Mononuclear Cell Transplantation Research Articles

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Abstract 2831: Development and application of the NCG-hIL2 mouse model: A humanized platform for enhanced NK cell evaluation in ADCC efficacy testing

Abstract Natural Killer (NK) cells play a pivotal role in immune surveillance and antitumor responses, making them valuable targets in cancer immunotherapy. The assessment of therapeutic antibodies' efficacy, particularly in the context of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), necessitates models that faithfully recapitulate human NK cell responses. Here, we introduce the NCG-hIL2 mouse model, designed to address these requirements and advance our understanding of NK cell development. The NCG-hIL2 mouse model was engineered to express human Interleukin-2 (IL2) cytokine, a critical factor in NK cell development and activation. This humanized platform allows for the robust reconstitution of human NK cells through the transplantation of peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs). In this study, we validate the NCG-hIL2 model's exceptional capability in supporting the reconstitution of human NK cells, highlighting the indispensable role of IL2 in NK cell development. Moreover, we demonstrate its utility in the evaluation of therapeutic antibodies, specifically Trastuzumab and the ADCC-enhanced Margetuximab, in the context of ADCC efficacy testing. Our findings reveal that the NCG-hIL2 model provides a unique advantage for assessing the impact of IL2 on NK cell development, thus facilitating a deeper understanding of the complex interplay between NK cells and therapeutic antibodies. The NCG-hIL2 mouse model serves as a valuable tool for studying NK cell development and evaluating the efficacy of immunotherapies, particularly in ADCC. By reconstituting human NK cells and leveraging the role of IL2, this model offers an enhanced platform for preclinical evaluation of therapeutic antibodies, ultimately advancing our ability to harness NK cell-mediated antitumor responses. It is poised to contribute significantly to the field of cancer immunotherapy and drug development. Citation Format: Yuan Fang, Hongyan Sun, Huiyi Wang, Jun Xing, Jialu Fan, Jing Zhao, Xiang Gao, Cunxiang Ju. Development and application of the NCG-hIL2 mouse model: A humanized platform for enhanced NK cell evaluation in ADCC efficacy testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2831.

The Effects of Busulfan on Xenogeneic Transplantation of Human Peripheral Blood Mononuclear Cells in Recipient Mice

Background. Humanized mouse models with engraftment of human peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) are effective tools for the study of human immunity. Busulfan has been used as a substitute for irradiation in human hematopoietic stem cell (HSC) transplantation models, but it has not been tested in human peripheral blood mononuclear cell (PBMC) transplantation models.Materials and Methods. This study evaluated PBMC engraftment using cytometry and enzyme-linked immunosorbent assay (ELISA) in female NOD.CB17/Prkdcscid/JKrb/ IL2 receptor γ−/− (NIG) mice treated with busulfan.Results. In this model, the percentage of human CD3+ T cell engraftment in the blood was 28.2%, with dominant infiltration of CD8+ cells in the spleen 3 weeks post PBMC transplantation. Production of human cytokines, including IL-12p70, IL-4, IL-5, IFN-γ, IL-6, IL-8, IL-22, TNF-α, and IL-10, was determined in mice treated with busulfan.Conclusions. Our findings demonstrate that busulfan treatment is a beneficial alternative for simple and efficient PBMC engraftment in a rodent model, possibly helping to evaluate human immunity in preclinical studies.

Follow-up of intramyocardial bone marrow mononuclear cell transplantation beyond 10 years

Bone marrow mononuclear cells (BMMCs) have been evaluated for their ability to improve cardiac repair and benefit patients with severe ischemic heart disease and heart failure. In our single-center trial in 2006–2011 we demonstrated the safety and efficacy of BMMCs injected intramyocardially in conjunction with coronary artery bypass surgery. The effect persisted in the follow-up study 5 years later. In this study, we investigated the efficacy of BMMC therapy beyond 10 years. A total of 18 patients (46%) died during over 10-years follow-up and 21 were contacted for participation. Late gadolinium enhancement cardiac magnetic resonance imaging (CMRI) and clinical evaluation were performed on 14 patients, seven from each group. CMRIs from the study baseline, 1-year and 5-years follow-ups were re-analyzed to enable comparison. The CMRI demonstrated a 2.1-fold larger reduction in the mass of late gadolinium enhancement values between the preoperative and the over 10-years follow-up, suggesting less scar or fibrosis after BMMC treatment (− 15.1%; 95% CI − 23 to − 6.7% vs. − 7.3%; 95% CI − 16 to 4.5%, p = 0.039), compared to placebo. No differences in mortality or morbidity were observed. Intramyocardially injected BMMCs may exert long-term benefits in patients with ischemic heart failure. This deserves further evaluation in patients who have received BMMCs in international clinical studies over two decades.

Dclre1c-Mutation-Induced Immunocompromised Mice Are a Novel Model for Human Xenograft Research.

Severe combined immunodeficient (SCID) mice serve as a critical model for human xenotransplantation studies, yet they often suffer from low engraftment rates and susceptibility to graft-versus-host disease (GVHD). Moreover, certain SCID strains demonstrate 'immune leakage', underscoring the need for novel model development. Here, we introduce an SCID mouse model with a targeted disruption of the dclre1c gene, encoding Artemis, which is essential for V(D)J recombination and DNA repair during T cell receptor (TCR) and B cell receptor (BCR) assembly. Artemis deficiency precipitates a profound immunodeficiency syndrome, marked by radiosensitivity and compromised T and B lymphocyte functionality. Utilizing CRISPR/Cas9-mediated gene editing, we generated dclre1c-deficient mice with an NOD genetic background. These mice exhibited a radiosensitive SCID phenotype, with pronounced DNA damage and defective thymic, splenic and lymph node development, culminating in reduced T and B lymphocyte populations. Notably, both cell lines and patient-derived tumor xenografts were successfully engrafted into these mice. Furthermore, the human immune system was effectively rebuilt following peripheral blood mononuclear cells (PBMCs) transplantation. The dclre1c-knockout NOD mice described herein represent a promising addition to the armamentarium of models for xenotransplantation, offering a valuable platform for advancing human immunobiological research.

Neuroprotective effects of syngeneic bone marrow‐derived mononuclear stem cell transplantation and intraperitoneal minocycline in the treatment of inherited photoreceptor retinal degeneration

Inherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects and Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors (fist rods following by cones), activation of microglial cells and gliosis increases of macroglial cell.The Royal college of Surgeon (RCS) rat is a well characterized animal models of RP which suffer a mutation of the MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus triggers the simultaneous degeneration of rods and cones. Recent studies have shown that microglial activation starts simultaneously with the beginning of photoreceptor death in the RCS retinas, suggesting that microglial cells play an important role in the retinal degeneration progression.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. These cells have shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations such us: improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein expression in Müller cells.On the other hands minocycline is a broad‐spectrum tetracycline antibiotic, of which it has been demonstrated has antimicrobial, anti‐inflammatory, antiapoptotic, neuroprotective, and microglial inhibition effects in central nervous system diseases.In RCS rat retina the neuroprotective effect of minocycline against photoreceptor apoptosis is can be explained by mainly two different mechanisms: (1) a direct antiapoptotic effect on photoreceptors and/or (2) an effect through its anti‐inflammatory properties reducing microglial cells activation and migration.The question arises whether the combination of intravitreal BM‐MNCs transplantation with minocycline, substances that increase survival presumably through different mechanisms, could further increase photoreceptor survival and therefore be used for the treatment of retinal degenerations.Our results showed that intravitreal transplantation of BM‐MNCs increase photoreceptor survival, improved the morphology of the photoreceptors outer segment, and reduce retinal thinning (in vivo). On the other hands intraperitoneally minocycline treatment, and reduced activation and migration of microglia and produced a significant rescue of photoreceptors. Finally, our result show that combination both treatments had higher neuroprotective effects in the retina, increasing photoreceptor survival, than each of the treatments alone.In conclusion in RCS rat animal model of photoreceptor degeneration, minocycline reduces microglial activation and migration, and intravitreal transplantation of BM‐MNCs and minocycline increase photoreceptor survival. The combination of both treatments shows greater neuroprotective effects than their isolated effects, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

Effects of Timing on the Efficacy of Stem Cell Transplantation after Acute Myocardial Infarction

Acute myocardial infarction (AMI) is the blocking of coronary arteries that prevents oxygenated blood from reaching the heart tissues, resulting in damage to the myocardium and affecting heart function. This condition affects millions of people every year and is detrimental to their quality of life. Several clinical trials have investigated the efficacy of using bone marrow-derived stem cells (BMSCs) to improve heart function after AMIs. However, different variables could impact the results of the trials, one of them being the injection time of cell therapy after reperfusion. This paper aims to investigate the short-term effects of timing on the efficacy of bone marrow-derived mononuclear cell transplantation after acute myocardial infarction. A systematic literature search of PUBMED, EMBASE, European Society of Cardiology, and American Heart Association databases was made on randomized controlled trials with at least 3-month follow-up data for patients with AMI undergoing percutaneous coronary intervention (PCI) and receiving intracoronary autologous BMSC transfer subsequently. A total of 12 trials with 1061 patients were selected for analysis. Compared to baseline level, BMSC transfer within 24 hours of PCI significantly improved left ventricular ejection fraction (LVEF; 3.44% increase, 95% confidence interval [CI]: 2.20%-4.68%, P< 0.00001). The “3-7 days after PCI” subgroup also showed notable improvements in LVEF (LVEF; 2.52% increase, 95% confidence interval [CI]: 1.01%-4.04%, P = 0.001). However, in the subgroups that received BMSC transplantation either 7-14 days after PCI or later than 15 days after PCI, there was no significant effect on treatment outcome.

Effects of transplantation of umbilical cord blood mononuclear cells into the scrotum on sexual function in elderly mice.

Aim: This study investigated the effect of allografting umbilical cord blood mononuclear cells (UCBMCs) into the scrotum on sexual function in male elderly mice. Methods: UCBMCs were injected once into the scrotal sheath cavity of elderly mice. Results: The transplanted UCBMCs survived in the scrotal sheath cavity for 1month. The micehad significantly increased blood testosterone concentrations, cyclic guanosine monophosphate (cGMP) levels and total nitric oxide synthase (T-NOS) activity in the corpus cavernosum and an increase inthe number of mouse matings within 30min (all p=0.000). Conclusion: Scrotum-implanted UCBMCs improve the sexual function of male elderly mice through testosterone production and the NOS/cGMP pathway, which may provide an innovative transplantation approach for thetreatment of erectile dysfunction.

G-CSF-mobilized peripheral blood mononuclear cells combined with platelet-rich plasma restored the ovarian function of aged rats

BackgroundRegenerative medicine with peripheral blood mononuclear cell (PBMC) transplantation sheds light on the issue of premature ovarian insufficiency (POI). However, the efficiency of PBMC treatment in natural ovarian aging (NOA) remains unclear. MethodsThirteen-month-old female Sprague–Dawley (SD) rats were used to verify the NOA model. Seventy-two NOA rats were randomly divided into three groups: the NOA control group, PBMC group, and PBMC+platelet-rich plasma (PRP) group. PBMCs and PRP were transplanted by intraovarian injection. The effects on ovarian function and fertility were measured after transplantation. ResultsTransplantation of PBMCs could restore the normal estrous cycle, consistent with the recovery of serum sex hormone levels, increased follicle numbers at all stages, and restoration of fertility by facilitating pregnancy and live birth. Moreover, when combined with PRP injection, these effects were more significant. The male-specific SRY gene was detected in the ovary at all four time points, suggesting that PBMCs continuously survived and functioned in NOA rats. In addition, after PBMC treatment, the expression of angiogenesis-related and glycolysis-related markers in the ovaries was upregulated, which indicated that these effects were associated with angiogenesis and glycolysis. ConclusionsPBMC transplantation restores the ovarian functions and fertility of NOA rats, and PRP could enhance the efficiency. Increased ovarian vascularization, follicle production, and glycolysis are likely the major mechanisms.

Safety and efficacy of intra-arterial bone marrow mononuclear cell transplantation in patients with acute ischaemic stroke in Spain (IBIS trial): a phase 2, randomised, open-label, standard-of-care controlled, multicentre trial

Pilot clinical trials have shown the safety of intra-arterial bone marrow mononuclear cells (BMMNCs) in stroke. However, the efficacy of different doses of intra-arterial BMMNCs in patients with acute stroke has not been tested in a randomised clinical trial. We aimed to show safety and efficacy of two different doses of autologous intra-arterial BMMNC transplantation in patients with acute stroke. The IBIS trial was a multicentre phase 2, randomised, controlled, investigator-initiated, assessor-blinded, clinical trial, in four stroke centres in Spain. We included patients (aged 18-80 years) with a non-lacunar, middle cerebral artery ischaemic stroke within 1-7 days from stroke onset and with a National Institutes of Health Stroke Scale score of 6-20. We randomly assigned patients (2:1:1) with a computer-generated randomisation sequence to standard of care (control group) or intra-arterial injection of autologous BMMNCs at one of two different doses (2 × 106 BMMNCs/kg or 5 × 106 BMMNCs/kg). The primary efficacy outcome was the proportion of patients with modified Rankin Scale scores of 0-2 at 180 days in the intention-to-treat population, comparing each BMMNC dose group and the pooled BMMNC group versus the control group. The primary safety endpoint was the proportion of serious adverse events. This trial was registered at ClinicalTrials.gov, NCT02178657 and is completed. Between April 1, 2015, and May 20, 2021, we assessed 114 patients for eligibility. We randomly assigned 77 (68%) patients: 38 (49%) to the control group, 20 (26%) to the low-dose BMMNC group, and 19 (25%) the high-dose BMMNC group. The mean age of participants was 62·4 years (SD 12·7), 46 (60%) were men, 31 (40%) were women, all were White, and 63 (82%) received thrombectomy. The median NIHSS score before randomisation was 12 (IQR 9-15), with intra-arterial BMMNC injection done a median of 6 days (4-7) after stroke onset. The primary efficacy outcome occurred in 14 (39%) patients in the control group versus ten (50%) in the low-dose group (adjusted odds ratio 2·08 [95% CI 0·55-7·85]; p=0·28), eight (44%) in the high-dose group (1·89 [0·52-6·96]; p=0·33), and 18 (47%) in the pooled BMMNC group (2·22 [0·72-6·85]; p=0·16). We found no differences in the proportion of patients who had adverse events or dose-related events, but two patients had a groin haematoma after cell injection in the low-dose BMMNC group. Intra-arterial BMMNCs were safe in patients with acute ischaemic stroke, but we found no significant improvement at 180 days on the mRS. Further clinical trials are warranted to investigate whether improvements might be possible at different timepoints. Instituto de Salud Carlos III co-funded by the European Regional Development Fund/European Social Fund, Mutua Madrileña, and the Regional Ministry of Health of Andalusia.

PROSPECTIVE SINGLE CENTER ANALYSIS OF OUTCOME STEM CELLS TRANSPLANTS IN PATIENTS WITH CEREBRAL PALSY.

Aim: To evaluate efficacy and safety of autologous bone marrow-derived mononuclear stem cell transplantation intrathecal in children with cerebral palsy. Materials and Methods: 35 children have levels I-V cerebral palsy aged 8-months to 8-years-old were enrolled from September (2021-2022) at Iraqi private hospital. Gross Motor Function was assessed by a pediatrician and neurologist specialist, 5 mcg/kg/day of G-CSF subcutaneous single injection daily for three consecutive days. Bone marrow harvested from posterior iliac crest under light general anesthesia. Bone marrow mononuclear cells (BMMNCs) separation was performed using density gradient centrifugation with Ficoll, the cell viability checked by propidium iodide dye in a TALI machine (Invitrogen) in average 98%. The viable BMMNCs injected intrathecal in L4-L5 over a period of 5-10 min. Results: Males accounted for 57.14% (20/35) while female 42.86% (15/35), and main neurological symptoms included spastic disorder spastic disorder (quadriplegia 24 (68.6), tetraplegia 2 (5.7), diplegia 5 (14.28), hemiplegia4 (11.42)). Gross Motor Function Classification System and Gross Motor Function Measure-66 (GMFM-66) showed II 10 (28.58), III 11(31.42) and IV 14 (40). On mean follow-up of 3 months post-stem cell transplant improvement was observed in 80% cases. The improvement showed in gross motor function (6/8) p=0.01, and speech (2/4) p=0.04, neck holding (5/5) p=0.0003, sitting balance (4/4) p=0.04, postural tone (5/5) p=0.0003, as well as significant reduction in seizure frequency (2/3) p=0.04 and improvement in cognition (6/7) p=0.01 were observed. Conclusion: Stem cell therapy for cerebral palsy shows a significant positive effect on the gross motor function, without long adverse effects.

The quality and quantity media-cultured mononuclear cell transplantation is safe and effective in ischemic hindlimb mouse model

ObjectiveThis study was conducted to investigate in vitro proangiogenic and anti-inflammatory phenotypes and functions and the in vivo efficacy and safety of quality and quantity (QQ) media-cultured mononuclear cells (MNCs) compared with standard cultured MNCs from the peripheral blood of patients with chronic limb-threatening ischemia (CLTI) with atherosclerotic risk factors. MethodsPeripheral blood MNCs (PBMNCs) from patients with CLTI were cultured in QQ culture media or standard culture media. Phenotypic analysis of progenitor cells (CD34+CD133+), M2 macrophages (CD206+), and inactivated T regulatory cells (CD4+CD25+CD127+), colony-forming assay, and tube formation assay of QQ media-cultured MNCs (QQMNCs) and PBMNCs, were conducted. Intramuscular transplantation of QQMNCs or PBMNCs was performed in the ischemic hindlimb model. The clinical appearance of ischemic limbs was observed, and blood flow in ischemic limbs was measured using a laser Doppler perfusion imager. Outcomes were compared between the QQMNC and PBMNC groups. ResultsTwenty patients with CLTI were included. The mean percentages of CD34+ cells, CD133+ cells, CD34+CD133+ progenitor cells, CD206+ cells, colony-forming cells, and tube formation were significantly higher in the QQMNCs. The mean percentage of CD4+CD25+CD127+ cells was significantly lower in QQMNC. The colony-forming unit count and Dil-acetylated low-density lipoprotein uptake were significantly greater in QQMNCs. The clinical appearance of post-QQMNC-injected limbs was less severe than the appearance of post-PBMNC-injected limbs. Limb perfusion was significantly better in the QQMNCs. ConclusionsProangiogenic and anti-inflammatory phenotypes of MNCs cultured in QQ culture media were reproducible. Intramuscular QQMNC transplantation was safe and resulted in better reperfusion of ischemic hindlimbs compared with PBMNCs.

Role of Vascular Endothelial Growth Factor-C during Stem Cell Therapy Using Autologous Bone Marrow Mononuclear Cells in Patients with Lower Limb Lymphedema

Introduction: Vascular endothelial growth factor-C (VEGF-C) is the primary lymphangiogenic factor that stimulates lymphangiogenesis by signaling via specific receptor, vascular endothelial growth factor receptor 3 (VEGFR3). This study was conducted to evaluate the change in the level of VEGF-C before and after autologous bone marrow mononuclear cell transplantation for treatment of Lower limb lymphedema. Patient and methods: Forty patients with lower limb lymphedema were divided into two groups. Group I included 20 patients with chronic lower limb lymphedema who underwent autologous bone marrow mononuclear cell transplantation. Group II included 20 patients with chronic lower limb lymphedema who were exposed only to compression therapy as a control group. VEGF-C level in the diseased limbs was measured in both groups at the beginning of the study then 3 and 6 months respectively. Results: Group I included 20 patients, 8 patients were male (40%) and 12 patients were females (60%) with mean age 29.5 ± 12.15 while group II included 20, 10 patients were male (50%) and 10 patients were females (50%) with mean age 39.5 ± 11.5. In group I, the specimens were taken at 3 and 6 months after transplantation showed a marked decrease in the VEGF-C level with statistically significant p value, 0.02 and 0.001 respectively. In group II the level of VEGF-C after compression therapy alone at 3 and 6 months interval showed fluctuation with statistically non-significant p value, 0.64 and 0.55 respectively. Conclusion: VEGF-C is essential for regulation of lymphangiogenesis. The level of VEGF-C was found elevated in patients with lymphedema and decrease after autologous mononuclear bone marrow cells, however these results were statically non-significant.

S100A9 upregulated by IFNGR signaling blockade functions as a novel GVHD suppressor without compromising GVL in mice

AbstractAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1−/− and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.

Neuroprotective effects of intraocular bone marrow‐derived mononuclear cell transplants in inherited retinal degenerations

AbstractInherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects. At present, only a minority are susceptible of genetic treatment and therefore are a frequent cause of blindness in infancy and adulthood.Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors, rods and then cones that may die secondarily to rod loss.The rats P23H and Royal college of Surgeon (RCS) are two well characterized animal models of RP because they bear genetic defects observed in RP patients. The P23H rat suffers an autosomal dominant mutation of the rhodopsin gene that causes protein misfolding and endoplasmic reticulum stress in rods, which die first followed by cones. The RCS rat has a mutation of MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus causing simultaneous rod and cone degeneration.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. The BM‐MNCs is a mixture of specific bone marrow cell subtypes in which is possible to find cells: Lin+, Lin−, CD34+, c‐Kit+, CD133+ or Sca‐1+ HSCs, endothelial precursor cells, and Mesenchymal Stem Cells.BM‐MNCs transplantation has shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations.In our studies we have performed a syngeneic intravitreal or subretinal transplantation of BM‐MNCs in a P23H‐1 and RCS rats, and we have documented that both increase photoreceptor survival. They also improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein (GFAP) expression in Müller cells. However, the transplanted cells did not penetrate the retina, only survived for up to 15 days, and did not modify microglial cell activation and migration or the electroretinographic responses. Their beneficial effects are maybe due to a paracrine trophic effect achieved through release of neurotrophic, anti‐angiogenic and/or immunomodulatory factors.In conclusion, the intra ocular transplantation BM‐MNCs shows photoreceptor neuroprotective actions in inherited retinal degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

The Long Non-Coding RNA MALAT1 Is Necessary for Hematopoietic Stem and Progenitor Cell Expansion and Preleukemic Myeloproliferation in TET2 Deficient Myeloid Neoplasms

Ten Eleven Translocation 2 (TET2) is a dioxygenase which regulates gene expression by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine. Loss of function mutations in TET2 occur in up to 58% and 30% of myeloid malignancies and clonal hematopoiesis respectively. Loss of TET2 function also occurs with isocitrate dehydrogenase 2 (IDH2) mutations in Acute Myeloid Leukemia (AML) via 2-hydroxyglutarate (2-HG). Genetically engineered murine models (GEMM) of Tet2 deficiency produce a Chronic Myelomonocytic Leukemia (CMML)-like phenotype with splenomegaly, granulomonocytic expansion and increased bone marrow hematopoietic stem and progenitors (LSK). Further, bone marrow mononuclear cell (BMNC) transplant models of Tet2-/- clonal hematopoiesis suggest that this myeloid expansion leads to enhanced inflammation and increased cardiovascular disease risk. However, the downstream effects of TET2 loss of function leading to this phenotype are unknown. Here we show that TET2-deficient granulomonocytic expansion and inflammation are mediated via MALAT1, a long non-coding RNA overexpressed in several malignancies. MALAT1 expression was increased in BMNCs from IDH2 mutated AML patients (n=152 WT n=17 mutant IDH2, p=0.04). Increased MALAT1 expression was also seen in human and murine models of TET2 deficiency including isogenic IDH2 mutated TF-1 cells (5 biological replicates p=0.005), 2-HG treated leukemia cell lines (2 biological replicates p<0.0001) and BMNCs from Tet2f/f mice (n= 7/grp p=0.005). To evaluate the impact of Malat1 on normal and Tet2 deficient hematopoiesis we utilized the Malat1tg overexpression and Tet2-/-Malat1-/-GEMM models. Similar to Tet2-/- mice, bone marrow (n=4/grp p=0.02) and spleen (n=8/grp p=0.004) LSK numbers were increased in Malat1tg mice compared to control. However, unlike Tet2-/-LSKs, MALAT1tgLSKs did not show increased self-renewal in serial in vivo competitive transplants. Further, depleting Malat1 in Tet2-/- mice did not rescue the increased self-renewal capacity seen in Tet2-/- LSKs. Collectively, these data suggest that Malat1 overexpression is sufficient for LSK expansion but does not impact LSK function. Splenomegaly, myeloproliferation and extramedullary hematopoiesis is a phenotype termed Preleukemic Myeloproliferation (PMP) and is predictive of myeloid neoplasm development in Tet2-/- models (Meisel M et al, Nature 2018). Consistent with a role of Malat1 in PMP (Fig A), Malat1 overexpression recapitulated PMP at 8 (n=10/grp p<0.0001) and 20 (n=10/grp p=0.0005) weeks. Malat1 overexpression was also associated with an increase in classical monocytes in the peripheral blood- a hallmark of CMML (8 weeks n= 7/grp p<0.0001, 20 weeks n=10/grp p=0.0007). This was recapitulated in Tet2-/-mice (n=3/grp p=0.001) and CMML patients with TET2 mutations (n=27 mutant TET2 v 109 WT, odds ratio 5.6 p= 0.001). Finally, depleting Malat1 in a Tet2-deficient GEMM rescued PMP (n=4 Tet2-/-Malat1-/-v 5 Tet2-/-p=0.01) and classical monocytosis (n=2/grp p=0.002) suggesting that Malat1 overexpression is both sufficient and necessary for PMP in Tet2 deficient leukemia. This impact was also observed in recipients in BMNC competitive transplant experiments (Fig B). Initial insights into the molecular mechanism underlying the role of Malat1 in PMP were derived from analyzing competitor and host cells during in vivo competitive transplants. Interestingly, transplant recipients with Malat1tg LSK demonstrated myeloid and granulomonocytic expansion in both donor and non-donor cells suggesting that the effects of Malat1 overexpression are, in part, cell extrinsic. Consistent with this hypothesis and the impact of NF-kB on inflammatory cytokine secretion and Tet2-/- PMP, we show that MALAT1 deletion in human leukemia attenuates NF-kB activity by enhancing the phosphatase activity of PP2A. Further, Interleukin 6 (IL-6), a major downstream target of NF-kB and potent activator of myelopoiesis in response to inflammatory stimuli, was increased in the plasma of MALAT1tg mice (n=8/grp p=0.01) suggesting a possible cell-extrinsic mechanism for Malat1 dependent PMP. Ongoing studies are focused on demonstrating that IL-6 is necessary for Malat1 dependent PMP. Our current data provides hitherto unknown insights establishing MALAT1 as a major effector of TET2 loss of function which may lead to novel therapeutic options in TET2 deficient myeloid neoplasms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transplantation of mesenchymal stem cells for prevention of acute myocardial infarction induced heart failure: study protocol of a phase III randomized clinical trial (Prevent-TAHA8)

BackgroundResults from recent clinical trials on bone marrow mononuclear cell (BM-MNC) transplantation show that this intervention can help reduce the incidence of heart failure (HF) after acute myocardial infarction (AMI). However, no study has evaluated the effect of the transplantation of mesenchymal stem cells (MSCs) on a clinical endpoint such as HF.MethodsThis single-blinded, randomized, multicenter trial aims to establish whether the intracoronary infusion of umbilical cord-derived Wharton’s jelly MSCs (WJ-MSCs) helps prevent HF development after AMI. The study will enroll 390 patients 3 to 7 days following AMI. Only patients aged below 65 years with impaired LV function (LVEF < 40%) will be included. They will be randomized (2:1 ratio) to either receive standard care or a single intracoronary infusion of 107 WJ-MSCs. The primary outcome of this study is the assessment of HF development during long-term follow-up (3 years).DiscussionData will be collected until Nov 2024. Thereafter, the analysis will be conducted. Results are expected to be ready by Dec 2024. We will prepare and submit the related manuscript following the CONSORT guidelines. This study will help determine whether or not the infusion of intracoronary WJ-MSCs in patients with AMI will reduce the incidence of AMI-induced HF.Trial registrationClinicalTrials.gov NCT05043610, Registered on 14 September 2021 - retrospectively registered.

Preventive Effect of Bone Marrow Mononuclear Cell Transplantation on Acute Myocardial Infarction-Induced Heart Failure: A Meta-analysis of Randomized Controlled Trials.

Heart failure (HF) is a major complication of acute myocardial infarction (AMI). Transplantation of bone marrow mononuclear cells (BM-MNC) in the setting of AMI has been proposed as a means for myocardial tissue regeneration. Several trials have explored the outcomes of these cells on surrogate end points such as left ventricular ejection fraction (LVEF) in patients with AMI. However, the data regarding the clinical efficacy are infrequent. Here, we performed a meta-analysis investigating the effect of BM-MNCs injection on the rate of hospitalization for HF in the long-term follow-up period. PubMed, Scopus, and Cochrane databases were queried with various combinations of keywords through May 2, 2022. A random-effects meta-analysis was performed to calculate risk ratio (RR) and 95% confidence interval (CI) of hospitalization for HF, all-cause mortality, and stroke rate. Subgroup analyses for hospitalization based on time and cell dose were performed. A total of 2150 patients with AMI across 22 trials were included for quantitative synthesis. At long-term follow-up, AMI patients treated with an intracoronary injection of BM-MNCs were less likely to be hospitalized for heart failure compared to the control group receiving standard treatment (RR = 0.54, 95% CI = [0.37; 0.78], p = 0.002). There was no association between BM-MNC therapy and all-cause mortality (RR = 0.69, 95% CI = [0.47; 1.01], p = 0.05) and stroke (RR = 1.12, 95% CI= [0.24; 5.21], p = 0.85). Autologous injection of BM-MNC in the setting of AMI may be associated with decreased risk of hospitalization of heart failure in the long term. However, its effect on all-cause mortality and stroke rate is questionable.

Local intramuscular transplantation of autologous bone marrow mononuclear cells for critical lower limb ischaemia.

Peripheral arterial disease is a major health problem, and in about 1% to 2% of patients, the disease progresses to critical limb ischaemia (CLI), also known as critical limb-threatening ischaemia. In a substantial number of individuals with CLI, no effective treatment options other than amputation are available, with around a quarter of these patients requiring a major amputation during the following year. This is the second update of a review first published in 2011. To evaluate the benefits and harms of local intramuscular transplantation of autologous adult bone marrow mononuclear cells (BMMNCs) as a treatment for CLI. We used standard, extensive Cochrane search methods. The latest search date was 8 November 2021. We included all randomised controlled trials (RCTs) of CLI in which participants were randomly allocated to intramuscular administration of autologous adult BMMNCs or control (either no intervention, conventional conservative therapy, or placebo). We used standard Cochrane methods. Our primary outcomes of interest were all-cause mortality, pain, and amputation. Our secondary outcomes were angiographic analysis, ankle-brachial index (ABI), pain-free walking distance, side effects and complications. We assessed the certainty of the evidence using the GRADE approach. We included four RCTs involving a total of 176 participants with a clinical diagnosis of CLI. Participants were randomised to receive either intramuscular cell implantation of BMMNCs or control. The control arms varied between studies, and included conventional therapy, diluted autologous peripheral blood, and saline. There was no clear evidence of an effect on mortality related to the administration of BMMNCs compared to control (risk ratio (RR) 1.00, 95% confidence interval (CI) 0.15 to 6.63; 3 studies, 123 participants; very low-certainty evidence). All trials assessed changes in pain severity, but the trials used different forms of pain assessment tools, so we were unable to pool data. Three studies individually reported that no differences in pain reduction were observed between the BMMNC and control groups. One study reported that reduction in rest pain was greater in the BMMNC group compared to the control group (very low-certainty evidence). All four trials reported the rate of amputation at the end of the study period. We are uncertain if amputations were reduced in the BMMNC group compared to the control group, as a possible small effect (RR 0.52, 95% CI 0.27 to 0.99; 4 studies, 176 participants; very low-certainty evidence) was lost after undertaking sensitivity analysis (RR 0.52, 95% CI 0.19 to 1.39; 2 studies, 89 participants). None of the included studies reported any angiographic analysis. Ankle-brachial index was reported differently by each study, so we were not able to pool the data. Three studies reported no changes between groups, and one study reported greater improvement in ABI (as haemodynamic improvement) in the BMMNC group compared to the control group (very low-certainty evidence). One study reported pain-free walking distance, finding no clear difference between BMMNC and control groups (low-certainty evidence). We pooled the data for side effects reported during the follow-up, and this did not show any clear difference between BMMNC and control groups (RR 2.13, 95% CI 0.50 to 8.97; 4 studies, 176 participants; very low-certainty evidence). We downgraded the certainty of the evidence due to the concerns about risk of bias, imprecision, and inconsistency. We identified a small number of studies that met our inclusion criteria, and these differed in the controls they used and how they measured important outcomes. Limited data from these trials provide very low- to low-certainty evidence, and we are unable to draw conclusions to support the use of local intramuscular transplantation of BMMNC for improving clinical outcomes in people with CLI. Evidence from larger RCTs is needed in order to provide adequate statistical power to assess the role of this procedure.