Allogeneic Marrow Stromal Cells Research Articles

Abstract 190: Allogeneic Marrow Stromal Cells Show Differences in Activation After Exposure to Blood From Healthy Subjects versus Stroke Patients

Background: Systemic administration of marrow stromal cells (MSCs) leads to the release of a broad panel of cytokines and growth factors that may stimulate repair after stroke. However, the activation of MSCs may differ depending on the external environment they encounter after systemic injection. We explored the question whether the secretomes of MSCs, once exposed to systemic blood, differs depending on whether blood is from stroke patients versus healthy controls. Methods: Clinical grade bone marrow MSCs from a healthy donor at passage 2 were thawed and re-suspended in PBS. Peripheral blood from healthy subjects (n=4) and stroke patients (n=3) was collected. Blood from stroke patients was collected at 24 hrs after symptom onset. Whole blood or plasma was added to wells. PBS was then added with or without 200,000 MSCs. At various time points, aliquots were collected and probed for cytokines using a MAGPIX reader. Results: Placing MSCs in blood from healthy or stroke patients led to a differential release of cytokines (Fig 1). There were no changes in cytokines levels over time from wells that contained MSCs plus blood derived from healthy controls. However, there were significant increases in IL-8, IL-10 and VEGF levels in wells that contained MSCs plus blood from stroke patients (Fig 1). The increase in TNF-α in wells that contained blood alone from stroke patients was reduced by the addition of MSCs. In wells that contained MSCs plus plasma derived from stroke patients, there were very similar cytokine changes compared with wells that contained MSCs plus plasma derived from healthy controls (data not shown). Conclusion: The components of circulatory blood in stroke patients may be important to activate MSCs to release soluble factors that could modify brain recovery after stroke. Upon systemic administration, the exposure of MSCs to the microenvironment of blood might lead to different activation patterns depending on the health status of the individual.

Allogeneic Mesenchymal Stem Cells for Treatment of Experimental Autoimmune Encephalomyelitis

The use of allogeneic "universal donor" mesenchymal stromal cells (MSCs) may be a substantial clinical convenience for treatment of autoimmune ailments such as multiple sclerosis. We therefore tested whether allogeneic MSCs can be exploited for treatment of experimental autoimmune encephalomyelitis (EAE) in mice with otherwise intact immune system. Administration of allogeneic Balb/c-derived MSCs to C57Bl/6 mice with pre-established EAE led to a significant decrease in disease score over time comparable to that achieved with syngeneic MSCs, and was correlated with a significant blunting of immune cell infiltration to the spinal cord and reduced circulating levels of interferon-gamma (IFN-gamma) and interleukin-17. Pretreatment of allogeneic MSCs with IFN-gamma increased the expression levels of CCL2 as well as major histocompatibility complex I (MHCI) and MHCII, but also led to complete loss of suppressive activity in vivo that correlated with immune rejection. In conclusion, allogeneic MSCs can suppress the manifestations of EAE, yet retain the potential for alloimmunization.

Autologous and Allogeneic Marrow Stromal Cells Are Safe and Effective for the Treatment of Acute Kidney Injury

Acute kidney injury (AKI) is a major clinical problem associated with high morbidity and mortality. Likely due to its complex pathophysiology, therapies with a single pharmacological agent have generally failed to improve outcomes. In contrast, stem cell-based interventions utilize these cells' ability to simultaneously target multiple pathophysiological components of AKI and thus represent a promising new tool for the treatment of AKI. The aims of the this study were to investigate the long-term outcome and safety of treatment with autologous and allogeneic mesenchymal stem cells (MSCs) after AKI and the role of vascular endothelial growth factor (VEGF) as one of the principal paracrine mediators of renoprotection of MSCs. MSC administration after AKI was not associated with adverse events and proved to be renoprotective in animals with severe renal failure. Identical doses of autologous MSC were more effective than allogeneic. At 3 months, MSCs were not engrafted in any tissues except in the bone marrow in 50% of animals given the highest allogeneic cell dose. There was no long-term fibrotic response in the kidneys attributable to MSC therapy, and animals with severe AKI were protected from development of fibrotic lesions after AKI. Furthermore, this study establishes VEGF as a critical factor mediating renal recovery. VEGF knockdown by small-interfering RNA reduced effectiveness of MSCs significantly and decreased survival. In summary, our results show that both autologous and allogeneic MSC are safe and effective in AKI, and importantly, reduce late renal fibrosis and loss of renal function in surviving animals and that VEGF is a critical factor in renoprotection by MSCs. Together, we posit that these data provide further justification for the conduct of clinical trails in which AKI is treated with MSC.

Treatment of acute renal failure with allogeneic marrow stromal cells: short and long term effects in experimental models and clinical trials

The Medical ProblemThe acute loss of kidney function (Acute Renal Failure caused by Acute Kidney Injury) due to shock, trauma, major cardiac surgery, toxic drugs and agents, results, if severe, in a patient mortality of > 50%. This unacceptable outcome has remained unchanged over the last 30 years, despite the availability of kidney replacement therapy (dialysis) and treatment in an Intensive Care Unit setting. This continued treatment resistance of AKI makes it urgent that new, effective treatment modalities are developed for this major medical problem.Pathophysiology of AKIAlthough the kidney has a robust capacity to repair itself after an acute insult, once its intrinsic regenerative capacity is exceeded, uremic, systemic complications develop and cause high levels of morbidity and mortality. In AKI, vascular and epithelial cells are injured, become dysfunctional, undergo apoptosis and necrosis, all associated with a major inflammatory component. These processes result in the abrupt loss of excretory and other functions of the kidney, functions that are only incompletely replaced by dialysis. Successful repair of the injured kidney and thus return of function depends directly on the survival of sublethally injured vascular and epithelial cells, and replacement of cells that have been permanently lost. Multiple pharmacological interventions that have been tested clinically have to date not succeeded to significantly improve patient survival and overall outcome.Pre‐Clinical DataExtensive work from our laboratory and other investigators has shown that bone marrow‐derived Multipotent Stromal Cells, also termed Mesenchymal Stem Cells (MSC) are largely immune privileged and can thus be used in allogeneic protocols, i.e., tissue typing or blood group matching is not required for their safe use in animals and patients. We demonstrated that the administration of allogeneic MSC to animals with severe ischemia/reperfusion‐induced AKI results in powerful protection of renal function, stimulated regeneration and excellent animal survival. Administered MSC are recruited to the injured kidney by specific homing signals that the organ elaborates. In the microvasculature of the kidney, MSC attach for 1‐3 days, respond to cues that are present in the injured microenvironment through complex paracrine mechanisms. These include anti‐inflammatory, anti‐apoptotic, immune modulating, anti‐thrombotic, anti‐fibrotic, angiogenic and mitogenic actions. Unexpectedly, MSC do not differentiate into renal target cells, and thus do not replace cells that have been lost. Importantly, early treatment of AKI with MSC results in well preserved renal function late after injury. This effect may be of major benefit clinically as well.Clinical TrialsMSC therapy in humans is being tested in patients with Graft versus Host Disease, Crohn's disease, osteogenesis imperfecta, ischemic heart disease and others. Our group is currently conducting a Phase I Clinical Trial in which allogeneic MSC are infused into patients who are at high risk for cardiac surgery‐induced AKI. So far, no safety issues have been encountered. We will conduct a second clinical trial in Europe, in which kidney transplant‐associated AKI will be treated with allogeneic MSC.

Correcting metabolic deficits with allogeneic bone marrow stem cells in the cat

We investigated the potential of allogeneic lipoprotein lipase (LpL) +/+ expressing bone marrow stromal cells (MSCs) to correct the metabolic deficiency in LpL‐/‐ cats. Bone marrow was harvested from LpL+/+ cats and fifty million MSCs injected into LpL‐/‐ cats twice using two week intervals. Plasma turbidity, triglycerides, cholesterol and lipase activity were monitored prior to and following the injection of the MSCs. Injection of MSCs resulted in a decrease in the plasma lipid profile three to five days following administration. Following the first injection, there was a modest decrease in plasma turbidity and lipids that correlated with a slight increase in LpL activity. The second injection resulted in a much larger increase in LpL activity and a greater decrease in plasma turbidity and lipids. Injection of allogeneic MSCs corrects the LpL‐/‐ genetic defect and transiently returns the plasma lipid profile to near normal in a dose dependent manner. The enhanced response following the second injection suggests an accumulation of LpL+/+ MSCs might occur. Using this dose and injection protocol, the bi‐phasic duration of therapeutic action appears to last approximately three months. This is a very powerful and robust animal model for studying cytokinetics and cytotherapeutics.This research was support by the Center for Companion Animal Research at U C Davis and the Winn Feline Research Foundation.

Marrow Stromal Cells as Universal Donor Cells for Myocardial Regenerative Therapy: Their Unique Immune Tolerance

Recently rodent and porcine bone marrow stromal cells (MSCs) have been reported to be uniquely immune tolerant. To confirm these findings in human cells, we tested whether human MSCs are also immune tolerant, such that they can be used as universal donor cells for myocardial regenerative therapy. Immunocompetent female rats underwent coronary ligations (n = 90). In group I, lacZ-labeled male human MSCs were implanted into the peri-infarcted area. In groups II, III, and IV, isogeneic rat MSCs, culture medium, or human fibroblasts were injected, respectively. Echocardiography was carried out to assess cardiac function, and the specimens were examined serially for up to 8 weeks with immunohistochemistry, fluorescent in situ hybridization, and polymerase chain reaction to examine MSCs survival and differentiation. Human MSCs survived within the rat myocardium for more than 8 weeks without immunosuppression. Furthermore, the implanted MSCs significantly contributed to the improvement in ventricular function and attenuated left ventricular remodeling. No cellular infiltration characteristic of immune rejection was noted in contrast to group IV. Human MSCs survived within this xenogeneic environment, and contributed to the improvement in cardiac function. Our findings support the feasibility of using these cells as universal donor cells for xenogeneic or allogeneic cell therapy, as they can be prepared and stored well in advance for urgent use. Allogeneic MSCs from healthy donors may be particularly useful for severely ill or elderly patients whose own MSCs could be dysfunctional.

Allogeneic marrow stromal cells are immune rejected by MHC class I– and class II–mismatched recipient mice

It has been suggested that marrow stromal cells (MSCs) may be immunoprivileged and can engraft in allogeneic recipients with intact immune systems. We determined if the implantation of murine MSCs engineered to release erythropoietin (Epo) would be feasible in major histocompatibility complex (MHC)-mismatched allogeneic mice without immunosuppression, and we monitored hematocrit (Hct) as a reporter of MSC graft survival. MSCs from C57Bl/6 mice were engineered to release murine Epo (Epo+ MSCs) and implanted subcutaneously in either syngeneic C57Bl/6 mice or MHC-mismatched Balb/c mice. In syngeneic recipients, the Hct rapidly rose from baseline level and remained higher than .88 (88%) for more than 200 days. However, in MHC-mismatched recipient Balb/c mice, the Hct rose transiently and rapidly declined to baseline values. Repeat implantations in these same mice were associated with an acquired refractoriness in the Hct response consistent with alloimmunization to donor Epo+ MSCs. Allogeneic MSC implants had an increased proportion of host-derived lymphoid CD8+, natural killer T (NKT), and NK infiltrating cells compared with syngeneic controls, and splenocytes isolated from Balb/c mice that had received implants also displayed a significant interferon-gamma (IFNgamma) response to C57Bl/6 MSCs in vitro. These results strongly suggest that MSCs are not intrinsically immunoprivileged and cannot serve as a "universal donor" in immunocompetent MHC-mismatched recipients.

Analysis of Allogeneic and Syngeneic Bone Marrow Stromal Cell Graft Survival in the Spinal Cord

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.

Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.

Marrow stromal cells (MSC) can differentiate into multiple mesenchymal tissues. To assess the feasibility of human MSC transplantation, we evaluated the in vitro immunogenicity of MSC and their ability to function as alloantigen presenting cells (APC). Human MSC were derived and used in mixed cell cultures with allogeneic peripheral blood mononuclear cells (PBMC). Expression of immunoregulatory molecules on MSC was analyzed by flow cytometry. An MSC-associated suppressive activity was analyzed using cell-proliferation assays and enzyme-linked immunoassays. MSC failed to elicit a proliferative response when cocultured with allogeneic PBMC, despite provision of a costimulatory signal delivered by an anti-CD28 antibody and pretreatment of MSC with gamma-interferon. MSC express major histocompatibility complex (MHC) class I and lymphocyte function-associated antigen (LFA)-3 antigens constitutively and MHC class II and intercellular adhesion molecule (ICAM)-1 antigens upon gamma-interferon treatment but do not express CD80, CD86, or CD40 costimulatory molecules. MSC actively suppressed proliferation of responder PBMC stimulated by third-party allogeneic PBMC as well as T cells stimulated by anti-CD3 and anti-CD28 antibodies. Separation of MSC and PBMC by a semipermeable membrane did not abrogate the suppression. The suppressive activity could not be accounted for by MSC production of interleukin-10, transforming growth factor-beta1, or prostaglandin E2, nor by tryptophan depletion of the culture medium. Human MSC fail to stimulate allogeneic PBMC or T-cell proliferation in mixed cell cultures. Unlike other nonprofessional APC, this failure of function is not reversed by provision of CD28-mediated costimulation nor gamma-interferon pretreatment. Rather, MSC actively inhibit T-cell proliferation, suggesting that allogeneic MSC transplantation might be accomplished without the need for significant host immunosuppression.