Bone Marrow Cell Transplantation Research Articles

Atorvastatin Treatment Significantly Increased the Concentration of Bone Marrow-Derived Mononuclear Cells and Transcutaneous Oxygen Pressure and Lowered the Pain Scale after Bone Marrow Cells Treatment in Patients with "No-Option" Critical Limb Ischaemia.

The present study investigated the outcomes and possible predictive factors of autologous bone marrow cells (BMCs) therapy in patients with "no-option" critical limb ischaemia (CLI). It was focused on exploring the clinical background and prior statin and renin-angiotensin system (RAS)-acting agents pharmacotherapy related to the therapeutic efficacy of BMCs treatment. In the present study, we reviewed thirty-three patients (mean age 64.9 ± 10 years; 31 males) with advanced CLI after failed or impossible revascularisation, who were treated with 40 mL of autologous BMCs by local intramuscular application. Patients with limb salvage and wound healing (N = 22) were considered as responders to BMCs therapy, and patients with limb salvage and complete ischemic wound healing (N = 13) were defined as super-responders. Logistic regression models were used to screen and identify the prognostic factors, and a receiver operating characteristics (ROC) curve, a linear regression, and a survival curve were drawn to determine the predictive accuracy, the correlation between the candidate predictors, and the risk of major amputation. Based on the univariate regression analysis, baseline C-reactive protein (CRP) and transcutaneous oxygen pressure (TcPO2) values were identified as prognostic factors of the responders, while CRP value, ankle-brachial index (ABI), and bone marrow-derived mononuclear cells (BM-MNCs) concentration were identified as prognostic factors of the super-responders. An area under the ROC curve of 0.768 indicated good discrimination for CRP > 8.1 mg/L before transplantation as a predictive factor for negative clinical response. Linear regression analysis revealed a significant dependence between the levels of baseline CRP and the concentration of BM-MNCs in transplanted bone marrow. Patients taking atorvastatin before BMCs treatment (N = 22) had significantly improved TcPO2 and reduced pain scale after BMCs transplant, compared to the non-atorvastatin group. Statin treatment was associated with reduced risk for major amputation. However, the difference was not statistically significant. Statin use was also associated with a significantly higher concentration of BM-MNCs in the transplanted bone marrow compared to patients without statin treatment. Patients treated with RAS-acting agents (N = 20) had significantly reduced pain scale after BMCs transplant, compared to the non-RAS-acting agents group. Similar results, reduced pain scale and improved TcPO2, were achieved in patients treated with atorvastatin and RAS-acting agents (N = 17) before BMCs treatment. Results of the Spearman correlation showed a significant positive correlation between CLI regression, responders, and previous therapy before BMCs transplant with RAS-acting agents alone or with atorvastatin. CRP and TcPO2 were prognostic factors of the responders, while CRP value, ABI, and BM-MNCs concentration were identified as predictive factors of the super-responders. Atorvastatin treatment was associated with a significantly increased concentration of BM-MNCs in bone marrow concentrate and higher TcPO2 and lower pain scale after BMCs treatment in CLI patients. Similarly, reduced pain scales and improved TcPO2 were achieved in patients treated with atorvastatin and RAS-acting agents before BMCs treatment. Positive correlations between responders and previous treatment before BMCs transplant with RAS-acting agents alone or with atorvastatin were significant.

Allogeneic transplantation of bone marrow versus peripheral blood stem cells from HLA-identical sibling donors for hematological malignancies in 6064 adults from 2003 to 2020: different impacts on survival according to time period

BackgroundMobilized peripheral blood stem cells (PBSC) have been widely used instead of bone marrow (BM) as the graft source for allogeneic hematopoietic cell transplantation (HCT). Although early studies demonstrated no significant differences in survival between PBSC transplantation (PBSCT) and BM transplantation (BMT) from human leukocyte antigen (HLA)-identical sibling donors to adults with hematological malignancies, recent results have been unclear. ObjectiveThe objective of this retrospective study was to compare overall survival (OS), relapse, non-relapse mortality (NRM), hematopoietic recovery and graft-versus-host disease (GVHD) between PBSCT and BMT according to the time period of HCT (2003–2008, 2009–2014, or 2015–2020). Study designWe retrospectively compared the outcomes after PBSCT versus BMT in 6064 adults with hematological malignancies using a Japanese registry database between 2003 and 2020. ResultsThe adjusted probability of OS was significantly higher in BMT recipients compared to PBSCT recipients during the early period of 2003–2008 (adjusted hazard ratio [HR], 0.79; 95% confidence interval [CI], 0.70–0.91; P < 0.001) and the middle period of 2009–2014 (adjusted HR, 0.80; 95% CI, 0.70–0.91; P < 0.001). However, during the late period of 2015–2020, the adjusted probability of OS was comparable between BMT and PBSCT recipients (adjusted HR, 0.94; 95% CI, 0.79–1.13; P = 0.564), which were mainly due to the reduction of NRM. There was no significant difference in the relapse rate between the groups, irrespective of the time period. Compared to BMT, PBSCT led to faster neutrophil and platelet recovery and the cumulative incidences of grades II–IV and grades III–IV acute and overall and extensive chronic GVHD were significantly higher in PBSCT recipients, irrespective of the time period. ConclusionsPBSCT and BMT had similar survival outcomes and relapse rates in adult patients with hematological malignancies during the late time period of 2015–2020 despite the hematopoietic recovery and acute and chronic GVHD being higher in PBSCT recipients in all time periods.

Aromatase deficiency in transplanted bone marrow cells improves vertebral trabecular bone quantity, connectivity, and mineralization and decreases cortical porosity in murine bone marrow transplant recipients.

Estradiol is an important regulator of bone accumulation and maintenance. Circulating estrogens are primarily produced by the gonads. Aromatase, the enzyme responsible for the conversion of androgens to estrogen, is expressed by bone marrow cells (BMCs) of both hematopoietic and nonhematopoietic origin. While the significance of gonad-derived estradiol to bone health has been investigated, there is limited understanding regarding the relative contribution of BMC derived estrogens to bone metabolism. To elucidate the role of BMC derived estrogens in male bone, irradiated wild-type C57BL/6J mice received bone marrow cells transplanted from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) mice. MicroCT was acquired on lumbar vertebra to assess bone quantity and quality. WT(ArKO) animals had greater trabecular bone volume (BV/TV p = 0.002), with a higher trabecular number (p = 0.008), connectivity density (p = 0.017), and bone mineral content (p = 0.004). In cortical bone, WT(ArKO) animals exhibited smaller cortical pores and lower cortical porosity (p = 0.02). Static histomorphometry revealed fewer osteoclasts per bone surface (Oc.S/BS%), osteoclasts on the erosion surface (ES(Oc+)/BS, p = 0.04) and low number of osteoclasts per bone perimeter (N.Oc/B.Pm, p = 0.01) in WT(ArKO). Osteoblast-associated parameters in WT(ArKO) were lower but not statistically different from WT(WT). Dynamic histomorphometry suggested similar bone formation indices' patterns with lower mean values in mineral apposition rate, label separation, and BFR/BS in WT(ArKO) animals. Ex vivo bone cell differentiation assays demonstrated relative decreased osteoblast differentiation and ability to form mineralized nodules. This study demonstrates a role of local 17β-estradiol production by BMCs for regulating the quantity and quality of bone in male mice. Underlying in vivo cellular and molecular mechanisms require further study.

Risk factors for mortality in children with hemorrhagic cystitis after hematopoietic transplant

Hemorrhagic cystitis (HC) is a devastating complication of bone marrow (BMT) and stem cell transplant (SCT). Much of the literature has focused on exclusively adult patient populations, with limited evidence regarding risk factors for mortality and morbidity among pediatric HC patients. To examine factors associated with all-cause mortality in children with HC after BMT/SCT. The Pediatric Health Information System database was queried for patients with ICD-9/10 codes for hematopoietic transplant and gross hematuria, hematuria unspecified, or cystitis with hematuria. Multivariable logistic regression examined association of medical and surgical interventions frequently employed for hemorrhagic cystitis with mortality and genitourinary morbidity, defined as having received instillation of any bladder medication or having undergone any genitourinary procedure. A total of 811 patients, mean age of 12.4 years and 62% male, were included. Primary diagnosis included 388 (49%) leukemia/lymphoma, 182 (22%) blood dyscrasia, 99 (12%) solid organ tumor, 27 (3%) metabolic disease, 115 (14%) unknown. Transplant type included 377 (46%) bone marrow, 329 (41%) stem cell, 105, and (13%) unknown. Performing any bladder instillation (p<0.0001) or any type of GU procedure (p<0.0001) was significantly associated with mortality. On multivariate analysis, dialysis (OR=10.7, 95% CI=5.7-20.2), genitourinary morbidity (OR=4, 95% CI=2.2-6.8) and intravenous cidofovir (OR=2.0, 95% CI=1.2-3.3) were significantly associated with all cause mortality. Having an underlying diagnosis of blood dyscrasia was protective against mortality (OR=0.425, CI=0.205-0.88). In this large retrospective study evaluating factors associated with mortality in children with HC, all cause mortality was found to be 11%. This is probably an underrepresentation of true mortality in this population, as many patients discharged from the hospital likely die outside the hospital at home or hospice care. This study supports the current literature that invasive GU procedures are not associated with increased survival in patients with severe HC. This study is limited by retrospective use of a billing database that has the potential for errors in data entry and missing data. Patients who were discharged from the hospital were not captured by the PHIS which only collects data from inpatient stays. Patients with HC who received dialysis, intravenous cidofovir, or underwent GU intervention had significantly higher all-cause mortality. High grade HC is a marker of disease severity and efforts should be made by urologists and oncologists to maximize quality of life and limit futile treatments in this patient population.

Effect of peptides of trophoblastic Β1 glycoprotein on peripheral and local T-regulatory lymphocytes level in Wistar rats with allogeneous bone marrow cell transplantation

Objective. The range of peptide drugs is expanding, but no drug with immunosuppressive activity has yet been found. Considering the fact that the trophoblastic β1-glycoprotein (PSG) is a fetoplacental protein with immunosuppressive activity, short peptide fragments of this protein were studied in the formation of an immune response in a situation of allogeneic cell transplantation. To study the effect of PSG peptides (YECE, YQCE, YVCS, and YACS) on the levels of peripheral and local T-regulatory cells (Treg) during the formation of an immune response to the introduction of allogeneic bone marrow cells (BM) in a dynamic experiment on Wistar rats.&#x0D; Materials and methods. We used an original host-versus-graft model in male Wistar rats without preconditioning of recipients. Animals were injected with PSG peptide fragment composition against a background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment, in which the following parameters were evaluated: the level of peripheral "true" Tregs (CD4+CD25+FOXP3+), CD4+CD25-FOXP3+ cells, and FOXP3 expression in mesenteric lymph nodes. Material was collected on days 3 and 21 of the experiment.&#x0D; Results. PSG peptide administration against a background of allogeneic BM cells was found to reduce the absolute and relative amount of Treg in the peripheral blood of rats on days 3 and 21 of the experiment. PSG peptides against the background of the introduction of allogeneic BM cells reduced the absolute and relative amounts of CD4+CD25-FOXP3+ cells on the day 3 of the experiment. The introduction of PSG peptides against the background of the introduction of BM cells resulted in a relative decrease in FOXP3 expression in the T zone of mesenteric lymph nodes on the day 21 of the experiment.&#x0D; Conclusions. Thus, the PSG peptides did not have the expected effect on the level of peripheral and local Treg cells; moreover, the presence of the peptides led to a decrease in the number of these cells.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites β-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages.

Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.

Impact of stem cell selection between bone marrow and peripheral blood stem cells for unrelated hematopoietic stem cell transplantation for hematologic malignancies: on behalf of the Donor/Source Working Group of the Japanese Society for Transplantation and Cellular Therapy

Background aimsThis study aimed to comprehensively assess the impact of stem cell selection between bone marrow (BM) and peripheral blood (PB) in unrelated hematopoietic stem cell transplantation (HSCT) for hematological malignancies. Our objective was to identify specific factors associated with better transplant outcomes. MethodsA retrospective analysis was conducted using data from the Japanese HSCT registry. Inclusion criteria were patients aged 0–70 years who underwent their first unrelated HSCT with BM or PB, with an 8/8 or 7/8 allele HLA match for hematological malignancies between 2010 and 2020. ResultsAmong 10 295 patients, no significant difference was observed in overall survival, relapse, graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS) or non-relapse mortality between the groups. Patients who received PB showed no clear difference in acute GVHD but had a greater rate of chronic GVHD, resulting in poor chronic GVHD-free, relapse-free survival (CRFS). Subgroup analyses highlighted the importance of patient-specific factors in source selection. Patients with non-Hodgkin lymphoma and a greater hematopoietic cell transplantation-comorbidity index showed better CRFS and GRFS when BM was the preferred source. Similar trends were observed among patients with standard-risk disease for CRFS. However, no such trends were evident among patients aged 0–24 years, indicating that both sources are viable choices for young patients. ConclusionsThis real-world retrospective analysis showed similar basic outcomes for BM and PB in an unrelated setting. The results support that BM may still be preferred over PB, especially when the long-term quality of life is a major concern. A consideration of individual factors can further optimize transplant success. Further research is warranted to explore the long-term implications of stem cell source selection.

Epigallocatechin-3-Gallate (EGCG) Ameliorates Ineffective Hematopoiesis of Myelodysplasia Syndrome (MDS)

Epigallocatechin-3-gallate (EGCG) is a gallate ester obtained by condensation of gallic acid with the (3R)-hydroxy group of (-)-epigallocatechin, extracted from green tea, exhibiting beneficial effects as an apoptosis and cell differentiation inducer helping counteract uncontrolled proliferation and impaired differentiation characteristic of leukemia and myelodysplastic syndrome. Clinical trials in patients with chronic lymphoid leukemia, low grade B-lymphomas and acute myeloid leukemia with myelodysplasia related changes were carried out showing positive results. This study aimed to evaluate the in vivo effect of EGCG in a mice model of myelodysplastic syndrome (NHD13 mice). B6.SJL-PtprcaPep3b/BoyJ mice (PepBoy) received 9,5Gy irradiation followed by transplantation of bone marrow cells from NUP98-HOXD13 mice by i.v. caudal vein injection; disease establishment was confirmed at day 15 by blood cytopenia. To assess the EGCG effects on MDS, 10-week-old female NUP98-HOXD13 mice (NHD13 +) and their littermate WT mice (NHD13 -) were treated for 4 weeks with EGCG (50mg/Kg/day 5x/week i.p.) or vehicle. The animals were then sacrificed, and bone marrow and spleen were obtained for flow cytometry and histology analyses. Automated hematological counts and clinical biochemistry parameter analysis were done. After signing the written informed consent, a 71-year-old man diagnosed with high-risk MDS, with 9% blasts in the bone marrow, underwent a daily oral 800mg EGCG treatment. This study was approved by the Ethical Committee of University of Campinas and by the National Research Ethics Committee ( CAAE: 40656720.5.0000.5404). During this period, the patient underwent weekly clinical assessments and laboratory studies to monitor any potential toxicity. Peripheral blood samples were obtained before and after treatment to observe any alteration in cell counts and immune cell composition. Before treatment, NDH13 + exhibited a reduction in number of leukocytes, platelets, and hemoglobin levels, in accordance with the literature. EGCG treatment ameliorated hematological parameters, showed by increased leukocyte numbers (monocytes and neutrophils) and platelets with no signals of liver and kidney toxicity detected by histology and serum biochemistry. Untreated mice showed increased osteoprogenitor, osteoblast and endothelial cells in the bone marrow, corroborating the literature. EGCG treatment did not alter the frequency of these cells, HSC or immature cells (CD34 + or CD117 +). However, EGCG induced extramedullary hematopoiesis in spleen and no changes in the percentage/frequency of Foxp3 + regulatory T cells (CD4 +CD25 + and/or CD4 +FOXP3 +). Additionally, the patient treated by ECGC for one month exhibited reduction of regulatory T cells CD4 +CD25 + and CD4 +FOXP3 +, and increased CD8 + T cells (CD3 +CD8 +) and natural killer cells (CD16 +CD56 +) accompanied by increased mature leukocytes (monocytes and neutrophils), with no changes in hemoglobin and platelet number. Our initial findings indicate that EGCG induces improved hematological parameters in MDS and might shift immune cells to a more cytotoxic phenotype. The extramedullary hematopoiesis observed in mice may be due to reduction of CXCR4 in bone marrow cells, as previously described by our group. In summary, EGCG might be a promising adjuvant in the treatment of MDS. Further research and clinical studies are warranted to validate and fully explore EGCG therapeutic benefits. Fig1a EGCG ameliorates NHD13 and transplanted mice hematological parameters. At the end of the EGCG treatment (50mg/kg/day i.p) or vehicle (Ctrl), NHD13 + and transplanted (PepBoy) mice were bled from the retro-orbital plexus. EGCG induced an increased the number of leukocytes ( A), monocytes ( B), neutrophils ( C) and the number of platelets ( D) using the CELL-DYN Emerald Hematology System counter. EGCG represented by the green column, and the vehicle by the gray color. Statistical significance ( Mann-Whitney test) is indicated; n=6 per group. Fig1b. EGCG improved the immune system of HR-SMD patient. A 71-years-old man with HR-SMD received EGCG treatment (800 mg/Kg/day) for 30 days. Peripheral blood counts were evaluated at day 0 and day 30. EGCG ameliorated hematological parameters - increased mature leukocytes (A) - monocytes (B) and neutrophils (C), with no changes in hemoglobin (D) and platelets

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4 +/+ and Lama4 -/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4 -/- recipients showed significantly increased platelet counts in blood compared to Lama4 +/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4 -/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1 + cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4 -/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4 -/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4 -/- recipients (33.6%) than that in Lama4 +/+ recipients (41.5%), raising a possibility that Lama4 -/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4 -/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

Cellular Barcoding of JAK2-V617F Hematopoietic Stem Cells Reveals No Substantial Preferences in the Contribution of Individual Stem Cell Clones to Erythroid Versus Megakaryocytic Lineages

JAK2-V617F is the most frequent driver gene mutation in patients with myeloproliferative neoplasms (MPN) but the phenotypic manifestation is heterogeneous with some patients presenting as polycythemia vera (PV), whereas others with essential thrombocythemia (ET) or myelofibrosis (MF). JAK2-V617F is acquired in a hematopoietic stem cell (HSC) and leads to clonal expansion of HSCs and progenitor cells that gain dominance over unmutated hematopoiesis. We tested the hypothesis that the preferential expansion of megakaryopoiesis in ET versus erythropoiesis in PV could be due to the acquisition of JAK2-V617F in different subsets of HSCs with inherent bias towards megakaryopoiesis or erythropoiesis. In addition, we addressed the question of whether sensitivity or resistance to interferon-α (IFNα), currently the only treatment that can induce a deep molecular remission in some MPN patients, could be also due to heterogeneity in HSC subpopulations. We used a mouse model of JAK2-V617F driven MPN in combination with molecular barcoding that allowed us to monitor expansion and lineage contribution of individual HSC subclones. First, we genetically barcoded HSCs from our tamoxifen-inducible SclCre;JAK2-V617F ( VF) mice with a lentiviral vector, and transplanted 2'000 HSCs into lethally irradiated recipient mice. After 12 weeks we sequenced the barcodes in bone marrow (BM) progenitors by next generation sequencing (NGS). We observed a strong selection of 1-2 HSCs clones per recipient mouse, which alone contributed to &amp;gt; 80% of all the myeloid cells. Since cytokine storm after irradiation and aplasia during the reconstitution with lentivirally transduced BM may favor such oligo-clonal dominance observed in this experiment, we characterized the clonal composition of JAK2-mutant HSCs in non-transplanted MPN mice. To this end, we crossed a CRISPR-based genetic barcoding mouse line ( CARLIN) (Bowling, S. et al, Cell 2020) with our VF mice to obtain VF;CARLIN mice. Cas9-mediated barcoding in VF;CARLIN or in CARLIN mice with wildtype Jak2(WT;CARLIN) was induced with doxycycline and immediately followed by the activation of the JAK2-V617F by tamoxifen (Figure 1A). After 12 weeks, when the mice developed full PV phenotype (hemoglobin 195 g/L; platelets 4.3x10 12 /L and neutrophils 10.5x10 9/L), we performed scRNAseq on lin- cKit+ BM cells using the 10X-platform to retrieve the cellular barcodes and transcriptomic profiles. MPN hematopoiesis in these non-transplanted VF;CARLIN mice was polyclonal (Figure 1B). A total of &amp;gt; 500 barcoded clones per mouse were detected and in both VF;CARLIN and WT;CARLIN mice, but ~5 individual barcodes accounted for 25% of the cells in all progenitor subsets analyzed (Figure 1B). Overall, clonal composition was similar between WT;CARLIN and VF;CARLIN mice, indicating that clonal selection was not more prominent in JAK2-mutant mice. Importantly, in the offspring of HSCs that accounted for 25% of the cells, we did not observe preferential presence of some barcodes in MkP versus EryP and other barcodes in EryP versus MkP, arguing against the model predicting that erythrocytosis and thrombocytosis originated in different subsets of biased HSCs. To investigate the question of whether sensitivity or resistance to IFNα could be also due to heterogeneity in HSC subpopulations, we transplanted BM cells from these barcoded VF;CARLIN mice together with an excess of WT competitor cells into irradiated WT recipient mice, and treated them with IFNα or vehicle for 16 weeks. IFNα normalized hemoglobin (122 g/L) compared to vehicle (177 g/L) and reduced VF;CARLIN chimerism in granulocytes from 85% to 44%. BM cells were harvested and the clonal composition is currently being analyzed by NGS in order to identify IFNα resistant and sensitive subclones. Our barcoding analyses of individual HSCs showed that, during recovery after BM transplantations, a few VF clones dominated, whereas induction of VF expression in situ without transplantation resulted in a polyclonal disease that showed a similar number of HSCs actively contributing to hematopoiesis as in a control experiment with WT mice. The lack of substantial preference in the clonal contribution of individual HSCs to erythropoiesis versus megakaryopoiesis in VF mice suggests that PV and ET are not caused by acquiring JAK2-V617F in HSCs with a pre-existing lineage bias.

ABO Incompatibility Haploidentical Peripheral Blood Stem Cell Transplantation Combined with a Single Cord Blood Unit for Severe Aplastic Anemia Patients

Background: For young severe aplastic anemia (SAA) patients without HLA matched donors, haploidentical hematopoietic stem cell transplantation can be used as an alternative selection. The grafts of classic Beijing protocol in China is a combination of bone marrow and peripheral blood stem cell. For patients with major or minor blood type incompatibility, bone marrow stem cells need to undergo red blood cell or plasma removal treatment. There was rare reports about haploidentical peripheral blood stem cell combined with a single unrelated cord blood unit for ABO incompatibility haploidentical stem cell transplantation in SAA patients. Objective: To observe the efficacy of ABO incompatibility haploidentcial peripheral blood hematopoietic stem cell transplantation combined with a single unrelated cord blood unit for SAA patients. Method s : Fifty-five SAA patients underwent stem cell transplantation from August 2018 to February 2022 in the First Affiliated Hospital of Xi'an Jiaotong Univeristy were divided into two groups based on donor-recipient ABO compatibility, the donor-recipient ABO matched group（BM+PB group）using haploidentical bone marrow and peripheral blood stem cell as grafts, and donor-recipient ABO mismatched group（CB+PB group）, using unrelated cord blood and haploidentical peripheral blood stem cell as grafts. The differences of hematopoietic reconstitution, acute and chronic GVHD, CMV and EB virus infection, PFS, and OS between the two groups were compared. Result s : There were 30 cases in CB+PB group and 27 cases in the BM+PB group. Except for differences in donor-recipient ABO compatibility, the other characteristics of the two groups of patients were comparable((P&amp;gt;0.05). One patient in the BM+PB group had primary graft failure, while the rest of the patients were successfully implanted. There were no significant statistical differences in neutrophil,platelet and red blood cells recovery rates, the incidence of acute and chronic GVHD, CMV, EB virus infection, and PTLD between the two groups of patients(P&amp;gt;0.05). There was no significant statistical difference in OS between the two groups of patients(P&amp;gt;0.05)(Figure 1). Conclusion: For SAA patients with donor-recipient ABO incompatibility could use haploidentical peripheral blood cell combined with unrelated cord blood for transplantation which had compared outcome with haploidentcal bone marrow and peripheral blood cell transplantation.

Signaling Pathway Mutations Cooperate with the PICALM/MLLT10 Fusion in a Knock-in AML Mouse Model

The PICALM/MLLT10 (or CALM/AF10; C/A) is a rare but recurring fusion gene detected in patients with T-cell acute lymphoblastic leukemia, malignant lymphoma and acute myeloid leukemia (AML). Our lab has previously established a C/A knock-in mouse model, where the fusion gene preceded by a loxP-flanked transcriptional stop cassette was inserted into the Rosa26 locus (the R26LSLCA strain). To achieve tissue-specific expression of C/A, R26LSLCA mice were crossed with three different Cre-inducer lines, namely the Vav-Cre, MB1-Cre and CD19-Cre. In the Vav-Cre strain, Cre recombinase is expressed in the entire hematopoietic system, while in the MB1-Cre and CD19-Cre lines, Cre is expressed in the B-cell compartment. Using this model, Dutta et al. showed that C/A leads to leukemia only when expressed in all hematopoietic cells, including hematopoietic stem cells and does not lead to induction of leukemia when expressed in B-cells alone ( Dutta et al., Leukemia 2016). To further characterise leukemias driven by the C/A fusion, we established new R26LSLCA and Vav-Cre crosses. Similar to what was found previously, the Vav-Cre/R26LSLCA mice developed leukemia with a long median latency of 1 year (366.5 days; N=24) and complete penetrance. On post-mortem analysis, the Vav-Cre/R26LSLCA mice were found to have splenomegaly and leucocytosis. Expression of the fusion gene was confirmed in these mice by RT-PCR on the cDNA from bone marrow (BM) cells. Immunophenotyping analysis of the BM cells and splenocytes of these mice revealed a predominantly myeloid phenotype. To assess the effect of expression of C/A on the transcriptome, we performed RNA-seq analysis using BM cells from 17 leukemic mice and 12 healthy controls. In accordance with reports on C/A patient samples, the Hoxa genes Hoxa7 and Hoxa9 as well as the Hox-cofactor Meis1 were significantly upregulated in our C/A expressing mice. In addition, genes involved in B-cell development, such as Cd19, Pax5 and Ikzf3, and Gata1, which is essential for the development of erythrocytes, were downregulated. To prove that the Vav-Cre/R26LSLCA mice were indeed leukemic and had the potential to propagate the disease, we serially transplanted BM cells from 4 Vav-Cre/R26LSLCA mice into congenic recipients. The secondary (N=20) and tertiary (N=19) transplanted mice developed leukemia with a significantly shorter median latency of 21 and 17 days, respectively. Our group and others have shown that a single genetic lesion is in most cases not sufficient to cause leukemia. Thus, to determine if additional somatic mutations were acquired by the C/A knock-in mice, we performed whole exome sequencing on the DNA from BM cells of the 4 knock-in mice, some of their daughter secondary and tertiary leukemias as well as the corresponding germline DNA samples (N=19). We identified between 2-32 mutations per leukemia exome, including mutations in genes and/or members of gene families that have been implicated in human leukemias. Mutations were detected in the transcription factors Sfpi1, Zeb1, and Sox9; Aff2, a member of the Af4/Fmr2 gene family; and Sf3a2, a component of the RNA-splicing machinery, among others. In addition, in two C/A sister secondary leukemias and the corresponding tertiary leukemias, we detected a missense mutation at amino acid 507 (G507A) of the Ptpn11 gene, which is homologous to G507 of human PTPN11, a known hotspot mutation site in AML and juvenile myelomonocytic leukemia. Interestingly, Dutta et al. also reported a G507 mutation (G507V) in Ptpn11 in a C/A knock-in mouse. PTPN11 regulates the RAS/MAPK signaling pathway. This finding is reminiscent of what we have previously reported in a C/A driven murine bone marrow transplantation model, where we detected somatic mutations in several AML-associated signal transduction genes, including Flt3, Cbl and Kras ( Desai et al., Leukemia 2020). Taken together, our results strongly suggest that mutations in genes involved in signaling cascades, particularly those in the RAS/MAPK pathway, cooperate with the C/A fusion and lead to complete malignant transformation. We aim to functionally validate these findings by establishing mouse models that harbor the C/A fusion as well as mutations in Ptpn11 and other signal transduction genes. These murine models will be immensely valuable for gaining a deeper insight into PICALM/MLLT10-mediated leukemogenesis, studying cooperating mutations and for testing new targeted therapies.

Novel Mouse Model That Recapitulates the Hematologic Phenotype of Diamond Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that presents during early childhood and is characterized by macrocytic anemia, congenital malformations, and predisposition to cancer. More than 90% of DBA patients are diagnosed during their first year of life (median age 12 weeks). The pathogenesis of DBA is linked to loss-of-function mutations in genes encoding ribosomal proteins (RP), although mutations in 3 non-RP genes ( GATA1, TSR2, and HEATR3) have been detected. DBA patients present with a high degree of clinical heterogeneity with varying severity and responses to steroid therapy. DBA provides a unique disease model to study how RP deficiency impacts the phenotype and pathogenesis of ribosomopathies. However, one challenge is the lack of animal models that faithfully recapitulate the clinical features of DBA in terms of disease severity and onset age. Heterozygous loss-of-function mutations in RPL11 are found in 5-20% of DBA patients, but previous mouse models carrying Rpl11 haploinsufficiency have only a mild anemia. Here, we report a novel mouse model of DBA with inducible haploinsufficient expression of Rpl11 which results in a severe macrocytic anemia in juvenile mice. Mx1-CreRpl11+/flox experimental mice were generated by cross-breeding homozygous Rpl11 flox/flox and Mx1-cre breeders. Haploinsufficiency of Rpl11 ( Rpl11+/Δ) was induced by intraperitoneal injection of polyinosinic-polycytidylic acidto mice with Mx1-CreRpl11 +/flox on postnatal day 8 and 10. Diseased mice with Rpl11+/Δ showed macrocytic anemia at 2 weeks (wks) post-induction with hemoglobin (Hb) &amp;lt; 10 g/dL, eventually dying of severe anemia (Hb&amp;lt; 2 g/mL) by 30 wks (median survival 27 wks, p&amp;lt;0.001, n=7 )( Figure 1). We observed a 50% decreased expression of Rpl11 in blood neucleated cells from Rpl11+/Δ mice and several characteristic clinical features of DBA. Erythrocyte adenosine deaminase was significantly elevated in the blood of all Rpl11+/Δ mice when compared to wild type (WT) littermates (median 5 vs 1 EU/g Hg, p&amp;lt;0.001, n=6). Similarly, EPO concentrations were significantly elevated in Rpl11+/Δ mice compared to WT mice (median 38752 vs 23 pg/mL, p&amp;lt;0.01, n=6). On postmortem examination, the DBA mice displayed splenomegaly. FACS analysis data demonstrated that the differentiation of erythropoiesis is significantly blocked in peripheral blood, bone marrow (BM), and spleen of Rpl11+/Δ mice compared to WT mice (p&amp;lt;0.05, n = 5). In addition, methylcellulose colony assays revealed that CFU-E colonies were significantly decreased in BM from Rpl11+/Δ mice in comparison to WT mice (median 23.5 vs 40.5 per 0.2 million BM cells in the presence of EPO at a concentration of 1 unit/mL, p&amp;lt;0.05, n=4). To determine whether BM hematopoietic stem cells/progenitor cells from Rpl11+/Δ mice can transfer the DBA phenotype, we transplanted bone marrow cells of Rpl11+/ Δ mice (CD45.2+) into sublethally irradiated WT mice (CD45.1+). Our data demonstrate that the BM cells from Rpl11+/Δ donor mice can induce lethal macrocytic anemia in WT recipient mice. Studies on the molecular mechanisms underlying the dysregulated erythropoiesis in Rpl11+/Δ mice are ongoing. In conclusion, we have successfully generated the first DBA mouse model that recapitulates the hematologic features of DBA patients. This model provides an ideal tool to study the pathogenesis of DBA and other ribosomopathies.

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic β1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

Effects of bone marrow cells transplantation for acute myocardial infarction on cardiac function, infarct size, and clinical events: a meta-analysis.

Effects of bone marrow cells transplantation for acute myocardial infarction on cardiac function, infarct size, and clinical events: a meta-analysis.

Sickle Cell Disease

Sickle cell disease and its variants constitute the most common inherited blood disorders affecting millions of individuals worldwide. Significant information regarding the nature of the genetic mutations and modifier genes that result in increased or decreased severity of the disease are available. In recent years, detailed data regarding molecular genetics, pathophysiology, mechanisms for the development of symptoms and side effects of sickle cell disease have been published. The relationship of physiological changes, cellular interactions, coexisting coagulation disorders, effects of association with other genetic disorders and a number of intervening factors have been explored. New techniques for pre-conception, prenatal, in utero, and neonatal screening are available. Means for prediction of the severity of the disease, clinical course of the disorder, and prevention of some of its major complications have been developed. The effects of psychosocial and environmental factors have been explored. Various therapeutic strategies including bone marrow and stem cell transplantation are currently employed in the treatment of patients with sickle cell disease. Recent progress in understanding the molecular pathways controlling mammalian erythropoiesis and globin switching, as well as advances in genome engineering, particularly the gene-editing techniques, have opened a venue for genetic-based treatment of the disease. Currently, sickle cell disease is often associated with a high rate of complications and mortality. The development of new pharmacological agents, methods for gene therapy, and alterations and modification of the coexisting genetic factors and modifiers for treatment of the disease are encouraging.