Injection Of Bone Marrow Cells Research Articles

Xenotransplantation of Human Hematopoietic Stem Cells into NBSGW Mice: A Basic Model for Preclinical Development of Gene Therapy Approaches

Background. The gene therapy based on hematopoietic cell xenotransplantation is becoming a powerful and universally applied therapeutic strategy in an ever-expanding range of human diseases. One of the current issues in implementing the techniques of genome modification in hematopoietic stem cells (HSCs) into clinical practice is to assure the quality and safety of gene and cell therapy products for human use. This is achieved by animal model testing at the stage of preclinical studies. With this purpose in view, NBSGW mice seem to be a unique and promising model for human HSC engraftment without pre-conditioning.
 Aim. To test the NBSGW mouse model for human HSC engraftment, to optimize the methods of assessing the state of the animals and monitoring the chimerism level for translational preclinical development of HSC-based products for gene and cell therapy.
 Materials & Methods. The xenograft models of NBSGW mice were generated using the samples of the selected peripheral blood CD34+ HSCs from a healthy donor. Serial transplantation was performed by intravenous injection of bone marrow cells from primary recipients with a high chimerism level. Engraftment efficiency was evaluated by flow cytofluorometry (FCF) and droplet digital PCR (ddPCR). Subpopulation pattern of human cell engraftment was assessed by FCF.
 Results. The tested HSC transplantation regimen is characterized by favorable toxicity profile. In the entire study sample of mice, the FCF analysis showed a long-term engraftment of human cells with a high chimerism level (23.5–93.6 %) in the bone marrow of the animals, also after serial transplantation, which was confirmed by ddPCR. The B-lineage differentiation cells predominated in all tested samples (of peripheral blood, bone marrow, and spleen) from mice after primary and serial transplantation. The ddPCR assay can be used as an additional tool for validating the level of human cell engraftment determined by FCF.
 Conclusion. NBSGW mice present a promising reference model for preclinical development of gene and cell therapy products based on human primary HSCs with a modified genome.

Additional improvement in regional myocardial ischemia after intracardiac injection of bone marrow cells during CABG surgery.

Post-procedure residual ischemia is associated with worse prognosis in patients with coronary artery diasease (CAD). We evaluated whether autologous bone marrow-derived cells (BMC) contribute to additional reduction in regional stress-induced myocardial ischemia (SIMI) in patients undergoing incomplete coronary artery bypass graft surgery (CABG). In a double-blind, randomized, placebo-controlled trial, we enrolled 143 patients (82% men, 58 ± 11 years) with stable CAD and not candidates for complete CABG. They received 100 million BMC (n = 77) or placebo (n = 66) injected into ischemic non-revascularized segments during CABG. The primary outcome was improvement on SIMI quantified as the area at risk in injected segments assessed by cardiovascular magnetic resonance (CMR) 1, 6, and 12 months after CABG. The reduction in global SIMI after CABG was comparable (p = 0.491) in both groups indicating sustained beneficial effects of the surgical procedure over 12 month period. In contrast, we observed additional improvement in regional SIMI in BMC treated group (p = 0.047). Baseline regional SIMI values were comparable [18.5 (16.2-21.0) vs. 18.5 (16.5-20.7)] and reached the lowest values at 1 month [9.74 (8.25; 11.49) vs. 12.69 (10.84; 14.85)] for BMC and placebo groups, respectively. The ischemia's improvement from baseline represented a 50% difference in regional SIMI in favor of the BMC transplanted group at 30 days. We found no differences in clinical and LVEF% between groups during the 12 month follow-up period. The 1 month rate of major adverse cerebral and cardiovascular events (MACCE) (p = 0.34) and all-cause mortality (p = 0.08) did not differ between groups 1 month post intervention. We provided evidence that BMC leads to additional reduction in regional SIMI in chronic ischemic patients when injected in segments not subjected to direct surgical revascularization. This adjuvant therapy deserves further assessment in patients with advanced CAD especially in those with microcirculation dysfunction. https://clinicaltrials.gov/, identifier NCT01727063.

Patient-derived intrafemoral orthotopic xenografts of peripheral blood or bone marrow from acute myeloid and acute lymphoblastic leukemia patients: clinical characterization, methodology, and validation

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are malignant clonal diseases of the hematopoietic system with an unsatisfactory overall prognosis. The main obstacle is the increased resistance of AML and ALL cells to chemotherapy. The development and validation of new therapeutic strategies for acute leukemia require preclinical models that accurately recapitulate the genetic, pathological, and clinical features of acute leukemia. A patient-derived orthotopic xenograft (PDOX) model is established using surgical orthotopic implantation. They closely resemble human tumor progression and microenvironment and are more reliable translational research tools than subcutaneous-transplant models. In this study, we established PDOX models by direct intrafemoral injection of bone marrow and peripheral blood cells from AML and ALL patients, characterized their pathology, cytology, and genetics, and compared the model's characteristics and drug responsiveness with those of the corresponding patients.

Prevention of acute graft-vs.-host disease by targeting glycolysis and mTOR pathways in activated T cells.

Graft-versus-host disease (GvHD) is a common life-threatening complication that can occur following allogeneic hematopoietic stem cell transplantation. This occurs if donor T cells recognize the host as foreign. During acute GvHD (aGVHD), activated T cells utilize glycolysis as the main source of energy generation. Therefore, inhibition of T cell glycolysis is a potential treatment strategy for aGVHD. In the present study, the effects of the combination of the glycolysis inhibitor 3-bromopyruvate (3-BrPA) and the mTOR inhibitor rapamycin (RAPA) on a mode of aGVHD were explored. In vitro mixed lymphocyte culture model was established by using splenocytes from C57BL/6 (H-2b) mice as responder and inactivated splenocytes from BALB/c (H-2d) mice as stimulator. In this model, 3-BrPA treatment (0-100 µmol/l) was found to suppress cell viability, increase cell apoptosis and reduce IFN-γ secretion, in a concentration-dependent manner. 3-BrPA treatment (0-100 µmol/l) was found to suppress cell viability, increase cell apoptosis and reduce IFN-γ secretion, in a concentration-dependent manner. In addition, combined treatment with 3-BrPA (0-100 µmol/l) alongside RAPA (20 µmol/l) exhibited synergistic effects on inhibiting cell viability and IFN-γ production, compared with those following either treatment alone. An aGVHD model was established by injection of bone marrow cells and spleen cells from the donor-C57BL/6(H-2b) mice to the receptor-BALB/c(H-2d) mice which were underwent total body irradiation first. In the aGVHD model, 3-BrPA (10 mg/kg/day), RAPA (2.5 and 5 mg/kg/day) and both in combination (5 and 2.5 mg/kg/day for 3-BrPA and RAPA, respectively) were all found to alleviate the damage caused by aGVHD, in addition to prolonging the survival time of mice with acute GvHD. In particular, the combined 3-BrPA and RAPA treatment resulted in the highest median survival time among all groups tested. In addition, the effects induced by combined 3-BrPA and RAPA treatment were found to be comparable to those in the 5 mg/kg/day RAPA group but superior to the 3-BrPA group with regards to the cumulative survival profile, GvHD score and lung histological score. The 3-BrPA and RAPA combination group also exhibited the lowest IFN-γ levels among all groups. Therefore, the combination of inhibiting both glycolysis and mTOR activity is a promising strategy for acute GvHD prevention.

Influence of PSG peptides on the morphofunctional state of the spleen in allogenous bone marrow cell transplantation

It is known that proteins associated with pregnancy provide immune tolerance during pregnancy. One of them is pregnancy-specific β1-glycoprotein (PSG), which has a pronounced immunomodulatory effect. Tetrapeptide fragments (YECE, YQCE, YVCS, and YACS) have been identified in the primary structure of PSG that have immunopharmacological potential. The aim of the study was to evaluate the effect of short peptide fragments of PSG on the morphological and immunohistochemical state of the spleen and its compartments during allogeneic transplantation of a suspension of red bone marrow cells in an in vivo experiment in Wistar rats. It was found that intraperitoneal injection of bone marrow cells in the dynamics of the experiment (35 days) caused splenic hyperplasia with accumulation of lymphoid cells and macrophages in the functional areas of the organ. Administration of short peptide fragments of PSG against the background of allogeneic bone marrow transplantation promotes activation of cells of the immune system in the spleen toward their proliferation (Ki-67) and differentiation, while reducing the content of macrophages (CD68). Thus, short peptide fragments of PSG stabilize proliferative processes and promote the development of adaptive responses in response to an allogeneic graft.

The Positive Impact of Donor Bone Marrow Cells Transplantation into Immunoprivileged Compartments on the Survival of Vascularized Skin Allografts

Using the vascularized skin allograft (VSA) model, we compared the tolerogenic effects of different allogeneic bone marrow transplantation (BMT) delivery routes into immunoprivileged compartments under a 7-day protocol immunosuppressive therapy. Twenty-eight fully MHC mismatched VSA transplants were performed between ACI (RT1a) donors and Lewis (RT11) recipients in four groups of seven animals each, under a 7-day protocol of alfa/beta TCRmAb/CsA (alpha/beta-TCR monoclonal antibodies/Cyclosporine A therapy). Donor bone marrow cells (BMC) (100 × 106 cells) were injected into three different immunoprivileged compartments: Group 1: Control, without cellular supportive therapy, Group 2: Intracapsular BMT, Group 3: Intragonadal BMT, Group 4: Intrathecal BMT. In Group 2, BMC were transplanted under the kidney capsule. In Group 3, BMC were transplanted into the right testis between tunica albuginea and seminiferous tubules, and in Group 4, cells were injected intrathecally. The assessment included: skin evaluation for signs and grade of rejection and immunohistochemistry for donor cells engraftment into host lymphoid compartments. Donor-specific chimerism for MHC class I (RT1a) antigens and the presence of CD4+/CD25+ T cells were assessed in the peripheral blood of recipients. The most extended allograft survival, 50–78 days, was observed in Group 4 after intrathecal BMT. The T cells CD4+/CD25+ in the peripheral blood were higher after intrathecal BMC injection than other experimental groups at each post-transplant time point. Transplantation of BMC into immunoprivileged compartments delayed rejection of fully mismatched VSA and induction of robust, donor-specific chimerism.

Diabetic Bone Marrow Cell Injection Accelerated Acute Pancreatitis Progression

Acute pancreatitis (AP) is one of the leading causes of hospital admission, 20% of which could progress to the severe type with extensive acinar cell necrosis. Clinical studies have reported that diabetes is an independent risk factor of the incidence of AP and is associated with higher severity than nondiabetic subjects. However, how diabetes participates in AP progression is not well defined. To investigate this question, wild-type (wt) and diabetic db/db mice at the age of 16 weeks were used in the study. AP was induced in wt recipients by 10 injections of 50 μg/kg caerulein with a 1 h interval. One hour after the last caerulein injection, bone marrow cells (BMC) isolated from wt and db/db mice were injected intraperitoneally into the recipients (1 × 107cells/recipient). The recipients with no BMC injection served as controls. Thirteen hours after BMC injection, serum lipase activity was 1.8- and 1.3-folds higher in mice that received db/db BMC, compared with those with no injection and wt BMC injection, respectively (p ≤ 0.02 for both). By H&E staining, the overall severity score was 14.7 for no cell injection and 16.6 for wt BMC injection and increased to 22.6 for db/db BMC injection (p ≤ 0.002 for both). In particular, mice with db/db BMC injection developed more acinar cell necrosis and vacuolization than the other groups (p ≤ 0.03 for both). When sections were stained with an antibody against myeloperoxidase (MPO), the density of MPO+ cells in pancreatitis was 1.9- and 1.6-folds higher than wt BMC and no BMC injection groups, separately (p ≤ 0.02 for both). Quantified by ELISA, db/db BMC produced more IL-6, GM-CSF, and IL-10 compared with wt BMC (p ≤ 0.04 for all). In conclusion, BMC of db/db mice produced more inflammatory cytokines. In response to acinar cell injury, diabetic BMC aggravated the inflammation cascade and acinar cell injury, leading to the progression of acute pancreatitis.

TREM2 promotes natural killer cell development in CD3\u2212CD122+NK1.1+ pNK cells

BackgroundTriggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer’s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells.ResultsHere, we demonstrated for the first time that CD3−CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice.ConclusionsCollectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

Influence of glycodelin on morphofunctional state of secondary immune organs in experimental model of allogeneic transplantation

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with a molecular weight of 42 to 56 kDa) is a marker of reproductive tissue receptivity. Immunoregulatory potential of glycodelin allows us to consider it among key factors that promote maternal immune tolerance to the developing embryo. In general, glycodelin has prospects for use in biomedicine as a biopharmaceutical to treat post-transplant complications. This work aimed to study the effect of glycodelin on morpho-functional state of the secondary organs of immune response in the course of in vivo experiment during allogeneic transplantation of bone marrow cell suspension to Wistar rats. The experiment was performed in white male Wistar rats aged 2-3 months (body mass, 250 g). The animals were kept at the Perm State National Research University animal clinic under GOST 33216-2014 “Rules for working with laboratory rodents and rabbits”. The animals were sacrificed on the 3rd, 7th, 14th, and 21st days according to international rules for working with experimental animals. In the control group, animals (n = 6) were injected once with a suspension of camptothecin-treated bone marrow cells; in experimental group, the animals (n = 12) were injected with a bone marrow cells in combination with glycodelin administered 4 times.We used recombinant glycodelin (MyBioSource, Germany), which was administered to the animals at a concentration corresponding to pregnancy state (0.75 μg/mL). Intraperitoneal injection of bone marrow cells simultaneously with glycodelin over the terms of experiment (21 days) showed that allogeneic transplantation caused hyperplasia of spleen and lymph nodes. In the functional zones of organs, proliferation and differentiation of immune cells developed. Injection of glycodelin at the early stages of experiment (3-7 days) caused an increase in proliferative processes in the organs of both T- and B-dependent immunity. Moreover, there were no signs of inflammation and apoptotic cell death in the organs. Since the 14th day of the experiment, eosinophilic infiltration of the organs was evident, being an indirect positive sign of response to the transplanted cells. By the end of study, differentiation processes dominated over proliferation in the organs. Thus, glycodelin stabilizes proliferative processes and promotes emergence of a new cell generation, thus supporting host response to the transplant. Apparently, glycodelin can participate in development of adaptive mechanism in secondary organs of immune system.

Long Term Results of Stem Cell Therapy in Patients with Peripheral Arterial Disease (PAD)

Peripheral Arterial Disease (PAD) is still a challenging condition affecting millions of people and leading to amputation or death. In patients with non reconstructable disease or failed revascularization, stem cell therapy (SCT) could help in promoting angiogenesis and limb salvage. However, there is limited data about the long-term benefits of SCT in such patients. Aim of this study is to evaluate the long-term survival and clinical outcomes in PAD patients who underwent stem cell therapy. All patients who underwent stem cell therapy using intramuscular injections of autologous bone-marrow mononuclear cells (BM-MNCs) for the treatment of PAD between January 2011 and November 2020 were include in the study. Electronic medical records (EMRs) and Telephonic phone follow up was used to get baseline clinical characteristics and follow up data. Out of 3627 PAD patient, 41 patients underwent stem cell therapy procedure between January 2011 to November 2020. Mean age was 48 years (range, 21 to 75 years) and 37 patients (90.24%) were males. The median follow-up period was 4.66 years (range, 6 months – 9.9 years). Indication for SCT was rest pain, gangrene or non-healing ulcer due to either non reconstructable disease or failed revascularization. All patients were given antiplatelets and statin therapy. Out of 41 PAD patients, 7 patients were lost to follow up. In the remaining, 34 patients, 29 (70.73%) patients were alive, and 5 patients were dead. Overall, 10 (24.39%) patients underwent ipsilateral amputation and 4 patients had persistent claudication pain. In the remaining patients’ ulcers healed and has no residual pain. Amputation free survival was 75.61%. The benefits of Autologous BM-MNCs stem cell therapy are sustained even in the long term with overall survival rate was 85.29% and the amputation free survival was 75.61% and should be considered for all CLI patients irrespective of age.

Analysis of curative effect of percutaneous autologous bone marrow cell transplantation for treating nonunion under laser positioning and navigation guidance

Fracture end cleaning, local bone grafting, and fixation are the classic treatment methods for fracture nonunion, but patients normally need to wait for a long time to ensure that there are signs of further healing. The purpose of this research is to verify whether precision injection improvement can be developed as treatment associated with autologous bone marrow cell injection to promoting fracture healing. The curative effect of traditional autologous bone marrow injection for the treatment of poor fracture healing is affected by injection accuracy and locally available cell retention the clinical. The curative effect of autologous bone marrow injection can be effectively improved through autologous bone marrow centrifugation and bone grinding at fracture end when combined with preoperative three-dimensional computed tomography (CT) planning. This minimally invasive treatment method is especially suitable for patients with delayed fracture healing within six months of presentation. Patients with nonunion over six months can be treated with improved autologous bone marrow injection. The convenient and minimally invasive method itself will not be difficult for patients, physically and mentally, and the most reliable treatment method for nonunion is to cut and clean hardened bone and implant autologous bone fixation.

Abstract 14451: Bone Marrow Cell Therapy Improves Cardiac Function, Survival, and Clinical Outcomes in Patients With Chronic Ischemic Heart Disease

Introduction: The effects of bone marrow cell (BMC) therapy in patients with chronic ischemic heart disease (CIHD) remain controversial. Hypothesis: We hypothesized that injection of BMCs in patients with CIHD would improve left ventricular (LV) structure and function. We also hypothesized that BMC therapy would improve clinical outcomes in CIHD patients. Methods: We performed a systemic review and meta-analysis of pooled data from published randomized controlled trials (RCTs) that evaluated the efficacy of BMC administration in patients with CIHD. The effects of BMC injection on LV ejection fraction (LVEF), LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), infarct size, and patient outcomes were analyzed using random-effects meta-analysis. Results: The literature search yielded 30 RCTs enrolling 1,548 patients. Transplantation of BMCs resulted in an improvement in LVEF in cell-treated patients compared with controls (2.89%; 95% confidence interval (CI): 1.80 to 3.97; P <0.001). There was a trend toward reduced LVESV (-4.96 ml; 95% CI: -11.64 to 1.71 ml; P = 0.14) and LVEDV (-5.95 ml; 95% CI: -12.09 to 0.18 ml; P =0.06). The improvement in LVEDV was more pronounced (-7.42 ml; 95% CI: -13.68 to -1.17ml; P =0.02) in patients with baseline LVEF <40%, indicating improved LV remodeling. BMC injection was also associated with marked reduction in the risk of all-cause mortality, rehospitalization due to heart failure, and ventricular arrhythmias in CIHD patients. Conclusions: BMC injection improves cardiac function and remodeling in patients with CIHD. These benefits are more pronounced in patients with LVEF <40% at baseline. Perhaps more importantly, BMC therapy also improves clinical outcomes that are critically important for this patient population with LV dysfunction, including survival, rehospitalization due to heart failure, and ventricular arrhythmias.

Relationship between cell number and clinical outcomes of autologous bone-marrow mononuclear cell implantation in critical limb ischemia

Cell therapy using intramuscular injections of autologous bone-marrow mononuclear cells (BM-MNCs) improves clinical symptoms and can prevent limb amputation in atherosclerotic peripheral arterial disease (PAD) patients with critical limb ischemia (CLI). The purpose of this study was to evaluate the effects of the number of implanted BM-MNCs on clinical outcomes in atherosclerotic PAD patients with CLI who underwent cell therapy. This study was a retrospective observational study with median follow-up period of 13.5 years (range, 6.8–15.5 years) from BM-MNC implantation procedure. The mean number of implanted cells was 1.2 ± 0.7 × 109 per limb. There was no significant difference in number of BM-MNCs implanted between the no major amputation group and major amputation group (1.1 ± 0.7 × 109 vs. 1.5 ± 0.8 × 109 per limb, P = 0.138). There was also no significant difference in number of BM-MNCs implanted between the no death group and death group (1.5 ± 0.9 × 109 vs. 1.8 ± 0.8 × 109 per patient, P = 0.404). Differences in the number of BM-MNCs (mean number, 1.2 ± 0.7 × 109 per limb) for cell therapy did not alter the major amputation-free survival rate or mortality rate in atherosclerotic PAD patients with CLI. A large number of BM-MNCs will not improve limb salvage outcome or mortality.

Comparison of the regenerative effects of bone marrow/adipose-derived stem cells in the Asherman model following local or systemic administration.

Cell therapy is a promising strategy for the treatment of Asherman's syndrome (AS), but the origin of these cells and injection route influence the therapeutic effect and complications of cell therapy. Herein, we compared the effects of systemic or local intrauterine injection of bone marrow or adipose-derived mesenchymal stem cells (BMSCs/AMSCs) on the endometrium in a rat model of AS. After induction of AS in adult Wistar rats, the CM-Dil-positive BMSCs or AMSCs were injected either locally or intravenously. After 3weeks, endometrial thickness, collagen deposition, cell migration, and VEGF expression were evaluated using histochemistry/immunofluorescence studies. In all stem cell-treated groups, an ameliorative effect on the damaged endometrium was noted. Collagen deposition diminished in both groups (IV and local injection) compared to the AS model. In rats injected locally with MSC, fibrosis decreased compared to the other groups. Moreover, endometrial thickness increased in the groups that received local injection of BMSCs and AMSCs more than the IV-transplanted AMSCs group. Immunofluorescent staining demonstrated that although the systemic transplantation of BMSCs was more effective than the other groups on VEGF expression, it led to the lowest number of CM-Dil+ stem cells in the damaged endometrium. Stem cell transplantation may reconstruct the damaged endometrium, but it is recommended to select the most effective stem cells and injection route. Because the removal of the fibrosis and the replacement of the epithelia cells is an effective therapeutic strategy for AS, in this study, we conclude that the local injection of AMSCs is more appropriate than BMSCs to treat AS.

Characterization of a Novel CD8+ B Cell Subset in Monkeys Tolerant of Heart Allografts

We have previously reported that transient mixed hematopoietic chimerism induced via donor bone marrow transplantation can achieve tolerance to cardiac allografts in non-human primates (NHPs). The purpose of this study was to measure the frequency and properties of a unique subset of CD8+ B cells, recently identified in our laboratory, in tolerant monkeys. Cynomolgus Macaques underwent organ transplantation followed by injection of donor bone marrow cells with mixed chimerism conditioning consisting of 3Gy TBI, 7Gy thymic irradiation, anti-thymocyte globulin, and a short course of anti-CD154 mAb and CyA therapy. The frequencies of different B cell subsets were measured in the peripheral blood of ten monkeys pre- and post-transplantation. In addition, PBMCs were isolated from peripheral blood of two tolerant animals (recipients of heart plus kidney or heart plus thymus cotransplants) 847 and 274 days post-bone marrow and FACS-sorted into five groups: T cells (CD3+), whole B cells (CD20+), mature B cells (CD20+CD8-CD27+), naïve B cells (CD20+CD8-CD27-), and newly identified CD8+ B cells (CD20+CD8+). mRNA was extracted from lysates of each cell population and sequenced using an Illumina NextSeq 500. During the reconstitution phase of the conditioning protocol, CD8+ B cells exhibited a phenotype reminiscent of transitional B cells (CD20+CD27-IgMhiCD38hi) and the percentage of these cells among peripheral B cells was much higher than that observed in naïve animals (23.4% vs 2.5% respectively; p<0.001). In the two tolerant animals, the CD8+ B cells gene expression suggested an immature profile which included the expression of CD21, CD38, and CD24. Interestingly, however, CD8+ B cells showed an increased expression of the gene encoding for the regulatory protein CD72. In addition, CD8+ B cells had greater expression of CD69 which is involved with TGFbeta production. The prevalence of CD8+ B cells expressing genes involved in regulatory immunity after bone marrow transplantation suggests that these cells may play a role in tolerance induction to heart allografts in NHPs. Work is in progress to further characterize these novel cells.

Combined intramuscular and intraspinal transplant of bone marrow cells improves neuromuscular function in the SOD1G93A mice

BackgroundThe simultaneous contribution of several etiopathogenic disturbances makes amyotrophic lateral sclerosis (ALS) a fatal and challenging disease. Here, we studied two different cell therapy protocols to protect both central and peripheral nervous system in a murine model of ALS.MethodsSince ALS begins with a distal axonopathy, in a first assay, we performed injection of bone marrow cells into two hindlimb muscles of transgenic SOD1G93A mice. In a second study, we combined intramuscular and intraspinal injection of bone marrow cells. Fluorescence-activated cell sorting was used to assess the survival of the transplanted cells into the injected tissues. The mice were assessed from 8 to 16 weeks of age by means of locomotion and electrophysiological tests. After follow-up, the spinal cord was processed for analysis of motoneuron survival and glial cell reactivity.ResultsWe found that, after intramuscular injection, bone marrow cells were able to engraft within the muscle. However, bone marrow cell intramuscular injection failed to promote a general therapeutic effect. In the second approach, we found that bone marrow cells had limited survival in the spinal cord, but this strategy significantly improved motor outcomes. Moreover, we also found that the dual cell therapy tended to preserve spinal motoneurons at late stages of the disease and to reduce microgliosis, although this did not prolong mice survival.ConclusionOverall, our findings suggest that targeting more than one affected area of the motor system at once with bone marrow cell therapy results in a valuable therapeutic intervention for ALS.

Effect of intracoronary bone marrow-derived mononuclear cell injection early and late after myocardial infarction on CMR-derived myocardial strain

BackgroundStudies indicate no clear impact of intracoronary injection of bone-marrow unselected mononuclear cells (BM-MNC) after acute myocardial infarction (AMI) on left-ventricular function (LVEF). Strain parameters by cardiovascular magnetic resonance (CMR) have been proposed to be more sensitive to functional changes of the heart. The aim of the present study was to assess changes of global longitudinal (GLS) and circumferential strain (GCS) in a group of patients treated with BM-MNC after AMI. MethodsOne-hundred and forty-nine patients with successfully reperfused AMI and LV dysfunction (LVEF<45%) were retrospectively included into this sub-study of the SWISS-AMI multicentre trial. Patients were divided into control (N = 54), early (5–7 days after AMI, N = 51) and late BM-MNC treatment groups (3–4 weeks, N = 44). The endpoint was the change of GLS and GCS as obtained from cine sequences 4 and 12 months after AMI using feature tracking algorithm. ResultsIn unadjusted analyses, the absolute change of GLS for the early treatment group from baseline to 4 months was 2.5 ± 4.3 (p < 0.01), to 12 months 2.7 ± 5.7% (p = 0.004). For late treatment, it was 1.5 ± 4.0% (p = 0.039, 4 months) and 2.5 ± 5.6% (p = 0.015, 12 months). For controls 0.7 ± 4.7% (p = 0.378), 0.8 ± 3.9% (p = 0.253) respectively. Adjusting for different baseline values, neither an overall treatment effect (both time-points) of BM-MNC nor a treatment time-related (only early or late) effect could be shown for all functional parameters. ConclusionsAmong patients after AMI with successful reperfusion and LV dysfunction, intracoronary infusion of BM-MNC early or late after AMI did not improve global strain parameters at 4- or 12-months follow-up.Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00355186.

Co-transplantation of Vascularized Thymic Graft with Kidney in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Using advanced gene editing technologies, xenotransplantation from multi-transgenic alpha-1,3-galactosyltransferase knockout pigs has demonstrated marked prolongation of renal xenograft survival, ranging from days to greater than several months for life-supporting kidneys and >2years in a heterotopic non-life-supporting cardiac xenograft model. However, continuous administration of multiple immunosuppressive drugs continues to be required, and attempts to taper immunosuppression have been unsuccessful. These data are consistent with previous reports indicating that the human-anti-porcine T cell response is similar or stronger than that across allogeneic barriers. Due to the strength of both the innate and adaptive immune responses in xenotransplantation, the level of continuous immunosuppression needed to control these responses and prolong xenograft survival has been associated with prohibitive morbidity and mortality. These facts provide compelling rationale to pursue a clinically applicable strategy for the induction of tolerance.Mixed chimerism and thymic tissue transplantation have both achieved xenogeneic tolerance in pig-to-mouse models, and both have recently been extended to pig-to-baboon models. Although these strategies are promising in small animal models, neither direct intravenous injection of porcine bone marrow cells nor direct fetal thymic tissue transplantation into recipients was able to achieve >2days chimerism following BM Tx or the engraftment of thymic tissues across xenogeneic barriers in pig-to-nonhuman primate models. Several innovative procedures have been largely developed by Kazuhiko Yamada to overcome these failures. These include vascularized thymic transplantation, combined with either thymokidney (TK) or vascularized thymic lobe (VTL) transplantation. Utilizing the strategy of transplanting vascularized thymic grafts with kidney from the same GalT-KO donor without further gene modification, we have achieved longer than 6 months survival of life-supporting kidneys in a baboon. Notably, the recipient became donor specific unresponsive and developed new thymic emigrants. In this chapter, we introduce a brief summary of our achievements to date toward the successful induction of tolerance by utilizing our novel strategy of vascularized thymic transplantation (including thymokidney transplantation), as well as describe the step-by-step methodology of surgical and in vitro procedures which are required for this experiment.

Hepatocyte CREBH deficiency aggravates inflammatory liver injury following chemokine-dependent neutrophil infiltration through upregulation of NF-κB p65 in mice.

Fulminant hepatitis is a serious inflammatory condition of the liver characterized by massive necrosis of liver parenchyma following excessive immune cell infiltration into the liver, and possibly causing sudden hepatic failure and medical emergency. However, the underlying mechanisms are not fully understood. Here, we investigated the role of cyclic AMP-responsive element-binding protein, hepatocyte specific (CREBH) in concanavalin A (ConA)-driven hepatitis-evoked liver injury. C57BL/6J (WT) and Crebh knockout (KO) mice injected with ConA (7.5 or 25mg/kg) and bone marrow (BM) chimeric mice, generated by injection of BM cells into sub-lethally irradiated recipients followed by ConA injection (22.5 or 27.5mg/kg) 8weeks later, were used for in vivo study. Primary mouse hepatocytes and HEK293T cells were used for a comparative in vitro study. Crebh KO mice are highly susceptible to ConA-induced liver injury and prone to death due to increased neutrophil infiltration driven by enhanced hepatic expression of neutrophil-attracting chemokines. Notably, BM chimera experiment demonstrated that Crebh-deficient hepatocytes have an enhanced ability of recruiting neutrophils to the liver, thereby promoting hepatotoxicity by ConA. Intriguingly, in vitro assays showed that p65, a subunit of NF-κB and common transcription factor for various chemokines, dependent transactivation was inhibited by CREBH. Furthermore, p65 expression was inversely correlated with CREBH level in ConA-treated mice liver and TNFα-stimulated primary mouse hepatocytes. This is the first demonstration that CREBH deficiency aggravates inflammatory liver injury following chemokine-dependent neutrophil infiltration via NF-κB p65 upregulation. CREBH is suggested to be a novel therapeutic target for treatment of fulminant hepatitis.

Autologous bone marrow expanded mesenchymal stem cells in patellar tendinopathy: protocol for a phase I/II, single-centre, randomized with active control PRP, double-blinded clinical trial

IntroductionPatellar tendon overuse injuries are common in athletes. Imaging may show a change in tissue structure with tendon thickening and disruption of the intratendinous substance. We wish to test the hypothesis that both autologous bone marrow expanded mesenchymal stem cells and autologous leukocyte-poor platelet-rich plasma (LP-PRP) implanted into the area of the disrupted tendinopathic patellar tendon will restore function, but tendon regeneration tissue will only be observed in the subjects treated with autologous bone marrow expanded mesenchymal stem cells.Methods and analysisThis is a single-centre, pilot phase I/II, double-blinded clinical trial with randomisation with active control. Twenty patients with a diagnosis of patellar tendinopathy with imaging changes (tendon thickening and disruption of the intratendinous substance at the proximal portion of the patellar tendon) will be randomised in a 1:1 ratio to receive a local injection of either bone-marrow autologous mesenchymal stem cells (MSC), isolated and cultured under GMP at The Institute of Biology and Molecular Genetics (IBGM) (Spain) or P-PRP. The study will have two aims: first, to ascertain whether a clinically relevant improvement after 3, 6 and 12 months according to the visual analogue scale (VAS), Victorian Institute of Sport Assessment for patellar tendons (VISA-P) and dynamometry scales (DYN) will be achieved; and second, to ascertain whether the proposed intervention will restore tendon structure as determined by ultrasonography (US), Doppler ultrasonography (DUS), and innovative MRI and ultrasound techniques: Magnetic Resonance T2 FAT SAT (UTE, Ultrashort Echo TE) sequence and Ultrasound Tissue Characterization (UTC). Patients who are randomised to the P-PRP treatment group but do not achieve a satisfactory primary endpoint after 6 months will be offered treatment with MSC.Trial registrationNCT03454737.