/JNK activity in the presence or absence of cisplatin. NF-ĸB activity was significantly higher in bcl-2-overexpressing cells than in control cells. These data show that activation of NF-ĸB mediates H2O2-induced necrotic injury, but inhibits apoptotic cell death in renal collecting duct cells, and that Bcl-2 selectively protects apoptotic cell death in M-1 cells.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.

Purpose To investigate the effect of an anti-inflammatory protein, TNF-α stimulated gene/protein (TSG)-6 and an antiapoptotic protein, stanniocalcin (STC)-1 on corneal endothelium in rabbits with transcorneal cryoinjury. Methods Transcorneal freezing (-80°C) was applied to rabbit corneas for 30 seconds. Immediately post injury, either TSG-6 (10 μg/100 μL), STC-1 (10 μg/100 μL), or the same volume of balanced salt solution (BSS) was injected into the anterior chamber. Each eye was examined for corneal opacity, corneal thickness, endothelial cell density, and endothelial hexagonality every 2 to 6 hours for 48 hours post injury. The concentrations of myeloperoxidase (MPO) and IL-1β were measured in the aqueous humor every 6 hours. At 48 hours post injury, each cornea was assayed for TNF-α, IL-1β, IL-6, and MPO, and histologically evaluated with alizarin red-trypan blue staining, hematoxylin-eosin staining, and immunostaining for neutrophils. Results Tumor necrosis factor-α stimulated gene/protein-6 significantly decreased the development of corneal opacity and edema after cryoinjury compared with STC-1 or BSS. The corneal endothelial cell density and hexagonality were markedly preserved by TSG-6. The mRNA levels of TNF-α, IL-1β, and IL-6 in the cornea and the protein levels of MPO and IL-1β in the aqueous humor and cornea were significantly lower in TSG-6-treated eyes than BSS-treated controls. Similarly, the expression of fibroblast growth factor-2 was reduced by TSG-6 treatment. Histologic evaluation demonstrated that neutrophil infiltration of the cornea was decreased in TSG-6-treated eyes. Conclusions Tumor necrosis factor-α stimulated gene/protein-6 protected corneal endothelial cells from transcorneal cryoinjury through suppression of inflammation.