Significant expansion of the donor pool achieved by utilizing islets of variable quality in the production of allogeneic "Neo-Islets", 3-D organoids of Mesenchymal Stromal and islet cells, a novel immune-isolating biotherapy for Type I Diabetes.

Novel biotherapies for Type 1 Diabetes that provide a significantly expanded donor pool and that deliver all islet hormones without requiring anti-rejection drugs are urgently needed. Scoring systems have improved islet allotransplantation outcomes, but their use may potentially result in the waste of valuable cells for novel therapies. To address these issues, we created "Neo-Islets" (NIs), islet-sized organoids, by co-culturing in ultralow adhesion flasks culture-expanded islet (ICs) and Mesenchymal Stromal Cells (MSCs) (x 24 hrs, 1:1 ratio). The MSCs exert powerful immune- and cyto-protective, anti-inflammatory, proangiogenic, and other beneficial actions in NIs. The robust in vitro expansion of all islet hormone-producing cells is coupled to their expected progressive de-differentiation mediated by serum-induced cell cycle entry and Epithelial-Mesenchymal Transition (EMT). Re-differentiation in vivo of the ICs and resumption of their physiological functions occurs by reversal of EMT and serum withdrawal-induced exit from the cell cycle. Accordingly, we reported that allogeneic, i.p.-administered NIs engraft in the omentum, increase Treg numbers and reestablish permanent normoglycemia in autoimmune diabetic NOD mice without immunosuppression. Our FDA-guided pilot study (INAD 012-0776) in insulin-dependent pet dogs showed similar responses, and both human- and canine-NIs established normoglycemia in STZ-diabetic NOD/SCID mice even though the utilized islets would be scored as unsuitable for transplantation. The present study further demonstrates that islet gene expression profiles (α, ß, γ, δ) in human "non-clinical grade" islets obtained from diverse, non-diabetic human and canine donors (n = 6 each) closely correlate with population doublings, and the in vivo re-differentiation of endocrine islet cells clearly corresponds with the reestablishment of euglycemia in diabetic mice. Conclusion: human-NIs created from diverse, "non-clinical grade" donors have the potential to greatly expand patient access to this curative therapy of T1DM, facilitated by the efficient in vitro expansion of ICs that can produce ~ 270 therapeutic NI doses per donor for 70 kg recipients.

Intraperitoneal administration of human "Neo-Islets", 3-D organoids of mesenchymal stromal and pancreatic islet cells, normalizes blood glucose levels in streptozotocin-diabetic NOD/SCID mice: Significance for clinical trials.

Globally, individuals with autoimmune Type 1 diabetes mellitus (T1DM) continue to depend for survival on insulin injections. While pancreas and intrahepatic pancreatic islet transplants can produce insulin-independence and ameliorate serious complications, both therapies depend on potentially toxic anti-rejection drugs. Furthermore, the scarcity of pancreas donors and islet transplant failures limit the general availability of such interventions. Recently, fetal and induced Pluripotent Stem Cells have been successfully differentiated to generate insulin producing ß-like cells that generate euglycemia in diabetic mice. However, their clinical use still depends on anti-rejection drugs or immune-isolating encapsulation systems. We reported recently that allogeneic "Neo-Islets" (NI), 3-D organoids of Mesenchymal Stromal and Islet Cells are immune protected and permanently correct autoimmune diabetes in NOD mice by omental engraftment and endocrine cell redifferentiation. This new "endocrine pancreas" delivers islet hormones physiologically into the hepatic portal vein. Furthermore, treatment of insulin-dependent dogs with allogeneic canine NIs (ongoing FDA-approved Pilot Study) consistently improved glycemic control without the need for antirejection drugs. As there remains a critical need for curative therapies of T1DM, we engineered human NIs and tested their ability, after i.p. administration, to reestablish euglycemia in streptozotocin (STZ)-diabetic NOD/SCID mice. This diabetes model reproduces, in part, the clinical situation in which recipients of allogeneic biotherapies must take potent anti-rejection drugs that similarly create a life-long immune-compromised status. The present study demonstrates that human NI therapy (2x10e5/kg bw NIs/mouse) of STZ-diabetic NOD/SCID mice (n = 6), compared to controls (n = 6) significantly improved glycemic control, and most importantly, that a second dose given to the initial group normalized blood glucose levels long-term. Conclusion: Despite the limitations of the utilized diabetic NOD/SCID mouse model, the obtained data show that human NIs are curative, an observation that has high translational relevance and significantly supports the planned conduct of clinical trials with human NIs.

Interim report on the effective intraperitoneal therapy of insulin-dependent diabetes mellitus in pet dogs using "Neo-Islets," aggregates of adipose stem and pancreatic islet cells (INAD 012-776).

We previously reported that allogeneic, intraperitoneally administered "Neo-Islets," composed of cultured pancreatic islet cells co-aggregated with high numbers of immunoprotective and cytoprotective Adipose-derived Stem Cells, reestablished, through omental engraftment, redifferentiation and splenic and omental up-regulation of regulatory T-cells, normoglycemia in autoimmune Type-1 Diabetic Non-Obese Diabetic (NOD) mice without the use of immunosuppressive agents or encapsulation devices. Based on these observations, we are currently testing this Neo-Islet technology in an FDA guided pilot study (INAD 012-776) in insulin-dependent, spontaneously diabetic pet dogs by ultrasound-guided, intraperitoneal administration of 2x10e5 Neo-Islets/kilogram body weight to metabolically controlled (blood glucose, triglycerides, thyroid and adrenal functions) and sedated animals. We report here interim observations on the first 4 canine Neo-Islet-treated, insulin-dependent pet dogs that are now in the early to intermediate-term follow-up phase of the planned 3 year study (> 6 months post treatment). Current results from this translational study indicate that in dogs, Neo-Islets appear to engraft, redifferentiate and physiologically produce insulin, and are rejected by neither auto- nor allo-immune responses, as evidenced by (a) an absent IgG response to the allogeneic cells contained in the administered Neo-Islets, and (b) progressively improved glycemic control that achieves up to a 50% reduction in daily insulin needs paralleled by a statistically significant decrease in serum glucose concentrations. This is accomplished without the use of anti-rejection drugs or encapsulation devices. No adverse or serious adverse events related to the Neo-Islet administration have been observed to date. We conclude that this minimally invasive therapy has significant translational relevance to veterinary and clinical Type 1 diabetes mellitus by achieving complete and at this point partial glycemic control in two species, i.e., diabetic mice and dogs, respectively.

Durable Control of Autoimmune Diabetes in Mice Achieved by Intraperitoneal Transplantation of "Neo-Islets," Three-Dimensional Aggregates of Allogeneic Islet and "Mesenchymal Stem Cells".

Novel interventions that reestablish endogenous insulin secretion and thereby halt progressive end-organ damage and prolong survival of patients with autoimmune Type 1 diabetes mellitus (T1DM) are urgently needed. While this is currently accomplished with allogeneic pancreas or islet transplants, their utility is significantly limited by both the scarcity of organ donors and life-long need for often-toxic antirejection drugs. Coadministering islets with bone marrow-derived mesenchymal stem cells (MSCs) that exert robust immune-modulating, anti-inflammatory, anti-apoptotic, and angiogenic actions, improves intrahepatic islet survival and function. Encapsulation of insulin-producing cells to prevent immune destruction has shown both promise and failures. Recently, stem cell-derived insulin secreting ß-like cells induced euglycemia in diabetic animals, although their clinical use would still require encapsulation or anti-rejection drugs. Instead of focusing on further improvements in islet transplantation, we demonstrate here that the intraperitoneal administration of islet-sized "Neo-Islets" (NIs), generated by in vitro coaggregation of allogeneic, culture-expanded islet cells with high numbers of immuno-protective and cyto-protective MSCs, resulted in their omental engraftment in immune-competent, spontaneously diabetic nonobese diabetic (NOD) mice. This achieved long-term glycemic control without immunosuppression and without hypoglycemia. In preparation for an Food and Drug Administration-approved clinical trial in dogs with T1DM, we show that treatment of streptozotocin-diabetic NOD/severe combined immunodeficiency mice with identically formed canine NIs produced durable euglycemia, exclusively mediated by dog-specific insulin. We conclude that this novel technology has significant translational relevance for canine and potentially clinical T1DM as it effectively addresses both the organ donor scarcity (>80 therapeutic NI doses/donor pancreas can be generated) and completely eliminates the need for immunosuppression. Stem Cells Translational Medicine 2017;6:1631-1643.

Carbamylated Erythropoietin Outperforms Erythropoietin in the Treatment of AKI-on-CKD and Other AKI Models.

Erythropoietin (EPO) may be a beneficial tissue-protective cytokine. However, high doses of EPO are associate with adverse effects, including thrombosis, tumor growth, and hypertension. Carbamylated erythropoietin (CEPO) lacks both erythropoietic and vasoconstrictive actions. In this study, we compared the renoprotective, hemodynamic, and hematologic activities and survival effects of identical EPO and CEPO doses in rat models of clinically relevant AKI presentations, including ischemia-reperfusion-induced AKI superimposed on CKD (5000 U/kg EPO or CEPO; three subcutaneous injections) and ischemia-reperfusion-induced AKI in old versus young animals and male versus female animals (1000 U/kg EPO or CEPO; three subcutaneous injections). Compared with EPO therapy, CEPO therapy induced greater improvements in renal function and body weight in AKI on CKD animals, with smaller increases in hematocrit levels and similarly improved survival. Compared with EPO therapy in the other AKI groups, CEPO therapy induced greater improvements in protection and recovery of renal function and survival, with smaller increases in systolic BP and hematocrit levels. Overall, old or male animals had more severe loss in kidney function and higher mortality rates than young or female animals, respectively. Notably, mRNA and protein expression analyses confirmed the renal expression of the heterodimeric EPO receptor/CD131 complex, which is required for the tissue-protective effects of CEPO signaling. In conclusion, CEPO improves renal function, body and kidney weight, and survival in AKI models without raising hematocrit levels and BP as substantially as EPO. Thus, CEPO therapy may be superior to EPO in improving outcomes in common forms of clinical AKI.

VEGF is a mediator of the renoprotective effects of multipotent marrow stromal cells in acute kidney injury.

Adult stem cell treatment of complex disorders is a promising therapeutic approach and multipotent marrow stromal cells (MSCs) have been shown to be effective in various animal models of diseases. Acute kidney injury (AKI) is a common and serious problem in hospitalized patients and bone marrow derived multipotent MSCs have been shown to be effective in different models of AKI. The mechanism of action of MSCs is complex but involves paracrine actions including growth factor secretion. Knockdown of vascular enthothelial growth factor (VEGF) by siRNA reduced effectiveness of MSCs in the treatment of ischemic AKI in a rat model. Animals treated with MSCs had increased renal microvessel density compared to VEGF knockdown MSC-treated and vehicle-treated animals. These results show that VEGF is an important mediator of the early and late phase of renoprotective action after AKI in the context of stem cell treatment.

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.

Bioluminescence imaging to monitor the in vivo distribution of administered mesenchymal stem cells in acute kidney injury.

Effective and targeted delivery of cells to injured organs is critical to the development of cell therapies. However, currently available in vivo cell tracking methods still lack sufficient sensitivity and specificity. We examined, therefore, whether a highly sensitive and specific bioluminescence method is suitable to noninvasively image the organ distribution of administered mesenchymal stem cells (MSCs) in vivo. MSCs were transfected with a luciferase/neomycin phosphotransferase construct (luc/neo-MSC). Bioluminescence of these cells was measured (charge-coupled device camera) after treatment with luciferin, showing a linear increase of photon emission with rising cell numbers. To track these cells in vivo, groups of mice were injected with 1 x 10(5) luc/neo-MSCs/animal and imaged with bioluminescence imaging at various time points. Injection of cells in the suprarenal aorta showed diffuse distribution of cells in normal animals, whereas distinct localization to the kidneys was observed in mice with ischemia- and reperfusion-induced acute kidney injury (AKI). Intrajugular infusion of MSCs demonstrated predominant accumulation of cells in both lungs. In animals with AKI, detectable cell numbers declined over time, as assessed by bioluminescence imaging and confirmed by PCR, a process that was associated with low apoptosis levels of intrarenally located MSCs. In conclusion, the described bioluminescence technology provides a sensitive and safe tool for the repeated in vivo tracking of infused luc/neo-MSCs in all major organs. This method will be of substantial utility in the preclinical testing and design of cell therapeutic strategies in kidney and other diseases.

Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury.

Acute kidney injury (AKI) is a major clinical problem in which a critical vascular, pathophysiological component is recognized. We demonstrated previously that mesenchymal stem cells (MSC), unlike fibroblasts, are significantly renoprotective after ischemia-reperfusion injury and concluded that this renoprotection is mediated primarily by paracrine mechanisms. In this study, we investigated whether MSC possess vasculoprotective activity that may contribute, at least in part, to an improved outcome after ischemia-reperfusion AKI. MSC-conditioned medium contains VEGF, HGF, and IGF-1 and augments aortic endothelial cell (EC) growth and survival, a response not observed with fibroblast-conditioned medium. MSC and EC share vasculotropic gene expression profiles, as both form capillary tubes in vitro on Matrigel alone or in cooperation without fusion. MSC undergo differentiation into an endothelial-like cell phenotype in culture and develop into vascular structures in vivo. Infused MSC were readily detected in the kidney early after reflow but were only rarely engrafted at 1 wk post-AKI. MSC attached in the renal microvascular circulation significantly decreased apoptosis of adjacent cells. Infusion of MSC immediately after reflow in severe ischemia-reperfusion AKI did not improve renal blood flow, renovascular resistance, or outer cortical blood flow. These data demonstrate that the unique vasculotropic, paracrine actions elicited by MSC play a significant renoprotective role after AKI, further demonstrating that cell therapy has promise as a novel intervention in AKI.

Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities.

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g., the brain) is higher than for treatment of anemia. Notably, a dose-dependent risk of adverse effects has been associated with rhEPO administration, especially in high-risk groups, including polycythemia-hyperviscosity syndrome, hypertension, and vascular thrombosis. Of note, several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity, primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover, we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here, we demonstrate that rhEPO administration in the rat increases systemic blood pressure, reduces regional renal blood flow, and increases platelet counts and procoagulant activities. In contrast, carbamylated rhEPO, a heteromeric receptor-specific ligand that is fully tissue protective, increases renal blood flow, promotes sodium excretion, reduces injury-induced elevation in procoagulant activity, and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands, which appear to elicit fewer adverse effects, may be especially useful in clinical settings for tissue protection.

Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury.

BACKGROUND: Stem cell and leukocyte migration during homeostasis and inflammation is regulated by a number of chemokines. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are important mediators of leukocyte homeostasis. The postischemic kidney has been shown to recruit different leukocyte populations, including bone marrow-derived stem cells. Therefore, we investigated the SDF-1/CXCR4 system in the kidney after ischemic acute renal failure (ARF). METHODS: We used immunohistochemistry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (RT-PCR) to detect SDF-1 and CXCR4 in the normal kidney and the kidney after ischemia/reperfusion (I/R) ARF. Mobilization was assessed by flow cytometry for CD34 and colony assays. RESULTS: We show that SDF-1 is expressed in the normal mouse kidney and tubular cells express CXCR4. SDF-1 expression in the kidney increases after I/R induced ARF and decreases in the bone marrow, thereby reversing the normal gradient between bone marrow and the periphery. This causes mobilization of CD34-positive cells into the circulation and their subsequent homing to the kidney with ARF. In vitro and in vivo chemotaxis of bone marrow cells toward damaged kidney epithelium is reversibly inhibited by anti-CXCR4 antibodies. CONCLUSION: Our data show that renal SDF-1 is a currently unrecognized mediator of homing to and migration of CXCR4 expressing cells in the injured kidney. Because certain cells that express CXCR4 may have renoprotective effects, our results suggest that SDF-1 may be a major signal involved in kidney repair.

Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.

Severe acute renal failure (ARF) remains a common, largely treatment-resistant clinical problem with disturbingly high mortality rates. Therefore, we tested whether administration of multipotent mesenchymal stem cells (MSC) to anesthetized rats with ischemia-reperfusion-induced ARF (40-min bilateral renal pedicle clamping) could improve the outcome through amelioration of inflammatory, vascular, and apoptotic/necrotic manifestations of ischemic kidney injury. Accordingly, intracarotid administration of MSC (approximately 10(6)/animal) either immediately or 24 h after renal ischemia resulted in significantly improved renal function, higher proliferative and lower apoptotic indexes, as well as lower renal injury and unchanged leukocyte infiltration scores. Such renoprotection was not obtained with syngeneic fibroblasts. Using in vivo two-photon laser confocal microscopy, fluorescence-labeled MSC were detected early after injection in glomeruli, and low numbers attached at microvasculature sites. However, within 3 days of administration, none of the administered MSC had differentiated into a tubular or endothelial cell phenotype. At 24 h after injury, expression of proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and inducible nitric oxide synthase was significantly reduced and that of anti-inflammatory IL-10 and bFGF, TGF-alpha, and Bcl-2 was highly upregulated in treated kidneys. We conclude that the early, highly significant renoprotection obtained with MSC is of considerable therapeutic promise for the cell-based management of clinical ARF. The beneficial effects of MSC are primarily mediated via complex paracrine actions and not by their differentiation into target cells, which, as such, appears to be a more protracted response that may become important in late-stage organ repair.

Membrane-bound matrix metalloproteinase-8 on activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant collagenase and serpinase.

Little is known about the cell biology or the biologic roles of polymorphonuclear cell (PMN)-derived matrix metalloproteinase-8 (MMP-8). When activated with proinflammatory mediators, human PMN release only approximately 15-20% of their content of MMP-8 ( approximately 60 ng/10(6) cells) exclusively as latent pro-MMP-8. However, activated PMN incubated on type I collagen are associated with pericellular collagenase activity even when bathed in serum. PMN pericellular collagenase activity is attributable to membrane-bound MMP-8 because: 1) MMP-8 is expressed in an inducible manner in both pro- and active forms on the surface of human PMN; 2) studies of activated PMN from mice genetically deficient in MMP-8 (MMP-8(-/-)) vs wild-type (WT) mice show that membrane-bound MMP-8 accounts for 92% of the MMP-mediated, PMN surface type I collagenase activity; and 3) human membrane-bound MMP-8 on PMN cleaves types I and II collagens, and alpha(1)-proteinase inhibitor, but is substantially resistant to inhibition by tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Binding of MMP-8 to the PMN surface promotes its stability because soluble MMP-8 has t(1/2) = 7.5 h at 37 degrees C, but membrane-bound MMP-8 retains >80% of its activity after incubation at 37 degrees C for 18 h. Studies of MMP-8(-/-) vs WT mice given intratracheal LPS demonstrate that 24 h after intratracheal LPS, MMP-8(-/-) mice have 2-fold greater accumulation of PMN in the alveolar space than WT mice. Thus, MMP-8 has an unexpected, anti-inflammatory role during acute lung injury in mice. TIMP-resistant, active MMP-8 expressed on the surface of activated PMN is likely to be an important form of MMP-8, regulating lung inflammation and collagen turnover in vivo.

Inducible expression of tissue inhibitor of metalloproteinases-resistant matrix metalloproteinase-9 on the cell surface of neutrophils.

Matrix metalloproteinase (MMP)-9 secreted by activated polymorphonuclear neutrophils (PMN) may play roles in mediating lung injury by degrading extracellular matrix proteins. However, the mechanisms by which MMP-9 retains activity in the presence of tissue inhibitors of metalloproteinases (TIMPs) are not known. We show that MMP-9 is also expressed on the cell surface of PMN, and proinflammatory mediators induce up to 10-fold increases in cell surface expression of MMP-9. Stimulated human PMN express active forms of cell surface MMP, which cleave the MMP substrate, McaPLGLDpaAR. Loss-of-function studies employing PMN from mice genetically deficient in MMP-9 (MMP-9-/-) demonstrate that membrane-bound MMP-9 contributes substantially to MMP-mediated surface-bound cleavage of McaPLGLDpaAR (approximately 50%) and gelatin (approximately 70%) by stimulated PMN. Like soluble MMP-9, membrane-bound MMP-9 cleaves McaPLGLDpaAR (Kcat/KM = 82,000 M-1s-1), gelatin, type IV collagen, elastin, and alpha1-proteinase inhibitor. However, in contrast to soluble MMP-9, membrane-bound MMP-9 is substantially resistant to inhibition by TIMPs. The IC50 for inhibition of membrane-bound MMP-9 by TIMP-1 and TIMP-2 are approximately 21-fold and approximately 68-fold higher, respectively, than those for inhibition of soluble MMP-9. The binding of MMP-9 to the plasma membrane of PMN enables it to evade inhibition by TIMPs, and thereby may alter the pericellular proteolytic balance in favor of extracellular matrix degradation. Membrane-bound MMP-9 on PMN may play pathogenetic roles in inflammatory lung diseases.