Combined Treatment of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Endothelial Cells Regenerate the Infarcted Heart in Mice and Non-Human Primates.

BACKGROUND: Remuscularization of the mammalian heart can be achieved after cell transplantation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). However, several hurdles remain before implementation into clinical practice. Poor survival of the implanted cells is related to insufficient vascularization, and the potential for fatal arrhythmogenesis is associated with the fetal cell-like nature of immature CMs. METHODS: We generated 3 lines of hiPSC-derived endothelial cells (ECs) and hiPSC-CMs from 3 independent donors and tested hiPSC-CM sarcomeric length, gap junction protein, and calcium-handling ability in coculture with ECs. Next, we examined the therapeutic effect of the cotransplantation of hiPSC-ECs and hiPSC-CMs in nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice undergoing myocardial infarction (n≥4). Cardiac function was assessed by echocardiography, whereas arrhythmic events were recorded using 3-lead ECGs. We further used healthy non-human primates (n=4) with cell injection to study the cell engraftment, maturation, and integration of transplanted hiPSC-CMs, alone or along with hiPSC-ECs, by histological analysis. Last, we tested the cell therapy in ischemic reperfusion injury in non-human primates (n=4, 3, and 4 for EC+CM, CM, and control, respectively). Cardiac function was evaluated by echocardiography and cardiac MRI, whereas arrhythmic events were monitored by telemetric ECG recorders. Cell engraftment, angiogenesis, and host-graft integration of human grafts were also investigated. RESULTS: We demonstrated that human iPSC-ECs promote the maturity and function of hiPSC-CMs in vitro and in vivo. When cocultured with ECs, CMs showed more mature phenotypes in cellular structure and function. In the mouse model, cotransplantation augmented the EC-accompanied vascularization in the grafts, promoted the maturity of CMs at the infarct area, and improved cardiac function after myocardial infarction. Furthermore, in non-human primates, transplantation of ECs and CMs significantly enhanced graft size and vasculature and improved cardiac function after ischemic reperfusion. CONCLUSIONS: These results demonstrate the synergistic effect of combining iPSC-derived ECs and CMs for therapy in the postmyocardial infarction heart, enabling a promising strategy toward clinical translation.

Helical TomoTherapy Total Lymphoid Irradiation and Hematopoietic Cell Transplantation for Kidney Transplant Tolerance in Rhesus Macaques.

Development of a post-transplant kidney transplant tolerance induction protocol involving a novel total lymphoid irradiation (TLI) conditioning method in a rhesus macaque model is described. We examined the feasibility of acheiving tolerance to MHC 1-haplotype matched kidney transplants by establishing a mixed chimeric state with infusion of donor hematopoietic cells (HC) using TomoTherapy TLI. The chimeric state was hypothesized to permit the elimination of all immunosuppressive (IS) medications while preserving allograft function long-term without development of graft-versus-host-disease (GVHD) or rejection. An experimental group of 11 renal transplant recipients received the tolerance induction protocol and outcomes were compared to a control group (n = 7) that received the same conditioning but without donor HC infusion. Development of mixed chimerism and operational tolerance was accomplished in two recipients in the experimental group. Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD. None of the animals in the control group achieved tolerance when IS was eliminated. This novel experimental model demonstrated the feasibility for inducing of long-term operational tolerance when mixed chimerism is achieved using a TLI post-transplant conditioning protocol in 1-haplotype matched non-human primate recipients of combined kidney and HC transplantation.

Targeted donor complement blockade after brain death prevents delayed graft function in a nonhuman primate model of kidney transplantation.

Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement-driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement-blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43-48 hours prior to implantation into ABO-compatible, nonsensitized, MHC-mismatched recipients. Animals were divided into 3 donor-treatment groups: G1 - vehicle, G2 - rhC1INH+heparin, and G3 - heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b-9, and reduced urinary neutrophil gelatinase-associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor-management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD-induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation.

Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia.

BACKGROUND: Nonhuman primates, particularly rhesus macaques, are ideal preclinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce chimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. METHOD: Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9-10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. RESULTS: The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50 µg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 h after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. CONCLUSION: Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Background: Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. Methods: A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Results: Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. Conclusions: These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Gut butyrate-producers confer post-infarction cardiac protection.

The gut microbiome and its metabolites are increasingly implicated in several cardiovascular diseases, but their role in human myocardial infarction (MI) injury responses have yet to be established. To address this, we examined stool samples from 77 ST-elevation MI (STEMI) patients using 16 S V3-V4 next-generation sequencing, metagenomics and machine learning. Our analysis identified an enriched population of butyrate-producing bacteria. These findings were then validated using a controlled ischemia/reperfusion model using eight nonhuman primates. To elucidate mechanisms, we inoculated gnotobiotic mice with these bacteria and found that they can produce beta-hydroxybutyrate, supporting cardiac function post-MI. This was further confirmed using HMGCS2-deficient mice which lack endogenous ketogenesis and have poor outcomes after MI. Inoculation increased plasma ketone levels and provided significant improvements in cardiac function post-MI. Together, this demonstrates a previously unknown role of gut butyrate-producers in the post-MI response.

Assessment of safety and immunogenicity of MHC homozygous iPSC-derived CD34+ hematopoietic progenitors in an NHP model.

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

Allogeneic MHC-matched T-cell receptor α/ß-depleted bone marrow transplants in SHIV-infected, ART-suppressed Mauritian cynomolgus macaques.

Allogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay. Thus, we infected four MCMs with CCR5-tropic SHIV162P3 and started them on ART 6-16 weeks post-infection (p.i.), maintaining continuous ART during myeloablative conditioning. To prevent graft-versus-host disease (GvHD), we transplanted allogeneic MHC-matched α/ß T cell-depleted bone marrow cells and prophylactically treated the MCMs with cyclophosphamide and tacrolimus. The transplants produced ~ 85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their blood post-transplant. However, SHIV-harboring cells persisted in various tissues, with detectable viral DNA in lymph nodes and tissues between 38 and 62 days post-transplant. Further, removing one MCM from ART at 63 days post-transplant resulted in SHIV rapidly rebounding within 7 days of treatment withdrawal. In conclusion, transplanting SHIV-infected MCMs with allogeneic MHC-matched α/ß T cell-depleted bone marrow cells prevented high-grade GvHD and decreased SHIV-harboring cells in the blood post-transplant but did not eliminate viral reservoirs in tissues.

Complement Blockade in Recipients Prevents Delayed Graft Function and Delays Antibody-mediated Rejection in a Nonhuman Primate Model of Kidney Transplantation.

BACKGROUND: Complement activation in kidney transplantation is implicated in the pathogenesis of delayed graft function (DGF). This study evaluated the therapeutic efficacy of high-dose recombinant human C1 esterase inhibitor (rhC1INH) to prevent DGF in a nonhuman primate model of kidney transplantation after brain death and prolonged cold ischemia. METHODS: Brain death donors underwent 20 h of conventional management. Procured kidneys were stored on ice for 44-48 h, then transplanted into ABO-compatible major histocompatibility complex-mismatched recipients. Recipients were treated with vehicle (n = 5) or rhC1INH 500 U/kg plus heparin 40 U/kg (n = 8) before reperfusion, 12 h, and 24 h posttransplant. Recipients were followed up for 120 d. RESULTS: Of vehicle-treated recipients, 80% (4 of 5) developed DGF versus 12.5% (1 of 8) rhC1INH-treated recipients (P = 0.015). rhC1INH-treated recipients had faster creatinine recovery, superior urinary output, and reduced urinary neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2-insulin-like growth factor-binding protein 7 throughout the first week, indicating reduced allograft injury. Treated recipients presented lower postreperfusion plasma interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, and IL-18, lower day 4 monocyte chemoattractant protein 1, and trended toward lower C5. Treated recipients exhibited less C3b/C5b-9 deposition on day 7 biopsies. rhC1INH-treated animals also trended toward prolonged mediated rejection-free survival. CONCLUSIONS: Our results recommend high-dose C1INH complement blockade in transplant recipients as an effective strategy to reduce kidney injury and inflammation, prevent DGF, delay antibody-mediated rejection development, and improve transplant outcomes.

Transplantation of T-cell receptor α/ß-depleted allogeneic bone marrow in nonhuman primates.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models. Here we evaluated the outcomes of major histocompatibility complex-matched T-cell receptor α/ß-depleted alloHSCT in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/ß depletion, bone marrow cells were transplanted into major histocompatibility complex-identical MCMs conditioned with total body irradiation. GvHD prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Posttransplant chimerism was determined by sequencing diagnostic single-nucleotide polymorphisms to quantify the amounts of donor and recipient cells present in blood. Animals treated posttransplant with sirolimus developed nearly complete chimerism with acute GvHD. In the cyclophosphamide and tacrolimus treatment group, animals developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. None of the animals developed cytomegalovirus infection. These studies indicate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. Further exploration of this model will provide a platform for elucidating the mechanisms of GvHD and GvHE and for testing novel alloHSCT modalities for HIV infection.

Brain Death Enhances Activation of the Innate Immune System and Leads to Reduced Renal Metabolic Gene Expression.

BACKGROUND: Brain death (BD)-associated inflammation has been implicated in decreased kidney allograft function and survival, but the underlying mechanisms have not been well distinguished from the conditions of critical care itself. We have developed a clinically translatable model to separate and investigate strategies to improve donor management and critical care. METHODS: Brain-dead (n = 12) and sham (n = 5) rhesus macaques were maintained for 20 hours under intensive care unit-level conditions. Samples were collected for immunophenotyping, analysis of plasma proteins, coagulation studies, and gene analysis for changes in immune and metabolic profile with comparison to naive samples (n = 10). RESULTS: We observed an increase in circulating leukocytes and cytokines, activation of complement and coagulation pathways, and upregulation of genes associated with inflammation in both brain-dead and sham subjects relative to naïve controls. Sham demonstrated an intermediate phenotype of inflammation compared to BD. Analysis of gene expression in kidneys from BD kidneys revealed a similar upregulation of inflammatory profile in both BD and sham subjects, but BD presented a distinct reduction in metabolic and respiratory processes compared to sham and naïve kidneys. CONCLUSION: BD is associated with activation of specific pathways of the innate immune system and changes to metabolic gene expression in renal tissue itself; however, sham donors presented an intermediate inflammatory response attributable to the critical care environment. The early onset and penetrating impact of this inflammatory response underscores the need for early intervention to prevent perioperative tissue injury to transplantable organs.

Guidelines for the management of a brain death donor in the rhesus macaque: A translational transplant model.

INTRODUCTION: The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature. METHODS: Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed. RESULTS: A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model. CONCLUSION: We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials.