[Show abstract][Hide abstract] ABSTRACT: Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.
Stem cell International 12/2012; 2012:392050. DOI:10.1155/2012/392050 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fibre bioreactor devices in which the cells, separated by a semipermeable polyethersulphone (PES) membrane, can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibres. The cells also preserved their immunophenotype under physiological fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per-cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow-fibre bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.
Journal of Tissue Engineering and Regenerative Medicine 05/2012; 6(5):369-77. DOI:10.1002/term.439 · 5.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin-18 (IL-18) is a potent proinflammatory cytokine that augments both innate and acquired immune responses. It is also a crucial regulator of lymphocyte production of interferon-γ (IFN-γ), which can promote acute cellular rejection of transplanted solid organs.
To evaluate the role of IL-18 in liver transplantation, we constructed an adenoviral vector encoding IL-18 binding protein (Adex-IL18bp), which specifically suppressed the biologic activity of IL-18, and examined the effect of this suppression on liver allografts by using a high-responder rat model (ACI to Lewis) of orthotopic liver transplantation (OLTx). Donor rats were given one intravenous injection of Adex-IL18bp or Adex-LacZ (control vector) 2 d before OLTx.
Seven days after OLTx, overexpression of IL-18bp resulting from the adenovirus gene transfer was associated with significantly decreased serum alanine aminotransferase levels and less histologic hepatic injury in recipient rats with Adex-IL18bp-pretreated donors compared with Adex-LacZ controls. Adex-IL18bp pretreatment also significantly prolonged rat/allograft survival, inhibited expression of IFN-γ, and reduced levels (versus control values) of both CXCL10 and CX3CL1, which can be induced by IFN-γ.
These results suggest that IL-18 has an important role in liver allograft rejection through IFN-γ and chemokines and that specific suppression of IL-18 may improve liver function early after transplantation.
Journal of Surgical Research 08/2011; 176(1):293-300. DOI:10.1016/j.jss.2011.07.053 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells represent an important barrier between the intravascular compartment and extravascular tissues, and therefore serve as key sensors, communicators, and amplifiers of danger signals in innate immunity and inflammation. Double stranded DNA (dsDNA) released from damaged host cells during injury or introduced by pathogens during infection, has emerged as a potent danger signal. While the dsDNA-mediated immune response has been extensively studied in immune cells, little is known about the direct and indirect effects of dsDNA on the vascular endothelium. In this study we show that direct dsDNA stimulation of endothelial cells induces a potent proinflammatory response as demonstrated by increased expression of ICAM1, E-selectin and VCAM1, and enhanced leukocyte adhesion. This response was dependent on the stress kinases JNK and p38 MAPK, required the activation of proinflammatory transcription factors NFκB and IRF3, and triggered the robust secretion of TNFα for sustained secondary activation of the endothelium. DNA-induced TNFα secretion proved to be essential in vivo, as mice deficient in the TNF receptor were unable to mount an acute inflammatory response to dsDNA. Our findings suggest that the endothelium plays an active role in mediating dsDNA-induced inflammatory responses, and implicate its importance in establishing an acute inflammatory response to sterile injury or systemic infection, where host or pathogen derived dsDNA may serve as a danger signal.
PLoS ONE 05/2011; 6(5):e19910. DOI:10.1371/journal.pone.0019910 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Excessive systemic inflammation following trauma, sepsis, or burn could lead to distant organ damage. The transplantation of bone marrow stromal cells or mesenchymal stem cells (MSCs) has been reported to be an effective treatment for several immune disorders by modulating the inflammatory response to injury. We hypothesized that MSCs can dynamically secrete systemic factors that can neutralize the activity of inflammatory cytokines. In this study, we showed that cocultured MSCs are able to decrease nuclear factor κ-B (NFκB) activation in target epithelial cells incubated in inflammatory serum conditions. Proteomic screening revealed a responsive secretion of soluble tumor necrosis factor (TNF) receptor 1 (sTNFR1) when MSCs were exposed to lipopolysaccharide (LPS)-stimulated rat serum. The responsive effect was eliminated when NFκB activation was blocked in MSCs. Intramuscular transplantation of MSCs in LPS-endotoxic rats decreased a panel of inflammatory cytokines and inflammatory infiltration of macrophages and neutrophils in lung, kidney, and liver when compared to controls. These results suggest that improvements of inflammatory responses in animal models after local transplantation of MSCs are at least, in part, explained by the NFκB-dependent secretion of sTNFR1 by MSCs.
[Show abstract][Hide abstract] ABSTRACT: The current state of the art for linear optimization in Flux Balance Analysis has been limited to single objective functions. Since mammalian systems perform various functions, a multiobjective approach is needed when seeking optimal flux distributions in these systems. In most of the available multiobjective optimization methods, there is a lack of understanding of when to use a particular objective, and how to combine and/or prioritize mutually competing objectives to achieve a truly optimal solution. To address these limitations we developed a soft constraints based linear physical programming-based flux balance analysis (LPPFBA) framework to obtain a multiobjective optimal solutions. The developed framework was first applied to compute a set of multiobjective optimal solutions for various pairs of objectives relevant to hepatocyte function (urea secretion, albumin, NADPH, and glutathione syntheses) in bioartificial liver systems. Next, simultaneous analysis of the optimal solutions for three objectives was carried out. Further, this framework was utilized to obtain true optimal conditions to improve the hepatic functions in a simulated bioartificial liver system. The combined quantitative and visualization framework of LPPFBA is applicable to any large-scale metabolic network system, including those derived by genomic analyses.
[Show abstract][Hide abstract] ABSTRACT: Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.
Nature medicine 07/2010; 16(7):814-20. DOI:10.1038/nm.2170 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury models induced by lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty-eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL-expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.
[Show abstract][Hide abstract] ABSTRACT: Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 microm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.
[Show abstract][Hide abstract] ABSTRACT: Cell-based technologies to support/restore organ function represent one of the most promising avenues in the treatment of acute liver failure (ALF). Recently, mesenchymal stem cells (MSCs) have been reported as a new therapeutic for inflammatory conditions. Here, we demonstrate the efficacy of MSCs, when cocultured with hepatocytes, to provide combination hepatic and antiinflammatory therapy in the setting of ALF. MSCs were shown to have multiple beneficial effects in vitro that were relevant in a therapeutic context, including (1) hepatocellular functional support, (2) secretion of molecules that inhibit hepatocyte apoptosis, and (3) modulation of an acute phase response by hepatocytes cultured in ALF-induced serum. In addition, we show that the MSC secretome is dynamically changed in response to serum exposure from ALF rats. We then conducted a therapeutic trial of liver assist devices (LADs). LADs containing cocultures of MSCs and hepatocytes provided a greater survival benefit compared to other coculture and monocellular control LADs. Treatment with MSC-hepatocyte devices was associated with specific improvements in hepatic functional and histological parameters as well as decreasing inflammatory serum cytokine levels, validating a combined therapeutic effect. Moreover, MSC coculture reduced the overall cell mass of the device by an order of magnitude. These findings demonstrate the importance of nonparenchymal cells in the cellular composition of LADs, and strongly support the integration of MSCs into hepatocyte-coculture-based LADs as a potential destination therapy for ALF.
Tissue Engineering Part A 05/2009; 15(11):3377-88. DOI:10.1089/ten.TEA.2008.0681 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue-engineered models that mimic in vivo tissue organization offer the potential of capturing complex signaling pathways in vitro. In the liver, hepatocytes and endothelial cells are closely associated but separated by the extracellular matrix of the space of Disse. This unique configuration was mimicked by embedding primary hepatocytes in collagen gel and overlaying the matrix with endothelial cells. We demonstrate that during the first few days of culture, the secretion of albumin and fibrinogen was 2-fold higher in cocultures compared to hepatocytes alone. Hepatocyte function in both cultures stabilized to a similar level during the second week, suggesting that endothelial cells can induce the early recovery of hepatocytes after isolation and seeding. Endothelial cell-conditioned medium reproduced the effect of coculture in a dose-dependent fashion, suggesting a role for endothelial cell-derived soluble factors. Endothelial cell-conditioned medium increased mRNA levels of various acute-phase proteins such as albumin, fibrinogen, transferrin, and alpha-macroglobulin in hepatocytes. Surprisingly, the effect of endothelial cell-conditioned medium was not mediated by growth factors or cytokines, or by secreted extracellular matrix, but by the release of the amino acid proline, which mediates endogenous collagen synthesis by hepatocytes. These findings suggest an important role for proline secretion by endothelial cells as a paracrine factor regulating hepatocyte function.
The FASEB Journal 03/2009; 23(7):2288-98. DOI:10.1096/fj.08-114934 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. To better understand the role of heterotypic cell-cell interactions on hepatocyte proliferation in cocultures, a cell patterning technique was used. Polydimethylsiloxane (PDMS) stencils with different circular hole sizes (300 or 1,000 μm in diameter) were made by the procedure modified from our previous report . Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.
 Jae-Sung Park, Cheul H. Cho, Natesh Parashurama, Yawen Li, Franois Berthiaume, Arno Tilles, Mehmet Toner, Martin Yarmush, "Microfabrication-based Modulation of Embryonic Stem Cell Differentiation", Lab on a Chip, 2007 (7): 1018-1028
[Show abstract][Hide abstract] ABSTRACT: In vitro models of liver tissue that mimics the in vivo arrangement of different cell types offer the possibility of capturing complex signaling environment found in vivo. In the liver, hepatocytes and endothelial cells are closely associated in their native environment separated by the extracellular matrix (the space of Disse). This unique configuration was mimicked by embedding primary hepatocytes in collagen gel and overlaying endothelial cells on top of the gel. We found that albumin and fibrinogen secretion was doubled in the co-cultures compared to hepatocytes cultured alone during first week after seeding. However during second week, albumin and fibrinogen secretion profile became similar in co-culture and monoculture indicating the importance of endothelial cells derived factors towards early recovery of hepatocytes after isolation. Endothelial cell-conditioned medium reproduced the effect of endothelial cell co-culture, suggesting a major contribution of soluble factors secreted by endothelial cells. Conditioned medium also increased mRNA levels of various acute phase proteins such as albumin, fibrinogen, transferrin, and -macroglobulin. The effect of endothelial cell-conditioned medium did not occur beyond 4 days of culture if hepatocytes were not embedded in collagen. This underscores the importance of synergism between the architecture of the culture and soluble factor secreted by endothelial cells. Currently work is in progress to identify the soluble factor responsible for enhanced function.
[Show abstract][Hide abstract] ABSTRACT: The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.
Biotechnology and Bioengineering 10/2008; 101(2):345-56. DOI:10.1002/bit.21911 · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell-based tolerogenic therapy is a relatively new approach for the treatment of autoimmune diseases. Mesenchymal stem cells (MSCs) have been shown to be potent immunomodulatory agents in a number of experimental and clinical scenarios; however, their use in various autoimmune diseases is undefined. Herein, we report the efficacy of MSC transplantation in a multiorgan autoimmunity model. Mice with defective peripheral tolerance caused by a deficiency in regulatory T cells were used as a testbed for therapy. After screening multiple target tissues of autoimmune attack, we observed an MSC-specific improvement in the histopathology of the distal ileum of treated mice. We then showed that MSCs can reduce mesenteric lymph node (MLN) cellularity in autoimmune mice during active disease and decrease activated T-cell populations in the MLN. Trafficking studies using enhanced green fluorescent protein (eGFP)-reporter MSCs revealed no appreciable engraftment in the intestine, but it did reveal the presence of eGFP+ cells organized in clusters within the MLN, as well as ancillary nodes. Semiquantitative analysis showed no difference in the number of clusters; however, eGFP+ cells in MLNs compared with ancillary nodes had distinct fibroblastoid morphology and formed a network with neighboring eGFP+ cells. Finally, we show evidence that transplantation of MSCs caused global immunosuppression, as measured by increased CD4+ CD8+ thymocyte production and serum interleukin-10 and decreased serum interferon-gamma. These data implicate the intestine as a new site of MSC tolerance induction and should motivate additional studies evaluating the use of MSCs as a treatment for autoimmune enteropathies.
[Show abstract][Hide abstract] ABSTRACT: Orthotopic liver transplantation is the only proven effective treatment for fulminant hepatic failure (FHF), but its use is limited because of organ donor shortage, associated high costs, and the requirement for lifelong immunosuppression. FHF is usually accompanied by massive hepatocellular death with compensatory liver regeneration that fails to meet the cellular losses. Therefore, therapy aimed at inhibiting cell death and stimulating endogenous repair pathways could offer major benefits in the treatment of FHF. Recent studies have demonstrated that mesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promote tissue repair in models of myocardial infarction, acute kidney failure, and stroke through the action of trophic secreted molecules. In this study, we investigated whether MSC therapy can protect the acutely injured liver and stimulate regeneration. In a D-galactosamine-induced rat model of acute liver injury, we show that systemic infusion of MSC-conditioned medium (MSC-CM) provides a significant survival benefit and prevents the release of liver injury biomarkers. Furthermore, MSC-CM therapy resulted in a 90% reduction of apoptotic hepatocellular death and a three-fold increment in the number of proliferating hepatocytes. This was accompanied by a dramatic increase in the expression levels of 10 genes known to be up-regulated during hepatocyte replication. Direct antiapoptotic and promitotic effects of MSC-CM on hepatocytes were demonstrated using in vitro assays. Conclusion: These data provide the first clear evidence that MSC-CM therapy provides trophic support to the injured liver by inhibiting hepatocellular death and stimulating regeneration, potentially creating new avenues for the treatment of FHF.
[Show abstract][Hide abstract] ABSTRACT: One of the major hurdles of cellular therapies for the treatment of liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm-like cells with 95% purity. Mouse ES cells cultured on top of collagen-sandwiched hepatocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm-like cells. The endoderm-like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte-like cells into a bioartificial liver device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic potential of these cells.
The FASEB Journal 04/2008; 22(3):898-909. DOI:10.1096/fj.06-7764com · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.
[Show abstract][Hide abstract] ABSTRACT: Recently there has been a paradigm shift in what is considered to be the therapeutic promise of mesenchymal stem cells (MSCs)
in diseases of vital organs. Originally, research focused on MSCs as a source of regenerative cells by differentiation of
transplanted cells into lost cell types. It is now clear that trophic modulation of inflammation, cell death, fibrosis, and
tissue repair are the main mechanisms of MSC therapy. Delivery of growth factors, cytokines, and other signaling molecules
secreted by MSCs is often sufficient to obtain the therapeutic effects. In this article, we provide an overview of the current
knowledge on trophic mechanisms of MSC therapy in disease models of vital organs. Important issues regarding the optimal
delivery methods of MSC therapy are discussed and critical gaps in our knowledge hampering experimental progress and clinical
implementation are identified.