Bone Marrow Cell Transplantation Research Articles

Promotion of Platelet Production by Co-Transplantation of Mesenchymal Stem Cells in Bone Marrow Transplantation.

Therapeutic strategies that can promote platelet production are in demand to enhance clinical outcomes of bone marrow transplantation (BMT). Our research group has studied human tonsil-derived mesenchymal stem cells (T-MSCs) and their effectiveness in promoting bone marrow (BM) engraftment. Here, we analyzed the effects of T-MSCs on platelet production and hemostasis. Donor BM cells (BMCs) were isolated from C57BL/6 mice and transplanted with or without T-MSCs to BALB/c recipient mice. Mice were sacrificed and blood cells were counted using an Auto Hematology Analyzer. Femur sections were stained with CD41 antibody to analyze megakaryocytes in the BM. Growth factor secretion from MSCs was analyzed using the Quantibody Array. Effects of T-MSC conditioned medium (CM) on megakaryopoiesis were investigated using the MegaCult assay. In a mouse model of BMT, T-MSC CM was injected with or without anti-placental growth factor (α-PlGF) blocking antibody, and blood cell numbers and coagulation were analyzed. T-MSC co-transplantation increased percent survival of BMT mice. Platelet numbers were significantly lower in the BMC-only group, whereas T-MSC co-transplantation restored circulating platelets to levels similar to those of the control group. Significantly reduced numbers of CD41 + megakaryocytes in Bu-Cy and BMC groups were increased by T-MSC co-transplantation. PlGF secretion from T-MSCs were detected and enhanced megakaryopoiesis, platelet production, and coagulation by T-MCS CM were disrupted in the presence of the α-PlGF blocking antibody. We demonstrated the effectiveness of T-MSC co-transplantation in promoting platelet production and coagulation after BMT. These findings highlight the potential therapeutic relevance of T-MSCs for preventing thrombocytopenia after BMT.

Adenosine A2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation.

Background: The role of adenosine A2A receptor (A2AR) in the ischemic white matter damage induced by chronic cerebral hypoperfusion remains obscure. Here we investigated the role of A2AR in the process of macrophage polarizations in the white matter damage induced by chronic cerebral hypoperfusion and explored the involved signaling pathways.Methods: We combined mouse model and macrophage cell line for our study. White matter lesions were induced in A2AR knockout mice, wild-type mice, and chimeric mice generated by bone marrow cells transplantation through bilateral common carotid artery stenosis. Microglial/macrophage polarization in the corpus callosum was detected by immunofluorescence. For the cell line experiments, RAW264.7 macrophages were treated with the A2AR agonist CHS21680 or A2AR antagonist SCH58261 for 30 min and cultured under low-glucose and hypoxic conditions. Macrophage polarization was examined by immunofluorescence. The expression of peroxisome proliferator activated receptor gamma (PPARγ) and transcription factor P65 was examined by western blotting and real-time polymerase chain reaction (RT-PCR). Inflammatory cytokine factors were assessed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR.Results: Both global A2AR knockout and inactivation of A2AR in bone marrow-derived cells enhanced M1 marker expression in chronic ischemic white matter lesions. Under low-glucose and hypoxic conditions, CGS21680 treatment promoted macrophage M2 polarization, increased the expression of PPARγ, P65, and interleukin-10 (IL-10) and suppressed the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The CGS21680-induced upregulation of P65 and IL-10 was abolished in macrophages upon PPARγ knockdown. The downregulation of TNF-α and IL-1β by CGS21680 was less affected by PPARγ knockdown.Conclusions: In the cerebral hypoperfusion induced white matter damage, A2AR signaling in bone marrow-derived cells induces macrophage M2 polarization and increases the expression of the anti-inflammatory factor IL-10 via the PPARγ-P65 pathway, both of which might explain its neuroprotective effect.

P107: A PHASE 2/3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY OF MITAPIVAT IN PATIENTS WITH SICKLE CELL DISEASE

Background: The key pathology in sickle cell disease (SCD), a life-threatening, hereditary hemoglobin (Hb) disorder, is red blood cell (RBC) sickling due to polymerization of deoxygenated sickle Hb (HbS), which can be exacerbated by increased levels of the glycolytic metabolite 2,3-diphosphoglycerate (2,3-DPG), and decreased ATP.1–3 Sickled RBCs are rigid, not deformable, and fragile, resulting in vaso-occlusion triggering pain and chronic hemolysis.4–6 SCD treatment options are limited, with an unmet need for safe and effective therapies to improve anemia and reduce pain. Mitapivat is an oral, activator of RBC pyruvate kinase (PKR), a key glycolytic enzyme. PKR activation decreases 2,3-DPG and increases ATP, which may reduce HbS polymerization, RBC sickling, and hemolysis in SCD.3,7–9 Data from the phase (ph) 1 National Institutes of Health multiple ascending dose study of up to 100 mg mitapivat twice daily (BID) in SCD (NCT04000165) showed that mitapivat was safe and tolerable, increased ATP and decreased 2,3-DPG in a dose-dependent manner, and improved anemia and hemolysis.8,10 Aims: To report the study design of RISE UP (NCT05031780, EudraCT: 2021-001674-34), a ph 2/3, double-blind, randomized, placebo-controlled, multicenter study evaluating the efficacy and safety of mitapivat in patients (pts) with SCD. Methods: Eligible: pts aged ≥16 yrs with documented SCD (HbSS, HbSC, HbSβ0/HbSβ+ thalassemia, other SCD variants), 2–10 sickle cell pain crises (SCPCs; acute pain needing medical contact, acute chest syndrome, priapism, hepatic or splenic sequestration) in the prior 12 months, and Hb 5.5–10.5 g/dL. If taking hydroxyurea (HU), the dose must be stable for ≥90 days before starting study drug. Ineligible: pts receiving regularly scheduled blood transfusions, with severe kidney disease or hepatobiliary disorders, currently receiving SCD therapies (excluding HU) or who have received gene therapy, bone marrow or stem cell transplantation. In the double-blind ph 2 part, 69 pts will be randomized (1:1:1) to 50 mg or 100 mg mitapivat, or placebo BID for 12 weeks (wks). The primary objective of ph 2 is to determine the recommended ph 3 mitapivat dose by evaluating anemia and safety vs placebo via the following endpoints: Hb response (≥1.0 g/dL increase in average Hb concentration over Wks 10–12 vs baseline [BL]), and type, severity, and relationship of adverse events and serious AEs. In the double-blind ph 3 part, 198 pts who did not participate in ph 2 will be randomized (2:1) to the selected ph 3 mitapivat dose or placebo BID for 52 wks, stratified by number of SCPCs in the prior yr (<5, ≥5) and HU use. The primary objectives of ph 3 are to determine the effect of mitapivat vs placebo on anemia, measured by Hb response (≥1.0 g/dL increase in average Hb concentration over Wks 24–52 vs BL), and the effect of mitapivat vs placebo on SCPC, measured by annualized rate of SCPCs. Key secondary endpoints include average change from BL in Hb concentration, indirect bilirubin, percent reticulocyte, and Pt-Reported Outcomes Measurement Information System® Fatigue 13a Short Form score over Wks 24–52; and annualized frequency of hospitalizations for SCPC. Pts who complete the double-blind period will be eligible to receive mitapivat in the open-label extension period in each ph. Results: Not yet available. Conclusion: This ph 2/3 study will investigate the efficacy and safety of mitapivat in pts aged ≥16 yrs with SCD. Enrollment is currently ongoing.

Residual effects of busulfan and irradiation on murine hematopoietic stem and progenitor cells

Exposure of young C57BL/6 (B6) mice to two courses of busulfan (BSF) injections or two rounds of sublethal total-body irradiation (TBI) induced significant damage to the function of hematopoietic stem and progenitor cells (HSPCs). Fifteen weeks after treatment, BSF- and TBI-treated mice had reduced white blood cells without significant change in red blood cells or platelets, indicating that BSF and TBI hematotoxicity was chronic, with leukocytes being the major targets. Hematopoietic damage induced by BSF or TBI persisted long term. Residual adverse effects were reflected by significantly decreased CD45R B cells and reduced recovery of total bone marrow cells, especially HSPCs carrying markers for KSL (Kit+Sca-1+Lin-) cells, multipotent progenitor (MPP) cells (KSLCD34+CD135+), myeloid progenitor (MP) cells (Kit+Sca-1-Lin-), and common lymphoid progenitor (CLP) cells 62 wk posttreatment. Transplantation of bone marrow (BM) cells from BSF and TBI donors at 49 weeks after treatment into lethally irradiated hosts resulted in decreased engraftment of CD45R B cells in blood and reduced reconstitution of BM HSPCs including KSL cells, short-term hematopoietic stem cells (KSLCD34+CD135-), MPP cells, and MP cell subsets. TBI donor had better reconstitution of CLP cells in recipients posttransplantation than did BSF donor, suggesting an impact of TBI and BSF on B cells at different development stages. In summary, BSF and TBI exposure produced long-lasting adverse effects on hematopoiesis with pronounced effects on mature B cells, immature ST-HSCs, and hematopoietic progenitor cells. Our results may have implications for therapy of human diseases.

Top 100 Cited Articles on Hematopoietic Stem Cell Transplantation: A Bibliometric Analysis

Background: Research performance is evaluated by bibliometric analysis of citations, and this can help to identify the most impactful articles in the field of hematopoietic stem cell transplantation (HSCT). We conducted a bibliometric analysis to identify scholarly impact and factors associated with the top 100 cited articles on HSCT.Methods: Thomas Reuters Web of Science core collection (WoS) was accessed in January 2021 and a title-specific search was conducted using the keywords “hematopoietic stem-cell transplantation ”, “transplantation, hematopoietic stem cell,” “stem cell transplantation, hematopoietic,” “bone-marrow transplantation,” “transplantation, bone marrow,” “grafting, bone marrow,” “transplantation bone marrow cell,” “bone marrow cell transplantation,” “stem cell transplant,” “allogeneic hematopoietic stem cell transplant,” “autologous hematopoietic stem cell transplant”. Non-Human and non-HSCT studies were excluded. A total of 39,406 records were identified and a list of the top 100 articles was made. Articles included in our study were characterized by the number of citations, year of publication, topic, study design, authors, h-index, and institutions. Data were analyzed using SPSS in April 2021. Bivariate analyses, using chi-square and t-test, and linear regression analyses were performed.Results: The 100 most cited articles in the field were published over 52 years from 1968 to 2020, with a maximum number of articles (n=40) published in the 1990s decade. Top-100 articles were cited 62,002 times with a median citation count of 465 (range 336-2240). The top-cited articles originated from 12 countries and the United States (US) contributed 69 articles. The University of Washington Fred Hutchinson Cancer Center (n=15) was the leading institution. Blood (n=32) and New England Journal of Medicine (n=31) made the greatest contribution, and 41 manuscripts were clinical trials. In a multivariate regression model, the first author's h-index (regression coefficient 5.46, 95% confidence interval 2.99-7.93, p<0.001) correlated with the citation count while gender, journal impact factor, years since publication, and the number of authors did not have a significant association.Conclusion: Our study highlights the most influential articles on clinical HSCT, centralization of research to North American and European institutions, and provides valuable insight for future research needs of the specialty. [Display omitted] DisclosuresAbhyankar: Incyte/Therakos: Consultancy, Research Funding, Speakers Bureau. McGuirk: Novartis: Research Funding; Pluristem Therapeutics: Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Astelllas Pharma: Research Funding; Magenta Therapeutics: Consultancy, Honoraria, Research Funding; Allovir: Consultancy, Honoraria, Research Funding; Gamida Cell: Research Funding; EcoR1 Capital: Consultancy; Bellicum Pharmaceuticals: Research Funding; Novartis: Research Funding; Fresenius Biotech: Research Funding; Kite/ Gilead: Consultancy, Honoraria, Other: travel accommodations, expense, Kite a Gilead company, Research Funding, Speakers Bureau.

IL13 Contributes to Fibrotic Progression of the Myeloproliferative Neoplasms

Pre-fibrotic-primary myelofibrosis PMF (Pre-PMF) is an indolent form of PMF that frequently progresses to overt-PMF. While both stages of the disease are characterized by the presence of dysplastic megakaryocytes, progression to fulminant disease is associated with significantly increased fibrosis in the bone marrow. We have previously shown that megakaryocyte maturation in overt-PMF is impaired due to a GATA1 deficiency. However, in Pre-PMF patients most megakaryocytes express GATA1. This raises the possibility that alterations in megakaryocyte development occur during the progression of the disease and may contribute to fibrosis. This progression in megakaryocyte defects is likely driven not only by the aberrant JAK/STAT signaling but also microenvironmental factors. To identify such factors, we performed studies in two mouse models of PMF (driven by JAK2V617 and MPLW515L mutations) before and after development of fibrosis. Using an unbiased approach, we measured the levels of different cytokines in the bone marrow, plasma, and spleen. In addition, we performed single cell RNAseq in bone marrow populations. We observed extensive changes in the level of cytokines in the bone marrow of the MPLW515L mouse model compared to the JAK2V617F model. We initially focused on those cytokines that are elevated in the bone marrow of both murine models, including IL13 (Figure 1A), because previous studies have shown that IL13 is elevated in PMF patients and that JAK2 inhibitors do not decrease IL13 (1-3). Moreover, elevated IL13 has been identified in patients who progress to secondary AML (2). How IL13 may contribute to the progression of the disease has not been investigated.We assayed the effect of IL13 on megakaryocytes in vitro and discovered that it promoted megakaryocyte differentiation in the absence of thrombopoietin (TPO) and potentiated the effect of TPO. This effect was observed in cultures of both wild-type and MPLW515L megakaryocytes. Next, we assayed for expression of the IL13 receptor (Il13ra1) in the bone marrow of JAK2V617F and MPLW515L mutant mice and found that it was highly upregulated compared to wild-type animals. IL13ra1 expression was particularly intense in the megakaryocyte lineage, and its expression increased with disease progression (Figure 1B). Next, we asked whether IL13 is essential for myeloproliferative neoplasm (MPN) development in vivo. To study this, we transplanted bone marrow cells from Il4/13 f/f Mx1-Cre mice expressing MPLW515L to irradiated recipients, waited until MPN developed, and then excised by pIpC injection. This experiment revealed that loss of IL13 and IL4 led to a profound reduction in disease burden (Figure 1C), decreased splenomegaly, and diminished degree of bone marrow fibrosis. Moreover, loss of IL13 and IL4 decreased the levels of pro-inflammatory cytokines in the bone marrow and spleen (Figure 1D). We attribute this effect to deletion of IL13 because IL4 was only moderately increased in the bone marrow of the MPLW515L mouse model, and because IL4 has been reported to not be altered in the MPNs.Finally, we performed single cell RNA-seq on bone marrow cells from mice transplanted with JAK2V617F or control progenitor cells early and late in the disease process (Figure 1E). Our results revealed that there was decreased myeloid progenitors but an enhancement in the mast cell lineage that tracked with the degree of fibrosis. We confirmed the presence of elevated numbers of mast cells in the bone marrow by immunohistochemistry (Figure 1F). Mast cells produce IL13, and therefore they are the likely source for the increased IL13. Finally, consistent with the observation that IL13 signaling is primarily mediated through STAT6, we found enrichment of STAT6 target genes in megakaryocyte progenitors from the late timepoint in our scRNAseq data (Figure 1G).In summary, our data demonstrate that IL13 is involved in the progression of PMF and that inhibition of the IL13 signaling pathway should be investigated as a therapeutic option in PMF.1. Tefferi A, et al. J Clin Oncol (2011)2. Fisher DAC, et al. Leukemia (2019)3. Chen P, et al. Front Med (2021) [Display omitted] DisclosuresCrispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy.

Phase II Clinical Trial of Abatacept for Steroid-Refractory Chronic Graft Versus Host Disease

Phase II Clinical Trial of Abatacept for Steroid-Refractory Chronic Graft Versus Host Disease

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

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

Ablation of collagen VI leads to the release of platelets with altered function

AbstractHemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI–related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI–null mouse model (Col6a1−/−), we found that collagen VI–null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1−/− megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1−/− mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1−/− mice. Peripheral blood platelets of patients affected by collagen VI–related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI–related diseases.

Long-term alterations in somatosensory functioning in survivors of childhood cancer.

Cancer and its treatment can have lasting consequences on somatosensation, including pain, which is often underrecognized and undertreated. Research characterizing the impact of cancer on pain and sensory processing in survivors of childhood cancer is scarce. This study aimed to quantify generalized differences in pain and sensory processing in survivors of childhood cancer compared with reference data using a standardized thermal and mechanical quantitative sensory testing (QST) protocol. The association between demographic, clinical (eg, leukemia vs other cancers and treatment exposures), and psychosocial (eg, anxiety and pain catastrophizing) variables and sensitivity to pain and sensory stimuli were also evaluated. Participants were 56 survivors of various types of childhood cancer (52% male, Mage = 13.5 years, SD = 3.2, range = 8-17 years). On average, children were 7 years (SD = 4.1, range = 1.2-16.5) post treatment. Almost all participants (86%) had at least 1 abnormal QST parameter compared with age- and sex-matched reference data; however, few participants self-reported the presence of sensory abnormalities. Generally, participants exhibited reduced sensitivity across the QST parameters examined (Ps < 0.05, ds = 0.40-3.45). A significant minority (45%) also exhibited pain sensitization (P <0.001, d = 0.42). Several risk factors for changes in sensory processing were identified, including current age, history of leukemia, certain treatment exposures (eg, vincristine cumulative dose, major surgery, and bone marrow or stem cell transplant), time off treatment, and higher anxiety and pain catastrophizing scores. Overall, this study demonstrated that somatosensory changes are prevalent in survivors of childhood cancer years after the completion of treatment. Future research is needed to understand long-term implications of altered somatosensation in this complex population.

Treatment-induced arteriolar revascularization and miR-126 enhancement in bone marrow niche protect leukemic stem cells in AML

BackgroundDuring acute myeloid leukemia (AML) growth, the bone marrow (BM) niche acquires significant vascular changes that can be offset by therapeutic blast cytoreduction. The molecular mechanisms of this vascular plasticity remain to be fully elucidated. Herein, we report on the changes that occur in the vascular compartment of the FLT3-ITD+ AML BM niche pre and post treatment and their impact on leukemic stem cells (LSCs).MethodsBM vasculature was evaluated in FLT3-ITD+ AML models (MllPTD/WT/Flt3ITD/ITD mouse and patient-derived xenograft) by 3D confocal imaging of long bones, calvarium vascular permeability assays, and flow cytometry analysis. Cytokine levels were measured by Luminex assay and miR-126 levels evaluated by Q-RT-PCR and miRNA staining. Wild-type (wt) and MllPTD/WT/Flt3ITD/ITD mice with endothelial cell (EC) miR-126 knockout or overexpression served as controls. The impact of treatment-induced BM vascular changes on LSC activity was evaluated by secondary transplantation of BM cells after administration of tyrosine kinase inhibitors (TKIs) to MllPTD/WT/Flt3ITD/ITD mice with/without either EC miR-126 KO or co-treatment with tumor necrosis factor alpha (TNFα) or anti-miR-126 miRisten.ResultsIn the normal BM niche, CD31+Sca-1high ECs lining arterioles have miR-126 levels higher than CD31+Sca-1low ECs lining sinusoids. We noted that during FLT3-ITD+ AML growth, the BM niche lost arterioles and gained sinusoids. These changes were mediated by TNFα, a cytokine produced by AML blasts, which induced EC miR-126 downregulation and caused depletion of CD31+Sca-1high ECs and gain in CD31+Sca-1low ECs. Loss of miR-126high ECs led to a decreased EC miR-126 supply to LSCs, which then entered the cell cycle and promoted leukemia growth. Accordingly, antileukemic treatment with TKI decreased the BM blast-produced TNFα and increased miR-126high ECs and the EC miR-126 supply to LSCs. High miR-126 levels safeguarded LSCs, as shown by more severe disease in secondary transplanted mice. Conversely, EC miR-126 deprivation via genetic or pharmacological EC miR-126 knock-down prevented treatment-induced BM miR-126high EC expansion and in turn LSC protection.ConclusionsTreatment-induced CD31+Sca-1high EC re-vascularization of the leukemic BM niche may represent a LSC extrinsic mechanism of treatment resistance that can be overcome with therapeutic EC miR-126 deprivation.Graphic abstract

Stem Cell Therapy for the Treatment of Myocardial Infarction: How Far Are We Now?

Myocardial infarction is one of the leading causes of death worldwide. Poor functional recovery of the myocardium is noticed after an event of myocardial infarction. Researchers and clinicians around the world have been engaged to regenerate the damaged human heart for a long time. Stem cell therapy is an exciting newer therapy to treat cardiovascular diseases.Various types of stem cells have been used to revive the damaged myocardium after myocardial infarction, and they have overall demonstrated safety and moderate efficacy. The specific mechanisms by which these cells help in improving cardiac function are still not completely known. There is growing evidence that intracoronary bone marrow cell transplantation in patients with myocardial infarction beneficially affects the remodeling of the damaged myocardium.Our systematic review article aims to assess the effects and the future of stem cell therapy in patients with myocardial Infarction. We searched articles in PubMed, ScienceDirect, and Google Scholar. Thirty-one studies that included 2171 patients in total were analyzed. Most of these studies showed stem cell therapy is safe and well tolerated in patients, and modest improvements are seen in left ventricular functions with no major adverse effects. However, some studies showed no positive and clinically significant outcomes. So, more high-quality studies on a larger scale are required to support and confirm its efficacy in remodeling damaged myocardium after myocardial infarction. We should also perform studies to determine the timing of cell delivery that is best suited for stem cell therapy.

Early Endothelial Activation in a Mouse Model of Graft vs Host Disease Following Chemotherapy.

Allogenic hematopoietic stem cell transplant (allo-HSCT) can lead to sinusoidal obstruction syndrome (SOS) and graft-versus-host disease (GvHD) in some individuals. GvHD is characterised by an immune triggered response that arises due to donor T cells recognizing the recipient tissue as “foreign”. SOS results in impaired liver function due to microvascular thrombosis and consequent obstruction of liver sinusoids. Endothelial damage occurs following chemotherapy and allo-HSCT and is strongly associated with GvHD onset as well as hepatic SOS. Animal models of GvHD are rarely clinically relevant, and endothelial dysfunction remains uncharacterised. Here we established and characterised a clinically relevant model of GvHD wherein Balb/C mice were subjected to myeloablative chemotherapy followed by transplantation of bone marrow (BM) cells± splenic T-cells from C57Bl6 mice, resulting in a mismatch of major histocompatibility complexes (MHC). Onset of disease indicated by weight loss and apoptosis in the liver and intestine was discovered at day 6 post-transplant in mice receiving BM+T-cells, with established GvHD detectable by histology of the liver within 3 weeks. Together with significant increases in pro-inflammatory cytokine gene expression in the liver and intestine, histopathological signs of GvHD and a significant increase in CD4+ and CD8+ effector and memory T-cells were seen. Endothelial activation including upregulation of vascular cell adhesion molecule (VCAM)- 1 and downregulation of endothelial nitric oxide synthase (eNOS) as well as thrombosis in the liver indicated concomitant hepatic SOS. Our findings confirm that endothelial activation is an early sign of acute GvHD and SOS in a clinically relevant mouse model of GvHD based on myeloablative chemotherapy. Preventing endothelial activation may be a viable therapeutic strategy to prevent GvHD.

Accommodating clinical trials and other externally manufactured cellular therapy products: challenges, lessons learned and creative solutions

Background aimsThe Division of Transfusion Medicine and Cellular Therapy at New York-Presbyterian/Columbia University Irving Medical Center (NYPH-CUIMC) comprises the immunohematology laboratory and blood bank (transfusion service), hemotherapy/apheresis and the cellular therapy laboratory (CTL). The CTL processes and stores hematopoietic progenitor cells for bone marrow transplantation from all currently acceptable sources, including bone marrow, peripheral blood and umbilical cord blood. The laboratory provides services for the pediatric and adult blood and marrow transplant programs at both NYPH-CUIMC and the Morgan Stanley Children's Hospital of New York-Presbyterian. The laboratory processes and stores approximately 200 cellular therapy products per year, and the division participates in numerous clinical trials within the institution and with external pharmaceutical manufacturing facilities. As a licensed tissue bank, commercial chimeric antigen receptor T-cell products and other cellular-based therapies that are approved by the US Food and Drug Administration (FDA) are routed through the CTL for storage, processing, coordination of transportation, chain of custody and, eventually, thaw and distribution. Currently, we are distributing four such products from Kite Pharma, Novartis and Bristol Meyers Squid. In comparison to the amount of work, we employ a small staff of only four full-time clinical laboratory technologists, one technical specialist, one quality and operations manager and one medical director. In recent years, the growing use of the hematology/oncology service and the introduction of new immunotherapies have put significant pressure on our division to accommodate and scale up operations. Accompanying this growth is a mounting administrative burden and upsurge in regulatory and protocol-specific oversight. The purpose of this article is to share lessons learned and creative solutions to help better accommodate the surge in development of novel cellular therapies. MethodsTo handle this increase in demand, the CTL worked to standardize administrative procedures, implement comprehensive document control solutions, digitize data collection and implement lean design concepts to increase effectiveness and mitigate risk. ResultsDistribution of a standard operating procedure for clinical trial management improved the “on-boarding” process and allowed the laboratory to have more influence in the decision-making process. Implementation of digital workflows and a comprehensive document control system allowed for improved organization of critical documents and proved to be flexible enough to accommodate various protocol-specific requirements. Introduction of visual ques and reorganization of the workspace facilitated better organization, mitigated risk and assisted the laboratory in maintaining regulatory compliance. ConclusionsBetter-defined, structured and controlled laboratory processes helped laboratory management implement new clinical trials and track critical data. Implementing digital solutions using widely available tools like Microsoft SharePoint has proven to be a very secure, low-cost and flexible solution to keeping us “up-to-date” and inspection-ready.

Transplantation of Bone Marrow Cells Preactivated With Sodium Nitroprusside Improves Acute Wound Healing in Rabbits.

The development of wound healing impairment mainly represents challenging clinical problems. The less and high concentrations of nitric oxide can influence angiogenesis, remodeling, and proliferation of skin cells. Delayed acute wounds generally have failed to progress via the normal stages of healing. Such wounds usually enter a state of pathological inflammation due to a postponed, incomplete, and uncoordinated healing process. This study aimed to investigate the effect of normal bone marrow cells (BMCs) and preconditioning of BMCs with minimum concentrations of sodium nitroprusside (NaNP) solution for acute wound healing. For acute wound healing, full-thickness dorsal wounds were created on rabbits. The acute wound of rabbits was treated with BMCs and preactivated BMCs with NaNP. Histological results showed that BMCs preactivated with NaNP could improve collagen deposition, enhanced reepithelization, and decreased inflammatory infiltration. Overall, BMCs treated with NaNP can help to improve acute wound healing in rabbits. The result strongly confirmed the beneficial effect in augmenting the wound healing process. The combination of BMCs with NaNP was safe and convenient for acute wound healing.

Bone Marrow Transfer in Relapsing-Remitting EAE Ameliorates Disease at First Remission, with No Synergistic Effect upon Co-Transplantation with Mesenchymal Stem Cells.

Multiple sclerosis (MS) is a neurological disorder characterized by an autoimmune response, demyelinating plaques and axonal damage. Intense immunosuppression (II) followed by autologous hematopoietic stem cell transplantation has been proposed as a treatment in severe forms of MS. We have used murine relapsing-remitting (RR) experimental autoimmune encephalomyelitis (RR-EAE) to evaluate the transplantation of syngeneic bone marrow cells (BMC) after II, in combination with mesenchymal stem cells (MSCs) as a new therapeutic adjunct capable of improving immune reconstitution. In EAE-affected mice treated with BMC alone, we observed a drastic reduction in the clinical course only during the early RR phase of the disease. There was no difference in the RR-EAE clinical course between mice treated with BMC alone and co-transplanted mice. To analyze the immune reconstitution, we quantified the circulating immune cells in naïve and RR-EAE-affected mice after II, with BMC alone or in combination with MSC. Although II resulted in reduced numbers of circulating immune cells, reconstitution did not differ in co-transplanted mice. During the early phase of the disease, IL-4 was significantly elevated in co-transplanted mice, as compared to those treated with BMC alone. These data suggest that BMC transplantation after II transiently ameliorates the clinical symptoms of RR-EAE, but that co-transplantation with MSC has no synergistic effect.

Serially Transplantable Patient-Derived Xenograft Model of Chronic Myelomonocytic Leukemia (CMML)

Introduction: The establishment of preclinical mouse models for MDS or MDS/MPN overlap syndromes from patient cells often fails or results in low engraftment. If successful, engrafting cells in primary mouse recipients usually do not engraft secondary or later recipients, thus precluding genetic manipulation of the cells for functional studies. We hypothesized that oncogenic complementation of patient cells would a) allow engraftment and serial transplantation, b) enable genetic modification of the cells for functional evaluation and c) preserve the disease-associated biology of the cell. To study these hypotheses we chose the MN1 oncogene for complementation of cells from a patient with chronic myelomonocytic leukemia (CMML). Importantly, healthy cord blood cells, when transduced with MN1, induce myeloproliferation in vivo, but cannot be serially transplanted and do not induce acute myeloid leukemia (Imren et al. Blood 2014).Aim: To establish a serially transplantable CMML patient-derived xenograft (PDX) model and perform a targeted in vivo shRNA screening in this model.Patient and Methods: Bone marrow (BM) cells were harvested from a CMML patient with 15% blasts in BM (CMML-2). BM cells were depleted for T cells and transduced with a lentiviral vector encoding MN1 and GFP. One million MN1-transduced or untransduced cells were injected intravenously in sublethally irradiated NOD/SCID IL-2Rγ-/- mice transgenic for SCF, IL3 and GM-CSF (NSGS). BM and spleen cells from engrafted mice were immunophenotyped and retransplanted in NSGS recipients up to 6 times. Mutations were evaluated by whole exome sequencing and confirmed by Sanger sequencing. A targeted shRNA library was generated and lentivirally transduced in serially transplantable CMML cells. Representation of transduced cells was monitored by next-generation sequencing of individual shRNAs.Results: MN1 transduction of human CMML cells resulted in mean engraftment of human CD45+ cells of 9% in peripheral blood (PB) and 41% in BM at week 12 after first transplantation. In contrast, untransduced CMML cells were hardly detected in PB and BM even 22 weeks after transplantation (mean engraftment 1.1% and 1.7%, respectively). Transplantation of BM cells from primary into secondary recipients led to stable engraftment of MN1-transduced CMML cells in PB (11%), spleen (4%) and BM (26%). In contrast, transplantation from primary to secondary recipients of untransduced CMML cells showed no engraftment in PB over a period of 40 weeks. Importantly, MN1+CMML cells have been serially transplanted up to the sixth recipient with good engraftment levels in PB, BM and spleen and could be re-derived from frozen cells. Overall, MN1+CMML cells could be expanded in vivo to allow engraftment in 33 mice so far.MN1+CMML cells in spleen and BM showed a myeloid phenotype expressing CD33, CD38 and CD14, but lacked expression of lymphoid markers CD3 and CD19. The stem and progenitor phenotype CD34+CD38- was found in 2.5% of these cells. By morphologic evaluation, monocytic cells prevailed in BM up to the third transplantation, while blast cells increased in later transplantations. Molecular analysis of primary patient cells identified mutations in BCOR, DNMT3A, U2AF1, NOTCH1 and NRAS . All mutations were still present in human cells isolated from recipient mice after the 4th and 6th transplantation, confirming the genetic stability of the model.A shRNA pool directed against the above mentioned mutated genes, MN1, and luciferase was lentivirally transduced into cells from the fifth recipient and transplanted into the sixth recipient. After 8 weeks, shRNAs against luciferase and BCOR were well preserved in vivo in MN1+CMML cells, while shRNAs against U2AF1 and MN1 were depleted. Thus, MN1+CMML cells remained dependent on at least one additional gene besides MN1, thus validating our hypothesis that the disease biology is preserved in our induced PDX model.Conclusion: We present a novel approach to establish serially transplantable MDS/MPN-PDX models by complementation with the MN1 oncogene. Our CMML model preserves the genetic and morphologic characteristics of the disease and allows dissection of the functional architecture of CMML and preclinical drug studies in this underserved disease. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stem Cells from Patients with Myelodysplastic Syndrome (MDS) Engraft and Migrate in-between the Humanized Niches in Immuno-Compromised Mouse Models

IntroductionMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized mainly by ineffective hematopoiesis. Xenograft models of MDS in immunodeficient mice have been instrumental in improving our understanding of clonal origin and evolution of this disease (Mian & Rouault-Pierre et al., Nat Commun. 2015; Rouault-Pierre & Mian et al Haematologica 2016; Leukemia, 2017); however, the low level of engraftment generally observed in these mouse models makes them impractical for testing therapeutic strategies. We used a 3D humanized scaffold in vivo system to test the MDS stem cell engraftment as well as study the cross talk between MDS-Initiating cells (MDS-ICs) and their niche.MethodologyIn this study bone marrow (BM) cells from 31 patients (27 MDS cases covering all WHO subtypes, 1 CMML, 1 MDS/MPD and 2 sAML) were studied. Initially, whole-exome sequencing was performed on all cases. Colony-forming assay was used to assess the colony forming ability of BM CD34+ cells from each patient. Mesenchymal stromal cells (MSCs) were isolated from the BM of the patient samples. We then used 3 immunodeficient mouse models (NSG, NSG-SGM3 and NBSGW) to study the engraftment kinetics and clonal evolution of the transplanted cells in gelatin-based 3D scaffolds coated with patient MSCs (RARS/RCMD, autologous; RAEB, autologous/allogenic).ResultsBM MSCs obtained from the MDS patients showed variable expansion levels, with the ability to differentiate into the adipogenic lineage whereas the osteogenic differentiation was highly aberrant.Next, 0.5x106 to 3x106 patient (n=31) mononuclear cells (MNCs T cell depleted) were seeded into 3D-scaffolds prior to implantation in NSG-SGM3 and/or NSG mice. We observed persistent long-term engraftment of hCD45+ cells ranging from 0.05% to 80% (up to 18 weeks). Importantly, 74% of the cases generated an engraftment level of >20% hCD45+ cells. Similar engraftment levels were observed in mice humanized scaffolds that were injected with BM cells from MDS/MPD, CMML and sAML (hCD45+ 10%, 22% and 86%, respectively). No difference in the hCD45+ cell engraftment between the patients with low-risk or high-risk MDS was observed.Remarkably, our 3D humanized scaffolds generated multi-lineage engraftment, with mice that received BM cells from RAEB cases yielding higher myeloid engraftment compared to RARS and RCMD (RARS hCD33+ 44%; RCMD hCD33+ 46.6%; RAEB hCD33+ 65%). Furthermore, transplantation of BM cells from 5 patients into NBSGW mice gave rise to engraftment levels of hCD45+ cells similar (hCD45+ cells, up to 40%) to the NSG and NSG-SGM3 mice. The MDS cells recovered from the mice maintained their genotypic characteristics, as demonstrated by the presence of the mutations that remained largely unchanged between the pre- and post-transplanted samples.Interestingly, Immuno-staining of the scaffolds revealed colonization by the host murine vasculature (mCD31+ endothelial cells). In line with this finding, mouse hematopoietic cells were also found to have migrated into the implanted humanized scaffolds. Surprisingly, we did not observe hCD45+ cell engraftment in the bone marrow of any of the xenotransplanted mouse strains (NSG or NSG-SGM3 mice with or without Irradiation and non-irradiated NBSGW mice). However, we demonstrated that MDS stem cells migrate by implanting a supplementary scaffold into NSG-SGM3 and NBSGW mice that was only coated with MDS-MSCs. Notably, these MDS-MSC seeded scaffolds were colonized by the circulating human MDS cells.Finally, we tested whether the cells recovered from the primary engrafted 3D humanized scaffolds would be able to generate secondary transplants. Our data shows that these cells were able to engraft in the secondary mice implanted with humanized 3D scaffolds.DiscussionOur model provides evidence that MDS BM cells are highly dependent on humanized MSC niche, confirming the importance of the cross-talk between MDS-ICs and MDS niche. Our data demonstrates that MDS stem cells do migrate out of the 3D Scaffold but require their “own niche” for colonization. Thus, our in vivo 3D model system, can be reliably used to better dissect the cross-talk between MDS stoma and MDS-ICs. This model requiring relatively low number of human MNCs, provides a robust preclinical assessment tool to study clonal evolution as well as to test therapeutic strategies on the MDS clonality prior to treatment of MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combined CCR5 and CXCR3 Blockade Attenuates Murine Agvhd through Alternating Donor-Derived T Cell Distribution and Function

Acute graft-versus-host disease (aGVHD) is one of the major complications and a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Chemokines and their cognate receptors play an important role in directing the migration of donor derived T cells to target organs. Chief among these chemokine receptors are CXCR3 and CCR5. Our previous study found that CCR5 blockade combined with cyclosporine A in a murine model could attenuate the severity of aGVHD in a less extent. This might be due to multiple expression of chemokine receptors on immune cell, only one kind receptor blockade might not completely block the migration of T cells to aGVHD target organs. In this study, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD, and also explored its immunologic mechanism.BALB/c(H-2Kd)recipients received 8.0 Gy of total body irradiation (60Coγsource) prior to transplantation followed by the infusion of bone marrow cells (BMCs) and splenic cells (SCs) from C57BL/6J(H-2Kb)or CCR5 knockout mice(H-2Kb)to establish an allogeneic bone marrow transplantation model. Body weight change, clinical scores of aGVHD, degree of histopathologic damage and survival rate were dynamically recorded to assess the efficacy of CCR5 and CXCR3 blockade in this transplantation model. Mechanically, donor derived lymphocytes were labeled with fluorescence dye to detect their distribution in vivo. Immunohistochemical analysis for CD4+ and CD8+ were performed on target organs of recipient mice at 7 days post-transplantation. Flow cytometry was used to calculate the absolute number of T cells in target organs, and to detect its proliferation, activation and differentiation in the spleen. ELISA was used to determine the concentration of T cells related cytokines.The results showed that both CCR5 and CXCR3 are enhanced during the aGVHD process. Although transplantation of allogeneic BMCs and SCs induced severe clinical aGVHD with high aGVHD scores (4.67±0.33) and a lethality of 90%, combined CCR5 and CXCR3 blockade attenuated clinical signs of aGVHD, reduced aGVHD scores (1.25±0.25, P< 0.001), reduced aGVHD-induced death (a lethality of 40%, P< 0.05). Compared with aGVHD group, combined CCR5 and CXCR3 blockade reduced liver histologic score (3.3±0.3 versus 1.3±0.3, P< 0.05), lung histologic score (3.6±0.3 versus 1.3±0.3, P< 0.05) and intestine histologic score (3.7±0.3 versus 1.3±0.3, P< 0.05). On days 7 after transplantation, more fluorescent signal emanated from the spleen and from lymph nodes after the use of CCR5 and CXCR3 blockade. Also combined CCR5 and CXCR3 blockade reduced the infiltration of CD4+ and CD8+ T cells into the liver and intestine, and retained part of CD4+ and CD8+ T cells within SLOs. As for the effector function of donor T cells, the results showed that combined CCR5 and CXCR3 blockade dampened T-cell activation, suppressed the percentage of Th1 and Tc1, and induced Treg induction. Additionally, combined CCR5 and CXCR3 blockade altered the concentrations of IFN-γ and IL10. Furthermore, the transplantation of CCR5-/- splenic cells with CXCR3 blockade showed a stronger protection effect on aGVHD.Combined CCR5 and CXCR3 blockade further attenuated aGVHD. aGVHD inhibition was associated with alternated donor T-cell distribution, limited donor T-cell activation and negatively impacted T-cell effector function in vivo. These data suggest that the combined use of CCR5 and CXCR3 blockade might be applicable for aGVHD prophylaxis in clinical settings. DisclosuresNo relevant conflicts of interest to declare.

Safety and Tolerability of PF-04447943 across a Clinical Trial Program Including 277 Patients

▪Background: Current treatment options (eg, hydroxyurea, chronic blood infusions, and bone marrow or stem cell transplantation) for the prevention of sickle cell disease (SCD) complications vary in effectiveness and are associated with safety risks/tolerability issues. PF-04447943, a selective inhibitor of the cyclic guanosine monophosphate−specific phosphodiesterase-9A enzyme, is being developed for prophylactic therapy to reduce the incidence and/or severity of vaso-occlusive crises in patients with SCD. Herein, we report on the safety and tolerability of PF-04447943, based on a large data set from the clinical trial program.Methods: The PF-04447943 safety database includes data from 8 phase 1 studies in healthy adult and elderly subjects, a phase 2 study in patients with Alzheimer's disease (AD) from a previous development program, and a phase 1b study in stable SCD patients. Study durations ranged from 1 day to 3 months. Data are presented descriptively.Results: Safety data for 277 patients receiving PF-04447943 were available. Frequently reported treatment-emergent adverse events (TEAEs) were headache, diarrhea, and nausea, which were generally mild in severity. In the phase 1 thorough QTc (TQT) study (N=44; study duration, 28 days), at therapeutic and supratherapeutic doses, PF-04447943 was not associated with QTc interval prolongation that met the threshold of clinical concern, based on ICH E14 criteria. In the phase 2 study of the AD development program (PF-04447943, n=91; placebo, n=100; study duration 12 weeks), the incidence of TEAEs was 63.7% with PF-04447943 and 58.0% with placebo. Incidence of all-causality AEs in the gastrointestinal disorders class (including abdominal pain, diarrhea, dry mouth, dyspepsia, gastroesophageal reflux disease, intestinal obstruction, nausea, and upper gastrointestinal hemorrhage) was higher with PF-04447943 (19.8%) vs placebo (5.0%); no other notable differences were observed. In the phase 1b study in SCD patients (safety analysis set: PF-04447943, n=22; placebo, n=7; study duration, 29 days plus 28 days follow-up), nervous system disorder TEAEs (eg, headache) occurred more frequently with PF-04447943 (25 mg BID, 60.0%; 5 mg BID, 57.1%) vs placebo (28.69%). In total, 6 serious AEs (sickle cell anemia with crisis, n=4 [one patient had 3 crises, of which 2 occurred during the follow-up period]; pneumonia, n=1; biliary colic, n=1) occurred in 3 patients receiving PF-04447943; all were consistent with the course of SCD and were not considered to be treatment related.Conclusion: Review of the safety database for PF-04447943, which includes 277 patients who received the study drug, indicates that PF-04447943 is generally well tolerated, with no major safety signals. Planned long-term, phase 2 and 3 studies of PF-04447943 in patients with SCD will provide additional safety data in this population. DisclosuresCharnigo:Pfizer: Employment. Tan:Pfizer Inc: Employment. Rybin:Pfizer: Employment. Pittman:Pfizer: Employment. Sivamurthy:Pfizer Inc: Employment. Beidler:Pfizer: Employment. Clarke:Pfizer Inc: Employment.