Engraftment Of Bone Marrow Cells Research Articles

Matrix Metalloproteinase 1 as a Marker of Tonsil-Derived Mesenchymal Stem Cells to Assess Bone Marrow Cell Migration.

To achieve optimal bone marrow engraftment during bone marrow transplantation, migration of donor bone marrow cells (BMCs) toward the recipient's bone marrow is critical. Despite the enhanced engraftment of BMCs by co-administration of mesenchymal stem cells (MSCs), the efficiency can be variable depending on MSC donor. The purpose of this study is to examine the functional heterogeneity of tonsil-derived MSCs (TMSCs) and to identify a marker to evaluate efficacy for the enhancement of BMC migration. To examine the donor-to-donor variation of TMSCs in potentiating BMC migration, we isolated TMSCs from 25 independent donors. Transcriptome of TMSCs and proteome of conditioned medium derived from TMSC were analyzed. Enhanced BMC migration by conditioned medium derived from TMSCs was variable depending on TMSC donor. The TMSCs derived from 25 donors showed distinct expression profiles compared with other cells, including fibroblasts, adipose-derived MSCs and bone marrow-derived MSCs. TMSCs were distributed in two categories: high- and low-efficacy groups for potentiating BMC migration. Transcriptome analysis of TMSCs and proteome profiles of conditioned medium derived from TMSCs revealed higher expression and secretion of matrix metalloproteinase (MMP) 1 in the high-efficacy group. MMP1 knockdown in TMSCs abrogated the supportive efficacy of conditioned medium derived from TMSC cultures in BMC migration. These data suggest that secreted MMP1 can be used as a marker to evaluate the efficacy of TMSCs in enhancing BMC migration. Furthermore, the strategy of analyzing transcriptomes and proteomes of the MSCs may be useful to set the standard for donor variation.

Characterization and Function of Cryopreserved Bone Marrow from Deceased Organ Donors: A Potential Viable Alternative Graft Source

Despite the readily available graft sources for allogeneic hematopoietic cell transplantation (alloHCT), a significant unmet need remains in the timely provision of suitable unrelated donor grafts. This shortage is related to the rarity of certain HLA alleles in the donor pool, nonclearance of donors owing to infectious disease or general health status, and prolonged graft procurement and processing times. An alternative hematopoietic progenitor cell (HPC) graft source obtained from the vertebral bodies (VBs) of deceased organ donors could alleviate many of the obstacles associated with using grafts from healthy living donors or umbilical cord blood (UCB). Deceased organ donor-derived bone marrow (BM) can be preemptively screened, cryogenically banked for on-demand use, and made available in adequate cell doses for HCT. We have developed a good manufacturing practice (GMP)-compliant process to recover and cryogenically bank VB-derived HPCs from deceased organ donor (OD) BM. Here we present results from an analysis of HPCs from BM obtained from 250 deceased donors to identify any substantial difference in composition or quality compared with HPCs from BM aspirated from the iliac crests of healthy living donors. BM from deceased donor VBs was processed in a central GMP facility and packaged for cryopreservation in 5% DMSO/2.5% human serum albumin. BM aspirated from living donor iliac crests was obtained and used for comparison. A portion of each specimen was analyzed before and after cryopreservation by flow cytometry and colony-forming unit potential. Bone marrow chimerism potential was assessed in irradiated immunocompromised NSG mice. Analysis of variance with Bonferroni correction for multiple comparisons was used to determine how cryopreservation affects BM cells and to evaluate indicators of successful engraftment of BM cells into irradiated murine models. The t test (with 95% confidence intervals [CIs]) was used to compare cells from deceased donors and living donors. A final dataset of complete clinical and matched laboratory data from 226 cryopreserved samples was used in linear regressions to predict outcomes of BM HPC processing. When compared before and after cryopreservation, OD-derived BM HPCs were found to be stable, with CD34+ cells maintaining high viability and function after thawing. The yield from a single donor is sufficient for transplantation of an average of 1.6 patients (range, 1.2 to 7.5). CD34+ cells from OD-derived HPCs from BM productively engrafted sublethally irradiated immunocompromised mouse BM (>44% and >67% chimerism at 8 and 16 weeks, respectively). Flow cytometry and secondary transplantation confirmed that OD HPCs from BM is composed of long-term engrafting CD34+CD38-CD45RA-CD90+CD49f+ HSCs. Linear regression identified no meaningful predictive associations between selected donor-related characteristics and OD BM HPC quality or yield. Collectively, these data demonstrate that cryopreserved BM HPCs from deceased organ donors is potent and functionally equivalent to living donor BM HPCs and is a viable on-demand graft source for clinical HCT. Prospective clinical trials will soon commence in collaboration with the Center for International Blood and Marrow Research to assess the feasibility, safety, and efficacy of Ossium HPCs from BM (ClinicalTrials.gov identifier NCT05068401).

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Research Highlights

Research Highlights

Irinotecan Induces Complete Remission in MLL -Rearranged Acute Lymphoblastic Leukemia Xenografts

BACKGROUND: Acute lymphoblastic leukemia (ALL) in infants (diagnosis < 1 year) is characterized by a high incidence of rearrangements of the MLL (KMT2A ) gene, occurring in ~80% of the cases. The resulting MLL fusions drive a leukemia biologically and clinically distinct from other subtypes. Therefore, while current ALL treatments achieve cure rates of 80-90%, MLL -rearranged (MLL -R) infant ALL is still beset by poor outcome, with a 5-year event-free survival of ~40%. Further intensification of current treatment is not possible, highlighting an urgent need for new therapeutic strategies.AIM: While novel drug classes have shown pre-clinical promise in MLL -R ALL, clinical implementation has become a major bottle-neck, as set-up of clinical trials with new drugs, as well as their FDA-approval, is a protracted process. Hence, we explored a drug repurposing approach, performing screens of >3000 FDA-approved drugs to identify compounds able to both target MLL -RALL cells and expeditiously transition into clinics.METHODS:MLL -RALL cell lines (SEM, KOPN8 and RS411) were screened using commercially available drug libraries comprising in total 3685 compounds; drug sensitivity to the initial concentration of 1 μM was assessed using 4-day MTS-assays. The most efficacious hits were further screened at 100 nM and 10 nM, to refine selection of potent candidate compounds. Among the top hits were the topoisomerase I inhibitor camptothecin and its derivatives, 10-hydroxycamptothecin and 7-ethyl-10-hydroxycamptothecin (SN38). The most potent derivative, SN38, was further analyzed in vitro to assess SN38-induced molecular changes in MLL -RALL cells using immunoblotting and cell death assays.To assess the anti-leukemic effects of SN38 in vivo , immunodeficient NOD.Cg- PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were injected with the luciferase -expressing MLL -RALL reporter cell line SEM-SLIEW, as well as primary leukemic cells of two MLL -Rinfant ALL patients. Mice were treated 3 times per week with either the pro-dug irinotecan (40 mg/kg), which is metabolised into SN38, or vehicle, for up to 4 weeks. Treatment was initiated after engraftment was confirmed by either bioluminescence imaging (SEM-SLIEW) or presence of >1% human leukemic cells in the blood was detected by multi-color flow cytometry (patient-derived xenografts, PDX). Disease progression over time was measured accordingly. To investigate the curative potential of irinotecan, treatment with the drug was initiated in a subset of mice displaying advanced, full-blown leukemia.At the end of treatment, mice were killed to assess the effect of irinotecan on human leukemic cell engraftment in bone marrow (BM), blood and spleen using flow cytometry and histochemistry. In addition, a subset of irinotecan-treated mice was kept alive for up to 42 days without treatment, to monitor for disease relapse.RESULTS: Camptothecins target MLL -RALL cells in vitro ; in particular SN38 had nanomolar IC50-values in both cell lines and primary leukemicpatient cells. Exposure with 25 nM SN38 inhibited proliferation and induced cell death of the MLL -RALL cell line SEM; characteristic DNA damage markers, such as accumulation of γ-H2AX and CHK2 phosphorylation could also be detected already after 2h of SN38 exposure. The anti-leukemic effect of SN38 could be validated in vivo in MLL -RALL xenografts, where the SN38 pro-drug irinotecan completely blocked leukemia expansion. At the experimental end-point, flow cytometric analyses showed human leukemic cells to be completely eradicated from the BM, blood and spleen in irinotecan-treated cell line xenografts, and to be sporadically present at minimal residual disease levels in irinotecan-treated PDX mice. Histochemistry stainings confirmed lack of leukemic infiltration in extramedullary tissues. Moreover, irinotecan induced complete remission in mice with advanced leukemia, as shown by flow cytometry as well as loss of bioluminescence. While ablation of human leukemic cells persisted in cell line-derived xenografts after treatment stop, PDX mice relapsed after 4 weeks, possibly due to presence of leukemic cells at low levels; this could possibly be improved by extending treatment duration.CONCLUSION: Irinotecan inhibits MLL -R ALL very potently, and as an FDA-approved drug already used in other pediatric cancers, it is an ideal candidate for repurposing and implementation in future MLL -R ALL treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Tonsil-derived mesenchymal stem cells enhance allogeneic bone marrow engraftment via collagen IV degradation

BackgroundCo-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified.MethodsMice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration.ResultsMice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro.ConclusionsCollectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Osteoclasts Derive Predominantly from Bone Marrow-Resident CX3CR1+ Precursor Cells in Homeostasis, whereas Circulating CX3CR1+ Cells Contribute to Osteoclast Development during Fracture Repair.

Osteoclasts (OC) originate from either bone marrow (BM)-resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX3CR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CX3CR1-EGFP/TRAP tdTomato mice, we found CX3CR1 expression in mononuclear cells, but not in multinucleated TRAP+ OC. However, CX3CR1-expressing cells generated TRAP+ OC on bone within 5 d in CX3CR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato+ OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CX3CR1+ cells migrated to bone, lost expression of CX3CR1, and became OC. These data demonstrate that OCP, but not mature OC, express CX3CR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CX3CR1+ cells can become OC.

PS991 CA‐4948, AN IRAK4/FLT3 INHIBITOR, SHOWS ANTILEUKEMIC ACTIVITY IN MOUSE MODELS OF FLT3 WILD‐TYPE AND FLT3 MUTATED ACUTE MYELOID LEUKEMIA (AML)

Background:AML is an aggressive hematopoietic malignancy that arises from a population of aberrant hematopoietic stem cells in the bone marrow (BM). Advances in understanding the molecular basis of AML has led to the development of new targeted therapies. CA‐4948 is a novel, oral IRAK4 kinase inhibitor with additional inhibitory activity against wild‐type (wt) and mutated FLT3 kinase. IRAK4 (Interleukin‐1 Receptor Associated Kinase 4) is required for interleukin 1 receptor (IL‐1R) and toll‐like receptor (TLR) innate immune pathway signaling, pathways that are frequently over activated in AML and myelodysplastic syndromes (MDS). For example, AML patients have increased IL‐1R agonist (IL‐1ß) levels that promote the survival of AML cells and IL‐1R KO represses AML cell growth in vitro and in vivo (Carey et al 2017). Dysregulation of the FLT3 signaling pathway is a well validated driver of AML. Constitutively activating mutations in FLT3 that comprise the ITD or the tyrosine kinase domain (KD) are frequently acquired late in AML disease and are poor prognostic factors with high relapse rates. FLT3 kinase inhibitors targeting FLT3‐ITD or ITD/KD double mutations show high remission rates; however, multiple resistance mechanisms have been reported in both nonclinical models and AML patients. CA‐4948 has both IRAK4 and FLT3 inhibitory activity, which may impart benefit to FLT3‐wt and FLT3‐mutant AML patients.Aims:Evaluate the ability of a novel IRAK4/FLT3 inhibitor, CA‐4948, to block IRAK4 and FLT3 in FLT3‐wt and FLT3‐mutant AML in vitro and in vivo.Methods:CA‐4948's kinome profile was assessed against 378 kinases and 9 FLT3 mutant variants (DiscoverX). CA‐4948's ability to inhibit TLR/IL‐1R or FLT3 signaling pathways was evaluated using NF‐kB reporter, cytokine production, or western blot. The growth inhibitory and pro‐apoptotic activity of CA‐4948 was tested in vitro against AML cell lines by viability assay or flow cytometry. For AML FLT3‐wt in vivo efficacy, THP‐1 cells were tail‐vein injected into mice and animal survival and degree of AML cell engraftment in BM were monitored in CA‐4948, FLT3i, or vehicle treated mice. For AML FLT3 mutant in vivo efficacy, subcutaneous MV4–11 and MOLM‐14 FLT3‐ITD and MOLM‐14 double FLT3‐ITD/KD tumor models were treated with CA‐4948 or other FLT3i.Results:CA‐4948 exhibited 23 nM (Kd) binding affinity for IRAK4, &gt;500‐fold selectivity against IRAK1, and high binding affinity for FLT3‐wt, ‐ITD, ‐ITD/D835 V, and ‐ITD/F691L at 8–31 nM (Kd). CA‐4948 blocked cellular TLR and IL‐1R‐stimulated signaling from 150–220 nM (IC50). Consistent with CA‐4948's additional FLT3 inhibitory activity, CA‐4948 induced apoptosis and exhibited cytotoxic activity against AML FLT3 mutant lines in vitro at 58–200 nM (IC50). In FLT3‐wt in vivo studies, CA‐4948 blocked THP‐1 bone marrow engraftment, while FLT3i quizartinib (Q) and midostaurin (M) had no inhibitory effect. In AML FLT3‐ITD tumors, CA‐4948 induced tumor regression equivalent to Q and M. CA‐4948 also induced tumor regression in FLT3‐ITD/D835Y tumors similar to M and showed greater efficacy in FLT3‐ITD/F691L tumors as compared to Q.Summary/Conclusion:These results demonstrate that targeting the IL‐1R/TLR signaling pathway with IRAK4 inhibitor CA‐4849 may be an effective therapeutic strategy in FLT3‐wt AML. Furthermore, CA‐4948's additional FLT3 inhibitory activity may repress the emergence of FLT3‐mutant AML clones. CA‐4948's dual IRAK4/FLT3 inhibitory activity merits further clinical investigation in FLT3‐wt and FLT3‐mutant AML/MDS.

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the egfp Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 106 on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.

PRO 140 Monoclonal Antibody to CCR5 Prevents Acute Xenogeneic Graft-versus-Host Disease in NOD-scid IL-2Rynull Mice

Graft-versus-host disease (GVHD) is a prevalent and potentially lethal complication of hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic GVHD are important tools used to study the human immune response in vivo. Here we used NOD-scid IL-2Rynull mice (NSG) transplanted with human bone marrow stem cells to evaluate the role of immune cell engraftment in the production of acute GVHD. PRO 140, a humanized monoclonal antibody targeting the chemokine receptor, CCR5, was used to evaluate its influence on bone marrow cell engraftment and modulation of acute GVHD. We evaluated the kinetics of engraftment by determining the percentage and absolute numbers of human CD45+ cells and CD3+ T cells from peripheral blood, spleen, and bone marrow in treated and control mice. With a dosing schedule of 2 mg of test or control antibody administered i.p. twice weekly, PRO 140–treated mice showed no signs of GVHD throughout the 70-day study period and gained weight until they were killed at 70 days for flow cytometry analysis. Control mice started losing weight after 25 days, showed classic signs of GVHD (ruffled fur, lethargy, severe hunching), and all were killed by day 54. The percentage and absolute numbers of human CD45+ cells in peripheral blood increased in both groups of mice throughout the 50-day comparison period and was lower in the PRO 140–treated mice at day 50. There was no difference in human CD45+ cells detected in bone marrow from control and PRO 140–treated killed mice. At this time point 76.1% and 68.2% of the hematopoietic cells from peripheral blood and from bone marrow, respectively, were of human lineage and 14.9% and 28%, respectively, were of mouse origin. With a schedule using 10-fold less dose of antibody (.2 mg i.p. twice weekly), PRO 140 still significantly modulated acute GVHD in terms of both weight loss and survival times, but no mice from either control or test group survived. By targeting the CCR5 chemokine receptor, PRO 140 modulated acute GVHD in a dose-response fashion in this xenogeneic mouse model without significantly altering engraftment.

A Critical Role for TGF-β/Fc and Nonlytic IL-2/Fc Fusion Proteins in Promoting Chimerism and Donor-Specific Tolerance.

Immunoglobulin-cytokine fusion molecules have been shown to be the new generation of immunomodulating agents in transplantation tolerance induction. In the present study, we tested whether immunoregulatory cytokine fusion proteins of IL-10/Fc, TGF-β/Fc, or IL-2/Fc would enhance allogeneic bone marrow cell (BMC) engraftment and promote tolerance induction. B6 (H2) mice were conditioned with anti-CD154 (MR1) and rapamycin (Rapa) plus 100 cGy total body irradiation (MR1/Rapa/100 cGy) and transplanted with allogeneic B10.D2 (H2) BMC. Recipients were treated with lytic IL-2/Fc, nonlytic IL-2/Fc, TGF-β/Fc, or IL-10/Fc fusion proteins to promote chimerism to induce tolerance. Donor chimerism was achieved in 20% of recipients conditioned with MR1/Rapa/100 cGy. The addition of TGF-β/Fc (5- or 10-day treatment) or nonlytic IL-2/Fc (10-day treatment) fusion proteins to the conditioning resulted in engraftment in nearly 100% of recipients. In contrast, lytic IL-2/Fc or IL-10/Fc had no effect. The combination of nonlytic IL-2/Fc and TGF-β/Fc had a synergistic effect to promote engraftment and resulted in significantly higher donor chimerism compared with recipients conditioned with TGF-β/MR1/Rapa/100 cGy. Engraftment was durable in the majority of chimeras and increased over time. The chimeras accepted donor skin grafts and promptly rejected third-party skin grafts. Moreover, specific T cell receptor-Vβ5.½ and TCR-Vβ11 clonal deletion was detected in host T cells in chimeras, suggesting central tolerance to donor alloantigens. Allogeneic BMC engraftment is enhanced with TGF-β/Fc fusion protein treatment. TGF-β/Fc and nonlytic IL-2/Fc exert a synergistic effect in promotion of alloengraftment and donor-specific transplant tolerance, significantly decreasing the minimum total body irradiation dose required.

Effect of the Wnt-Signaling Inhibitor Sclerostin and Bone Marrow Adipocytes on Multiple Myeloma

Multiple Myeloma (MM) is a plasma cell tumor that originates and expands within bone marrow (BM). MM occurs primarily later in life with a median age at diagnosis in the seventh decade characterized by loss of bone tissue due to osteolytic lesions. Recent studies have suggested a positive correlation of MM with obesity. BM fat cells originate, as bone cells, from a common progenitor called Mesechymal Stem Cell (MSC). MSC commitment into osteoblastic or adipose lineage is regulated by two major signaling pathways: Wnt signaling, which promotes bone formation, and PPARγ signaling, responsible for adipocyte differentiation. Adipogenesis and osteogenesis are inversely correlated processes, thus a signaling imbalance favoring differentiation towards one lineage will tilt this critical balance. In fact, the amount of marrow adiposity increases with age such that it is 30% of the BM volume in young adults but rises up to 70% in elderly people while bone formation reduces with age. We have recently demonstrated that MM patients have increased levels of a Wnt-signaling inhibitor sclerostin (SOST) and patient MSC differentiation into osteoblastic cells is improved in the presence of a SOST neutralizing antibody (Eda et al. JBMR 2015). Sclerostin is secreted by osteolineage cells and has also been shown to increase adipogenesis of an adipogenic cell line 3T3-L1 (Ukita et al. JCB 2016). However, the role of SOST on BM adipose tissue in MM patients has not been investigated. Here we show that elevated SOST levels, induced by MM cells, increase BM adipogenesis which, in turn, supports MM progression.To assess the role of MM cells on MSC differentiation into adipocytes, MSCs from patients and normal donors (ND) were differentiated in vitro in the presence or absence of MM.1S, a human MM cell line. Presence of MM.1S cells significantly reduced osteogenic differentiation of MSCs as assessed by quantitative and qualitative Alizarin Red S staining; by contrast, the presence of MM.1S cells significantly enhanced adipogenesis in the MSCs as assessed by Oil Red O staining quantification. At the molecular level, we observed a 2-fold increase in PPARγ gene expression in MSCs from MM patients when compared to ND samples at baseline and a 5-fold increase when MSCs from patients and NDs were cultured for 72 hours in the presence of MM.1S in trans-well. To evaluate the role of SOST on BM adiposity, we intraperitoneally injected recombinant SOST, or PBS as control, into wild-type (WT) mice daily for 3 weeks. At the end of the treatment, mice treated with SOST showed a significant increase in BM adiposity. To validate MM cell induced adipogenesis in in-vivo settings, we injected MM.1S cells into the SCID-hu immunodeficient mouse model. Four weeks after cell injection, increased BM adipocytes were observed in MM.1S treated immunodeficient mice. Interestingly, when mice injected with MM.1S cells were treated for 3 weeks with SOST neutralizing antibody the level of BM adiposity returned to the healthy control levels. Finally, MM cell engraftment and tumor development was analyzed in SOST knock-out (KO) mice. While both SOST KO and WT animals showed MM cell engraftment and extramedullary plasmacytoma formation, preliminary results suggest a lower level of MM cell engraftment in BM of SOST KO mice.Our data suggest that sclerostin secretion increases BM adipogenesis supporting MM cell growth and survival and therefore may play a critical role in the development and progression of MM. DisclosuresRaje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding.

Bone Marrow Is a Reservoir for Cardiac Resident Stem Cells.

Resident cardiac stem cells (CSCs) represent a responsive stem cell reservoir within the adult myocardium and have a significant function in myocardial homeostasis and injury. However, the distribution, origin, homing and possible therapeutic benefits of CSCs are still under discussion. Here we investigated whether bone marrow (BM) stem cells could contribute to repopulating the pool of CSCs in heart. The engraftment of BM cells in heart was detected at a low level after BM transplantation (BMT) and ischemia/reperfusion (I/R) could increase BM cells engraftment but not significant. We clarified that more than 50% CSCs are derived from BM and confirmed that BM-derived CSCs have similar characteristics with the host CSCs. Furthermore, we transplanted BM-derived CSCs into heart ischemia models and presented evidence for the first time that BM-derived CSCs can differentiate into cardiomyocytes in vivo. In conclusions, BM stem cells could be a potential back-up source of CSCs for restoring heart function after injury or maintaining homeostasis of CSCs.

Leukemia Cell Xenograft in Ovo Enables Real-Time Testing of Response to Therapy

Background:Acute myeloid leukemia (AML) is most common in people over the age of 60 where it remains an almost incurable malignancy with a grim prognosis. Evaluation of new therapeutic agents in vitro and in vivo is critical for drug development, yet there are few in-vivo models for studying human leukemia and its therapy. The best model used is the high cost immune-deficient mice and that require several weeks to assess drug response. To complicate matters, AML almost certainly relapses with cells that are not necessarily exactly identical to the original malignant clone, often limiting therapeutic options. The development of anti-leukemia therapies could be facilitated by a rapid and cost effective in vivo system for evaluating response to new drugs. Additionally, decisions regarding personalized treatment for relapsed or refractory leukemia patients must be rapid, and produce results within several days, as longer time periods can be detrimental. Herein, we describe a fast, economical, in ovo turkey embryo model, which provides a unique system to meet these requirements. The model can be used for the assessment of human leukemia infiltration in medullary and extramedullary tissues and more importantly for rapid testing of anti- leukemic agents within the bone marrow (BM). This system can be applied for new drug development and for personalized real time response of patient cells to potential leukemia therapies.Methods:BCR/Abl+ AML lines K562 and LAMA-84 , c-Kit+ CHRF-4288, fresh AML patient and Raji Burkit lymphoma cells (5x106) were injected into turkey egg chorioallantoic membrane (CAM) veins on embryonic day E11 previously optimized (1). Engraftment in BM was detected by flow cytometry (FC) using anti-human CD71 or anti- human CD33 for AML and anti human CD45 for Raji cells, or by Quantitative real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human /avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®), Doxorubicin or dexamethasone, 48H after grafting cells. Drug levels were precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control (Taizi M et al Exp Hem 34:1698,2006, Grinberg I et al, Leuk Res. 33:1417, 2009). Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis.Results:The kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver after cell injection on day E11 was assessed to determine the optimal treatment and readout times. The highest engraftment level in BM and liver was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. We quantitatively compared the engraftment of AML cells at E18-20 without and with drug treatment that was administered IV 48 hours after cell injection. The average engraftment (±SD.) in the BM after one week was 4.5%+1.7 K562, 5.83% +0.88 LAMA-84, 11.2%+3.5 CHRF-4288, 8.9% +1.6 Raji (n=7-15 per group) and 2.5% fresh leukemia cells detected by FC and confirmed by Q-PCR. A single dose of either 0.75 mg Imatinib or 50 mg Doxorubicin /embryo previously calibrated to be non toxic to the embryos reduced engraftment of AML cells in BM and in several other organs by more than ten fold. A similar effect was also obtained by a single dose of 5mg dexamethasone in Raji injected embryos. Treatment with 0.5 mg Imatinib on injected ARH-77 (multiple myeloma) or Raji cells had no effect on cell engraftment, while treatment with a single non toxic dose of Revlimid as previously described eliminated engraftment of ARH77 cells (1), clearly demonstrating the specificity of the drugs and utility of the system.Conclusion:Our study demonstrates the potential utility of a practical and unique avian embryo model for testing drug activity on human AML cells in vivo. This system, under preclinical development, is expected to provide a new xenograft platform for real time affordable testing leukemia therapies. More importantly, this may open a new venue for individualized screening for response or resistance to specific therapeutic agents for the relapsed or refractive patient and may lead to better optimization of practical and applicable therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

Engraftment of autologous bone marrow cells into the injured cranial cruciate ligament in dogs

Current research indicates that exogenous stem cells may accelerate reparative processes in joint disease but, no previous studies have evaluated whether bone marrow cells (BMCs) target the injured cranial cruciate ligament (CCL) in dogs. The objective of this study was to investigate engraftment of BMCs following intra-articular injection in dogs with spontaneous CCL injury. Autologous PKH26-labelled BMCs were injected into the stifle joint of eight client-owned dogs with CCL rupture. The effects of PKH26 staining on cell viability and PKH26 fluorescence intensity were analysed in vitro using a MTT assay and flow cytometry. Labelled BMCs in injured CCL tissue were identified using fluorescence microscopy of biopsies harvested 3 and 13 days after intra-articular BMC injection.The intensity of PKH26 fluorescence declines with cell division but was still detectable after 16 days. Labelling with PKH26 had no detectable effect on cell viability or proliferation. Only rare PKH26-positive cells were present in biopsies of the injured CCL in 3/7 dogs and in synovial fluid in 1/7 dogs. No differences in transforming growth factor-β1, and interleukin-6 before and after BMC treatment were found and no clinical complications were noted during a 1 year follow-up period. In conclusion, BMCs were shown to engraft to the injured CCL in dogs when injected into the articular cavity. Intra-articular application of PKH26-labelled cultured mesenchymal stem cells is likely to result in higher numbers of engrafted cells that can be tracked using this method in a clinical setting.

Loss Of Canonic Notch Signaling In The Microenvironment Causes a Myeloproliferative Syndrome Via Derepression Of The miR155 Promoter

The Notch signaling pathway is an important regulator of normal hematopoiesis, and is involved in leukemogenesis. Although the cell-autonomous roles of Notch signaling on hematopoietic cells have been extensively studied, its contribution to the regulation of the bone marrow (BM) niche has not been investigated yet. In our study, we used a Notch loss-of-function model in which deletion of the DNA-binding domain of RBPJ by Mx1-Cre results in the loss of signaling from all Notch receptors, in both hematopoietic cells and stromal cells of the BM microenvironment. In this model, we observed that loss of RBPJ-dependent Notch signaling in the hematopoietic microenvironment leads to a lethal myeloproliferative-like disease. Transplantation studies revealed that the microenvironment (ME) of RBPJ KO mice is necessary for the development and maintenance of the disease. Kinetics of engraftment of BM cells by intravital microscopy of the BM niche showed that WT donor cells engrafted more rapidly and had a greater expansion when transplanted into the RBPJ KO ME than in WT ME. Analysis of the serum and of supernatants from BM stroma showed increased cytokine levels in the RBPJ KO mice compared to controls, in particular G-CSF and TNF-alpha. Histological analysis of BM and spleen of RBPJ recipients showed increased microvascular density and increased CD31+ endothelial cells (EC) by IHC, preceding the onset of disease, suggesting an activated state of the endothelium. Specific deletion of RBPJ in the endothelium by using Tie2CreER model confirmed a critical contribution of EC to the myeloproliferative disease. We observed also that RBPJ KO BM-derived stroma exhibit increased expression of inflammatory microRNAs, in particular of miR155, a microRNA involved in inflammation and cancer. miR155 promoter analysis indicated the existence of two RBPJ binding sites, and direct interaction of RBPJ and the miR155 promoter was confirmed by ChIP assay. Loss of transcriptional repression by RBPJ in BM stromal cells induced miR155 upregulation and increased cytokine levels supporting greater expansion of myeloid progenitors. Double knock-out of RBPJ-/- and miR155-/- mice validated the requirement of miR155 for the hematopoietic changes due to RBPJ deletion. Finally, Analysis of patient's samples affected by myeloproliferative neoplasia showed elevated levels of miR155 in the BM, suggesting miR-155 as a potential therapeutic target in myeloproliferative disorders. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

RationaleStage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.ObjectiveWe generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.Methods and ResultsLethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.ConclusionsThese results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.

Bone Marrow Cell Recruitment to the Brain in the Absence of Irradiation or Parabiosis Bias

The engraftment of bone marrow-derived cells has been described not only during diseases of the central nervous system (CNS) but also under healthy conditions. However, previous studies pointing to an ample bone marrow cell engraftment used irradiation-induced bone marrow chimeras that evoked severe alterations of the CNS micromilieu including disturbances of the blood brain barrier (BBB), damage of endothelial cells and local induction of proinflammatory cytokines. On the other hand, parabiosis experiments using temporarily joined circulatory systems generally yielded low levels of myeloid cell chimerism thereby potentially underestimating bone marrow cell turnover with the CNS. To avoid these drawbacks we established a protocol using the alkylating agent busulfan prior to allogenic bone marrow transplantation from CX3CR1GFP/+ donors. This regimen resulted in a stable and high peripheral myeloid chimerism, significantly reduced cytokine induction and preserved BBB integrity. Importantly, bone marrow cell recruitment to the CNS was strongly diminished under these conditions and only weakly enhanced during local neurodegeneration induced by facial nerve axotomy. These results underscore the requirement of local CNS conditioning for efficient recruitment of bone marrow cells, establish busulfan as an alternative treatment for studying bone marrow chimeras and suggest a critical re-evaluation of earlier chimeric studies involving irradiation or parabiosis regimens.

Nrf2 Deficiency Leads to Reduced Hematopoietic Stem Cell Self-Renewal and Increased Sensitivity to Genotoxic Stressors Through Impaired P53 Function

Abstract 397Nrf2 is a basic region-leucine-zipper transcription factor that regulates expression of numerous genes required for oxidative stress response, drug and toxin detoxification, protein ubiquitination, and proteasomal degradation of damaged proteins. Therefore Nrf2 represents a major cellular defense mechanism against oxidative and toxic stresses. We have shown that CD34+ hematopoietic stem/progenitor cells (HSPC) drawn from peripheral blood stem cell (PBSC) autografts of lymphoma patients who develop therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) after autologous hematopoietic cell transplantation (aHCT) demonstrate significantly reduced expression of Nrf2-mediated stress response pathway genes compared to cells from patients who do not develop t-MDS/AML (Cancer Cell 20, 591–605). We have extended these findings to study the role of Nrf2 in HSPC regulation and the response to genotoxic chemotherapeutic agents by using an Nrf2 knockout (KO) mouse model. Nrf2 KO mice demonstrated impaired bone marrow (BM) long-term hematopoietic stem cell (LT-HSC) function, manifested by significantly reduced competitive engraftment of BM cells in recipient mice at 16 weeks compared to wild-type (WT) controls (42.5±2.8% vs. 57.2±3.3% peripheral blood engraftment for KO and WT donors respectively, p<0.01). An even more significant defect in engraftment of KO cells was seen after transplantation to secondary recipients (16.4±1.7% vs. 61.9±1.5% peripheral blood engraftment at 8 weeks for KO and WT donors respectively, p<0.01). Nrf2 KO mice also demonstrated increased sensitivity to ENU treatment compared to WT mice. ENU (100mg/kg) treatment resulted in severe anemia at 7 months in 100% of KO mice compared with 36% of WT mice (p<0.001). Furthermore, anemia developed at a median of 5 months in Nrf2 KO mice compared to 10 months in WT controls. Pathological examination of BM from anemic mice revealed reduced erythropoiesis with impaired erythroid maturation and expansion of myeloid cells, consistent with myeloid dysplasia. Q-RT-PCR analysis demonstrated significantly reduced expression of Nrf2 target genes, including Hmo-1, Nqo-1, Gclc and Gpx1, in BM c-kit+ HSPC from KO mice compared to WT mice (p<0.05). Interestingly, expression of several P53 target genes including Bax, Puma, Cdkn1a and Necdin were also significantly reduced in BM c-kit+ cells from Nrf2 KO compared to WT mice. We also observed reduced P53 target gene expression in Nrf2 KO c-Kit cells compared with WT cells 4 hours after treatment with ENU (p<0.05), indicating impaired P53 response in Nrf2 deficient HSPC. To examine the P53 response to DNA damaging agents in LT-HSC, we exposed Nrf2 KO and WT mice to 2Gy irradiation and selected BM LT-HSC (Lin-Sca-1+c-kit+Flt-3-CD150+CD48-) using flow cytometry 12 hours after irradiation. Q-RT-PCR analysis showed significantly reduced expression of P53 target genes Bax, Puma and Necdin (p<0.05), and a trend towards reduced expression of Cdkn1a (p=0.08), in Nrf2 KO compared to WT LT-HSC. Immunofluorescence microscopy showed significantly reduced nuclear and increased cytoplasmic localization of P53 in Nrf2 KO compared to WT LT-HSC. Western blotting analysis showed increased levels of ubquitinated proteins in BM c-kit+ HSPC from Nrf2 KO compared to WT mice, and immunoprecipitation followed by Western blotting for P53 revealed increased high molecular weight bands indicative of ubiquitinated P53 in Nrf2 KO HSPC. Nrf2 is a key regulator of protein ubiquitination and proteasomal degradation, and these results suggest that abnormal cytoplasmic accumulation of ubiquitinated P53 may contribute to altered P53 function in Nrf2 deficient HSPC. In conclusion, our results show that Nrf2 deficiency results in impaired HSC self-renewal capacity under physiological conditions, and significantly increased sensitivity to genotoxic stress, potentially explained by altered P53 protein modulation in Nrf2 deficient HSPC. These observations suggest that Nrf2 deficiency could contribute to development of t-MDS/AML following exposure to genotoxic agents, and support further evaluation of Nrf2 as a potential target of chemopreventive efforts. Disclosures:No relevant conflicts of interest to declare.

Homing and engraftment of bone marrow cells derived from different donors in a murine model of sensitization

Sensitized recipients are at a high risk of graft rejection in hematopoietic stem cell transplantation. To explore the trace of donor cells, we tried to explore homing and engraftment of bone marrow cells (BMCs) derived from different donors in a murine model of sensitization. Sensitized BALB/c mice were used as transplanted recipients, which received BMCs derived from C57BL/6 or BALB/c donors after lethal irradiation. The homing study showed that the donor cells decreased along with time in recipients of the C57BL/6 donor group, but the donor cells increased along with time in recipients of the BALB/c donor group. For the engraftment assay, all the sensitized recipients transplanted with BMCs derived from C57BL/6 donors died after lethal irradiation. In contrast, all the recipients transplanted with BMCs derived from BALB/c donors got long-term survival. Our results suggest that it is crucial to have human leukocyte antigen identical donors for sensitized recipients during hematopoietic stem cell transplantation.