Bone Marrow Of Recipient Mice Research Articles

C/EBPβ Promotes Initial Expansion and Exhaustion of Hematopoietic Stem and Progenitor Cells in Response to Hematopoietic Stresses

Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein (C/EBPb), a leucine zipper transcription factor, in granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPb is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem and progenitor cells (HSPCs) (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPb is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and exhaustion of leukemic stem cells in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPb plays important roles in regulation of normal and leukemic HSPCs. In this study, we focus on the functions of C/EBPb in normal HSPCs under stressed conditions.At steady state, the frequencies of HSPCs in the bone marrow (BM) of C/EBPb knockout (KO) mice were identical to those in the BM of wild type (WT) mice. It suggests that C/EBPb has little impact on the emergence or maintenance of HSPCs during steady state.To investigate function of C/EBPb in HSPCs, competitive repopulation assay was performed. Total BM cells from either WT or KO mice (CD45.2+) and the equal number of competitor cells from the BM of CD45.1+ WT mice were transplanted into lethally irradiated recipient WT mice (CD45.1+), and the chimerism of CD45.2+ cells in the peripheral blood (PB) of the recipient mice was monitored once a month. Chimerism of KO cells in the recipient mice was significantly lower than that of WT cells at 1 month after transplantation (52.2 ± 10.3% vs 37.8 ± 8.8%, p < 0.0000001, n = 37 vs 36) and the differences were maintained thereafter (Figure 1), suggesting that C/EBPb is required at early time points after transplantation.In order to elucidate the early events which make difference in the chimerism, homing ability was assessed first. Sixteen hours after transplantation of lineage depleted WT or KO BM cells (CD45.2+) together with lineage negative CD45.1+ WT BM cells, the frequencies of CD45.2+ WT and KO donor cells in the c-kit+ Sca1+ lineage- (KSL) fraction were identical. Then we compared the initial expansion of HSPCs. Purified 1000 KSL cells from either WT or KO mice (CD45.2+) were transplanted to lethally irradiated recipient WT mice (CD45.1+ / CD45.2+) together with the equal number of competitor KSL cells from WT mice (CD45.1+). The ratio of CD45.2+ KO cells to CD45.1+ competitors in the KSL fraction of the recipient mice was significantly lower than that of CD45.2+ WT cells at 4 weeks after transplantation (6.76 ± 2.35 vs 2.84 ± 1.16, p = 0.040, n = 4 vs 4). These results suggest that C/EBPb is required for initial expansion of HSPCs rather than for homing after transplantation.Next, we investigated the roles of C/EBPb in maintenance of HSPCs under stressed conditions. By staining of intracellular C/EBPb in combination with multi-color flow cytometric analysis, we found that C/EBPb is upregulated at protein level in KSL cells of WT mice 5 days after intraperitoneal injection of 5-fluorouracil (5-FU). Then the recipient mice were repetitively administered with 5-FU (150mg/kg i.p.) after BM transplantation in a competitive way. As mentioned above, the chimerism of KO cells in PB of recipient mice was significantly lower than those of WT mice at 1 month after transplantation. Interestingly, the chimerism of KO cells gradually increased by repetitive administration of 5-FU and even overtook those of WT cells 5 months after transplantation (Figure 2). In accordance with the changes observed in the PB, the chimerism of KO cells in the KSL fraction in the BM of recipient mice was significantly higher than those of WT cells (70.7 ± 25.3% vs 12.1 ± 9.78%, p = 0.016, n = 5 vs 4) 5 months after transplantation, suggesting that WT HSPCs exhausted earlier than KO HSPCs in response to hematopoietic stress.From these findings, we conclude that C/EBPb is required for initial expansion and exhaustion of HSPCs after hematopoietic stresses. We are currently investigating the molecular targets of C/EBPb and its clinical significance in the pathogenesis of leukemia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rictor is required for early B cell development in bone marrow.

The development of early B cells, which are generated from hematopoietic stem cells (HSCs) in a series of well-characterized stages in bone marrow (BM), represents a paradigm for terminal differentiation processes. Akt is primarily regulated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2, and Akt signaling plays a key role in hematopoiesis. However, the role of mTORC2 in the development of early B cells remains poorly understood. In this study, we investigated the functional role of mTORC2 by specifically deleting an integral component, Rictor, in a hematopoietic system. We demonstrated that the deletion of Rictor induced an aberrant increase in the FoxO1 and Rag-1 proteins in BM B cells and that this increase was accompanied by a significant decrease in the abundance of B cells in the peripheral blood (PB) and the spleen, suggesting impaired development of early B cells in adult mouse BM. A BM transplantation assay revealed that the B cell differentiation defect induced by Rictor deletion was not affected by the BM microenvironment, thus indicating a cell-intrinsic mechanism. Furthermore, the knockdown of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) promoted the maturation of B cells in the BM of recipient mice. In addition, we revealed that treatment with rapamycin (an mTORC1 inhibitor) aggravated the deficiency in B cell development in the PB and BM. Taken together, our results provide further evidence that Rictor regulates the development of early B cells in a cell-intrinsic manner by modifying the expression of FoxO1 and Rag-1.

Candida albicansstimulatesin vivodifferentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2-dependent signalling

Toll-like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2-dependent signalling. Purified lineage-negative cells (Lin(-)) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or in response to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin(-) cells from TLR2(-/-) mice did not give rise to macrophages, whereas Lin(-) cells from TLR4(-/-) mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin(-) cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Our results indicatethat TLR-mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that TLR-mediated signalling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen.

Dynamic Imaging of Marrow-Resident Granulocytes Interacting with Human Mesenchymal Stem Cells upon Systemic Lipopolysaccharide Challenge

Human mesenchymal stem cells (hMSCs) have gained intense research interest due to their immune-modulatory, tissue differentiating, and homing properties to sites of inflammation. Despite evidence demonstrating the biodistribution of infused hMSCs in target organs using static fluorescence imaging or whole-body imaging techniques, surprisingly little is known about how hMSCs behave dynamically within host tissues on a single-cell level in vivo. Here, we infused fluorescently labeled clinical-grade hMSCs into immune-competent mice in which neutrophils and monocytes express a second fluorescent marker under the lysozyme M (LysM) promoter. Using intravital two-photon microscopy (TPM), we were able for the first time to capture dynamic interactions between hMSCs and LysM+ granulocytes in the calvarium bone marrow of recipient mice during systemic LPS challenge in real time. Interestingly, many of the infused hMSCs remained intact despite repeated cellular contacts with host neutrophils. However, we were able to observe the destruction and subsequent phagocytosis of some hMSCs by surrounding granulocytes. Thus, our imaging platform provides opportunities to gain insight into the biology and therapeutic mechanisms of hMSCs in vivo at a single-cell level within live hosts.

Rescue of Lethal Hypophosphatasia Mice by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells.

Abstract 3154Hypophosphatasia (HPP) is an inherited skeletal disease caused by genetic defects of tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme which is attached to the outside plasma membrane via a GPI anchor and plays an essential role in bone mineralization. The major symptoms are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Severe HPP is often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Although previous trials of ERT using various forms of soluble ALP showed no clinical benefit, it was recently demonstrated that TNALP with deca-aspartates at the C terminus (TNALP-D10) had a high affinity for bone tissue and repeated injections of TNALP-D10 successfully rescued lethal HPP mice. HPP mice were generated by knockout the mouse TNALP gene (Akp2) and phenotypically mimic to severe infantile HPP and develop hypomineralization, growth failure and epileptic seizures after birth. The plasma ALP activity in HPP mice was less than 0.01 U/ml (approx. 0.1 U/ml in wild type (wt) mice) and the average life span of non-treated HPP mice is about 20 days. We have also shown that a single intravenous injection of either lentiviral or AAV vector expressing TNALP-D10 resulted in prolonged survival and phenotypic correction of HPP mice. In this in vivo gene therapy, bone cells were not efficiently transduced, but the plasma ALP activity derived from TNALP-D10 secreted from transduced liver or muscle cells was maintained at extremely high levels (10 to 100 folds higher than that of wt mice). As an alternative approach, we are studying the feasibility of hematopoietic stem cells (HSC) based ex vivo gene therapy for HPP. After homing of HSC to the bone marrow, local expression of TNALP in the bone should be beneficial to improve bone mineralization. Other potential advantages of this strategy compared with an in vivo systemic gene therapy include lifelong expression of TNALP, no risk of germline gene transfer, and no immunoreaction against viral vector.Lineage negative bone marrow cells (BMC) were harvested from B6.CD45.1 mice (Ly5.1) using the Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD Bioscience) and incubated with lentiviral vector expressing GFP or TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6. Transduction efficiency assessed by GFP expression was approximately 40 % under the condition used. Recipient neonatal mice (Ly5.2) were sub-lethally irradiated at 4Gy and received BMC (1 × 106′ cells) through the jugular vein on day 2. Irradiated neonatal wt mice showed a slight reduction of the growth rate but normal physical activity and healthy appearance. GFP positive donor cells migrated to the bone marrow in recipient mice. FACS analysis of the peripheral blood samples 4 to 12 weeks after transplantation demonstrated that approximately 30 % of Ly5.1 donor cells were stably detected in all lineage blood cells of recipient mice. After treatment of neonatal HPP mice with TNALP-D10 expressing BMC, the plasma ALP activity was elevated to 1 to 2 U/ml at 4 weeks of age and remained at this level during the observation period. The treated mice actively moved in the cage without epileptic seizures and the life span was prolonged over 3 months. X-ray examination of the skeleton showed that mineralization was significantly improved compared to non-treated HPP mice, but not completely normalized compared to age matched wt mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for continuous supply of TNALP-D10 to rescue lethal HPP mice. However, the concentration of TNALP-D10 in the bone may not be sufficient for complete correction of skeletal abnormalities. Further optimization of gene transfer and neonatal BMT is under way to increase the plasma ALP activity. HSC mediated ex vivo gene therapy is now being applied to treat not only hematological diseases but also neurological disorders such as adreno leukodystrophy and metachromatic leukodystrophy. Hypophosphatasia, a systemic bone disease, is also an important target for ex vivo gene therapy. Disclosures:No relevant conflicts of interest to declare.

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Well MET: Exosomes promote metastasis through transfer of MET from melanoma to bone marrow progenitors

Well <scp>MET</scp>: Exosomes promote metastasis through transfer of <scp>MET</scp> from melanoma to bone marrow progenitors

Direct Toll-Like Receptor-Mediated Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Macrophages

As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin(-) ) as well as HSPCs (identified as Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-) [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin(-) and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2(-/-) , TLR4(-/-) , or MyD88(-/-) mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen-derived products directly during infection, inducing a rapid generation of cells of the innate immune system.

Single line or parallel lines: NK cell differentiation driven by T-bet and Eomes

Single line or parallel lines: NK cell differentiation driven by T-bet and Eomes

Bone Marrow-Engrafted Cells after Mice Umbilical Cord Blood Transplantation Differentiate into Osteoblastic Cells in Response to Fracture and Placement of Titanium Screws

As the in vivo function of bone marrow-engrafted umbilical cord blood (UCB)-derived mesenchymal cells (UCBCs) after UCB transplantation is unknown, we examined in vivo osteoblastic differentiation using mouse UCB transplantation and fracture models. UCBCs obtained from GFP transgenic mice were intravenously injected into irradiated C57BL/6 mice. After three months, the in vivo osteoblastic differentiation potential of bone marrow-engrafted UCBCs was examined histologically using a mouse fracture model. GFP-positive UCBCs were detected in the bone marrow of recipient mice. On day 7, UCBCs were observed in the fracture gap and surrounding the titanium screws of the fixation device. The UCBCs were also positive for alkaline phosphatase and von Kossa staining. By day 14, UCBCs were observed around and within a formed intramedullary callus. The newly formed woven bone consisted of ALP- and von Kossa-positive cells. Our findings suggest that UCBCs contribute to the fracture healing process after bone marrow engraftment and that UCBC transplantation can fully reconstruct not only hematopoietic cells but also mesenchymal cell lineages.

Acute Myeloid Leukemia Cells Generate Leukemic Endothelial Cells with Leukemogenic Potential: Blood Vessels As Sanctuaries for Leukemia Relapse

Abstract 241Human hematopoietic stem cells (HSCs) possess hemangioblast activity, which is defined as the ability to generate both blood and endothelium. Whether malignant HSC counterparts such as acute myeloid leukemia (AML) also display this bipotentiality remains to be defined. To test the hemangioblast potential of AML cells we first cultured primary human AML bone marrow in conditions established by Yoder and colleagues that support the growth of functional endothelial cell (EC) progenitors. AML cultured in endothelial colony forming cell (ECFC) media generated endothelial progenitor cell colonies that showed uptake of acetylated LDL and expressed several EC surface proteins, including CD105, CD146, UEA-1 and CD144. Importantly, ECFCs derived from AML bone marrow no longer expressed CD45 or myeloid surface proteins such as CD14. When placed in Matrigel, these AML derived ECFC generated capillary-like, tubular structures. Moreover, these ECFCs contained cytogenetic mutations associated with their parental leukemias. Thus, under the appropriate conditions, AML bone marrow cells can generate cells with an endothelial-like phenotype and harboring leukemia specific mutations that will be referred to as ‘L-ECFC.’ To functionally define leukemia hemangioblast activity, a xenograft model of AML was employed. Sublethally irradiated NOD/scid/IL2Rγ−/− (NSG) mice were transplanted with primary human AML cells and then sacrificed at 8–36 weeks after transplant. Significant accumulations of human AML cells were found in perivascular regions of the liver. Both tight coupling and bona fide cell fusion between AML and ECs was observed. AML derived EC that were integrated into portal vein endothelium showed induction of CD105 expression Follow-up AML xenotransplant experiments with BrdU labeling revealed almost four-fold fewer (6%) of the AML cells incorporated within blood vessels were BrdU+, as compared to AML cells not integrated in blood vessels (22%) (P=0.01). These results suggest that AML cell incorporation within the endovascular lining induces cell quiescence. Thus, leukemia-integrated ECs may be less susceptible to cell cycle active agents like cytarabine. Results from these experiments also raised the possibility that AML cells adopting an endothelial-like phenotype may serve as a reservoir for leukemic relapse. To test this hypothesis, we injected CD105+CD45- L-ECFC derived from AML patients into NSG mice. These L-ECFC generated colonies of human CD105+CD45- within spleens and bone marrow of recipient mice. We also found a distinct population of human CD45+CD19- cells comprising 5–10% of bone marrow cells. Leukemia-derived cells were confirmed by detection of cytogenetically mutant cells consistent with the parent leukemia (e.g., MLL duplications). In conclusion, this study demonstrates that AML cells can functionally generate leukemic ECs that become quiescent after incorporation in blood vessel networks and can re-emerge with a leukemogenic phenotype. Together, our results raise the strong possibility that AML cells exhibit functional hemangioblast activity and that vascular endothelium may serve as a clinically important sanctuary for occult leukemia. Our data also support endothelial cell targeting strategies as a means to eradicate AML. Disclosures:No relevant conflicts of interest to declare.

Glycosylation Engineering Combined with CD26 Inhibitor Enhances Umbilical Blood Cells Engraftment In the NOD/SCID Mouse

AbstractAbstract 4687Given the tremendous need for and potential of umbilical cord blood (UCB) to be utilized as a donor source for hematopoietic stem cell (HSC) transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited HSC and hematopoietic progenitor cell (HPC) numbers available for HSC transplantation from a single collection. Hematopoietic stem cell homing is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in clinical setting. We have previously described the functional defect of partial homing receptor in cord blood stem cell (the higher activity of CD26 and the lower expression of P/E-selectin ligand) in vitro. Respectively, we had confirmed that using of glycosylation engineering and CD26 inhibitor can fix the defect of stem cell homing. However, there is no evidence can prove whether the combination would improve the ability of homing. In present study, to explore the use of glycosylation combined with CD26 inhibitor as a method of improving the transplant efficiency of CB CD34+ hematopoietic stem cells (expressing CD45), we utilized the xenotransplanted nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model in vivo. 6–8 weeks age of NOD/SCID mice were divided into 5 groups (8 mice per group): blank control group (without stem cells infusion); control group (CD34+ cells infusion alone); glycosylation engineering group; CD26 inhibitor group and combination group (glycosylation engineering combined CD26 inhibitor group). Purified CD34+ stem cells (5×105 per mice) derived from fresh UCB by immunomagnetic beads were injected into lethally irradiated NOD/SCID mice intravenously. To investigate the effect of early engraftment in vivo, we monitored the expression of CD45 in peripheral blood and the overall viability rate weekly until the fourth week by flow cytometry. One week after transplantation, there is no expression of CD45 in peripheral blood in any group, but we observed 2.52±1.41% and 4.8% expression in bone marrow of recipient mice respectively in control and glycosylation engineering groups. All mice in the blank control group were dead and overall viability rate were almost the same for the other four groups (80% in control group,80% in glycosylation engineering group, 100% in CD26 inhibitor group and 100% in combination group). The second week, there is only expression of CD45 in recipient mice for combination group 0.63±0.37%. The overall survival rate among control were significantly lower than the other four groups (40% in control group,80% in glycosylation engineering group, 100% in CD26 inhibitor group and 100% in combination group, P<0.05). In the third week, all groups except the blank control group expressed CD45 in peripheral blood, we observed 0.81±0.12%, 2.81±0.14%, 3.12±0.53% and 7.05±0.87% in peripheral blood of recipient mice for control, glycosylation engineering, CD26 inhibitor treated and combination group. The overall survival rate among CD26 inhibitor treated and combination groups were significantly higher than the other three groups. (40% in control group,40% in glycosylation engineering group, 80% in CD26 inhibitor group and 80% in combination group, P<0.05). In the fourth week, we observed 15.87±0.21%, 30.51±0.62%, 34.33±0.82% and 40.22±1.62% in peripheral blood of recipient mice for control, glycosylation engineering, CD26 inhibitor treated and combination group. The overall survival rate among CD26 inhibitor treated and combination groups were significantly higher than the other three groups. (40% in control group,40% in glycosylation engineering group, 80% in CD26 inhibitor group and 80% in combination group, P<0.05). These results provide the evidence that the combination of two methods have better effect on early implantation than use them respectively. Our results also supported the potential use of CD26 inhibitor combined with glycosylation engineering to treat the umbilical blood which the number of stem cell is less than bone marrow and peripheral blood during hematopoietic stem cell transplantation as a method to improve the transplant efficiency. Furthermore, we aimed to investigate the long-term implantation effect of combination of glycosylation engineering and inhibition of CD26 in vivo. Disclosures:Sun:International Cooperation Research Fund of Anhui Provincial Scientific and Technologic Committee (08080703026): Research Funding; Fund of the Key Medical Project of Anhui Provincial healthy department (2010A005): Research Funding; Anhui Provincial “115” Industrial Innovation Program: Research Funding.

Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

AbstractAbstract 1548Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34-lineage-Sca-1+c-Kit+), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage-Sca-1+c-Kit+; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs. Disclosures:No relevant conflicts of interest to declare.

CD26 Inhibitor Treated and CD26−/− Recipient Mice Exhibit Improved Transplant Efficiency as Quantitated by Increased Short-Term Homing and Long-Term Engraftment

A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/ HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in the clinical setting. Through the use of CD26 inhibitors and CD26 deficient mice (CD26−/−), we have previously generated data in mice suggesting that suppression of CD26/DPPIV (dipeptidylpeptidase IV) enzymatic activity on the transplant donor cell population can be utilized as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al, Science 2004. 305:1000–3). However, the clinical importance of the transplant recipient should not to be overlooked given the potential importance of the bone marrow microenvironment in regulating the transplant process. We therefore investigated here whether inhibition or loss of CD26 activity in recipient mice would have an effect on transplant efficiency utilizing an in vivo congenic mouse model of transplantation. The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26−/− mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant respectively. Twenty-four hours post-transplant of 20×106 BoyJ mononuclear cells, we observed 8.85±0.58%, 10.69±1.01%, and 12.45±1.33% donor derived Sca-1+lin− cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26−/− recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p=0.01) with CD26 inhibitor treatment and a 40.7% increase (p£0.05) resulting from the use of a CD26−/− recipient in short-term homing (N=5 mice per group). Six months post-transplant of 1×105 BoyJ mononuclear cells, we observed 39.90± 4.38%, 70.22± 3.72%, and 92.51± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26−/− recipient mice respectively. This represents a 76.0% increase (p£0.01) with CD26 inhibitor treatment and a 131.9% increase (p£0.01) as a result of the CD26−/− recipient in long-term engraftment as compared to control recipient mice (N=14 mice per group). These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency. Lastly, our use of inhibitor treated C57BL/6 and CD26−/− recipient mice, which are also on a C57BL/6 background, in conjunction with a congenic model of transplantation provides a accurate and convenient model system for the in vivo testing of the efficacy of existing and new CD26 inhibitors in transplant recipients.

Lyl1 and Scl Dosage Is Critical for Adult Hematopoietic Stem Cell Function and Survival

The Stem Cell Leukemia gene (Scl) and the Lymphoblastic Leukemia gene 1 (Lyl1) genes encode two related basic-helix-loop-helix transcription factors that are involved in T-cell acute lymphoblastic leukemia via chromosomal translocations or aberrant expression due to other genetic or epigenetic abnormalities. Previous studies have shown that Scl is necessary for embryonic hematopoiesis and erythropoiesis, but dispensable for adult hematopoietic stem cell (HSC) function. Lyl1 on the other hand is important for adult HSC function and B-cell development. In order to elucidate in more detail the functions of Lyl1 and Scl in adult HSCs, we generated double knock-out mice and studied the function of their HSCs. Double knock-out mice demonstrate a striking genetic interaction between the two genes, with a clear dose-dependence for the presence of Scl or Lyl1 alleles for HSC function.Specifically, we carried out competitive fetal liver transplantation assays using mice with different combinations of Lyl1 and Scl alleles and analyzed their potential to repopulate the bone marrow of recipient mice. Peripheral blood chimerism of recipient mice shows that HSC function is lost with progressive loss of Lyl1 or Scl dosage, suggesting an overlap of function of the two genes in the HSCs (Figure)We have further investigated the function of Lyl1 and Scl in adult hematopoiesis by generating Lyl1/Scl-conditional double knock-out mice and studied adult HSC function measured by bone marrow repopulation assays. Donor engraftment dramatically decreased after deletion of the floxed Scl allele, demonstrating rapid loss of adult HSC function. Furthermore, Scl deletion in a Lyl1−/− background causes hematopoietic progenitors to rapidly undergo apoptosis in a cell-intrinsic fashion. These results demonstrate that at least one allele of either Lyl1 or Scl must be present for adult HSC survival, and show that their functions partly overlap in HSCs, suggesting that Lyl1 and Scl are crucial regulators of adult, as well as embryonic, hematopoiesis. [Display omitted]

Leukemic Stem Cells and Progenitors Demonstrate Impaired Interaction with the Hematopoietic Microenvironment in Vivo in An Inducible Murine Model of Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a stem cell malignancy induced by p210 BCR-ABL and characterized by myeloproliferation in BM and egression of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSC in patients under Imatinib suggests inhibition of ABL-kinase alone is not sufficient to eliminate the LSC/P. One of the major hallmarks of CML induced by signaling downstream BCR-ABL is the loss of control of the hematopoietic microenvironment on LSC/P. Expression of p210 BCR-ABL has been associated with loss of adhesion to the bone marrow, impaired migration in response to CXCL12 and decreased retention in the BM. In order to study the putative LSC/P niches in steady-state chronic-phase leukemia, we have analyzed the ability of LSC/P to proliferate and get retained in the bone marrow (BM) in an inducible model of CML. Binary transgenic SCL-tTA/TRE-BCR-ABL mice (Koschmieder S et al., Blood 2005) express p210 BCR-ABL in LSC/P upon doxycycline withdrawal (CML mice). Induced myeloproliferation was associated with activation of the downstream signaling effectors CrkL and p38-MAPK and expansion of circulating (Table 1) and splenic LSC/P but not in BM, suggesting massive LSC/P egression from the marrow (Table 2). Proliferation analysis showed that myeloid expansion in the spleen was secondary to increased cycling of Lin−Sca1+c-Kit+ (LSK) cells (3.1-fold increase in S-phase cells, P<0.05), but not in Lin−/c-Kit+ (LK) population, compared with the control spleens. In agreement with the LSC/P BM content data, the frequency of BM-derived LSK and LK cells incorporating BrdU in CML and in control mice remained similar, suggesting a specific egression of LSC/P from the BM to extramedullary sites. To test whether this model truly represented a model of BM LSC/P egression, we compared the splenic and BM LSC/P compared with their controls regarding their adhesion molecule expression, interaction with the hematopoietic microenvironment (HM) and homing to the overall marrow cavity and endosteal space. Splenic, but not BM-derived, LSK and LSK CD34+ ST-HSCs had increased cell surface expression of CD44 compared to controls (1.35 fold, P=0.006 and 1.23 fold, P<0.05 respectively) and decreased expression of L-selectin (8.7 fold, P<0.05) while expression of CXCR4, α4β1 and α5β1 integrins remain similar in bone marrow and splenocytes from CML and control mice. CML BM progenitors also showed 18-fold reduced adhesion to fibronectin and 1.4-fold increased migration towards CXCL12 compared to control BM progenitors. Myeloproliferative disease was transplantable into non-transgenic littermates and homing of CML BM progenitors was increased (4.3 fold, P<0.005) in myeloablated littermate recipient BM. However, lineage-negative leukemic BM-derived cells which had increased homing in BM of recipient mice had an impaired ability to migrate to the BM endosteal space compared with their littermate controls (control: 31 ± 18% vs CML mice: 17.6 ± 17%), suggesting an specific impairment to lodge in specialized anatomically-defined hematopoietic “niches”. Altogether, this murine model may represent an adequate in vivo system to analyze the ability of p210 BCR-ABL-expressing LSC/P to interact with BM niches and study the control of the hematopoietic microenvironment on LSC/P survival, proliferation and retention.Table 1 Increase in circulating LSC/P in the CML mice after withdrawal of doxycyclinPeripheral BloodLSK (×103)Cells/mL Blood P<0.05LT-HSC(×103)Cells/mL Blood P<0.05CFU-GM+BFU-E/mL Blood P<0.05Control1.56 ± 0.250.459 ± 0.2960.86 ± 51.09CML mice3.56 ± 1.522.159 ± 2.03869.6 ± 628.4Table 2. Immunophenotypic analysis of BM and splenocytes in control and CML micePopulationBM (Cells ×104) (Control)BM (Cells x104) (CML)SP (Cells ×104) (Control)SP (Cells x104) (CML)C-Kit+Sca1+24.3 ± 9.921.3 ± 116.8 ± 4.530.1 ± 12.3 (P<0.05)Mac1+Gr1+1779 ± 3071583 ± 26578.4 ± 32608 ± 377 (P<0.05)CFU-C/105Cells342 ± 66334 ± 9963.3 ± 7.0979 ± 6.54 (P<0.05)

Transduction of Human CD34+ Cells with Recombinant TAT-NF-YA Protein Enhances Their Ex Vivo Expansion and Repopulating Potential in NOD/SCID Mice

Protein transduction offers great promise for clinical applications as a safe alternative to viral gene delivery. We have previously shown that NF-YA transcription factor which regulates the expression of several genes implicated in hematopoietic stem cell (HSC) renewal and differentiation, including the Hox4 paralogs HoxB4, HoxC4, HoxD4, LEF1, Notch1, p27 and telomerase (Zhu J. et al. PNAS , 102:11728–11733; 2005) can be delivered as a TAT-NF-YA fusion protein to primitive human hematopoietic CD34+ cells (Domashenko A. et al. Blood, 106:144B; 2005). In these earlier studies transduced human NF-YA protein demonstrated functional activity by upregulated HOXB4 mRNA, as measured by real time PCR. Based on these results we have now asked whether TAT-NF-YA treatment of CD34+ cells:stimulates the ex vivo expansion of hematopoietic precursor and progenitor cells andmaintains and/or expands the repopulating capacity of HSC in the NOD/SCID mouse model.Human CD34+ cells from peripheral blood or bone marrow were treated with IL3/IL6/SCF/FL/TPO, with or without TAT-NF-YA protein, and grown for up to two weeks in a serum free media. Fifteen days after fusion protein delivery cell growth in treated culture was 4-fold higher than in control cultures without added TAT-NF-YA. Cells plated on methylcellulose at different time points of cell growth showed an increase of colony-forming progenitor cells (CFC) in culture exposed to TAT-NF-YA primarily due to increase of GEMM population: ~3 fold at day 4 and 7-fold day 9 after protein delivery. These results suggest that TAT-NF-YA promotes cell proliferation by maintaining the most primitive population in an undifferentiated state, biasing HSC toward self-renewal rather than differentiation. To measure more directly the engraftment potential of TAT-NF-YA treated cells, bone marrow CD34+ cells cultured for 3 or 6 days after exposure to the protein were transplanted into NOD/SCID mice. After 9 weeks, human CD45+ donor cells were analyzed for myeloid and lymphoid surface markers in the bone marrow of recipient mice by flow cytometry with anti-human CD33 and CD19 antibodies. TAT-NF-YA plus cytokine treated cells demonstrated significantly higher long-term repopulating capacity of cultured cells compared to cells cultured with cytokines alone, averaging a 4-fold increase for 3-day and a 3-fold increase for 6-day cell culture in the percent of huCD45+ cells recovered from the circulation and spleen of NOD/SCID mice. The proportion of lymphoid and myeloid engrafted populations was not significantly different in control and protein treated cells. These data provide evidence that NF-YA delivered as a TAT fusion protein to primary hematopoietic cells may stimulate progenitor/stem cell expansion, homing and/or repopulating ability. More generally, these results indicate that protein transduction has significant potential for various applications in manipulation of HSC, thereby offering a potentially safer alternative to viral gene therapy; both because the protein has a shot half-life in the targeted cells in vivo and because there is opportunity for insertional mutagenesis.

Critical Role of the Th1 Transcription Factor T-Bet in An Animal Model of Immune-Mediated Bone Marrow Failure

Patients with aplastic anemia have elevated T-bet, a Th1 transcription factor, in peripheral blood CD4 and CD8 T cells, suggesting that T-bet over-expression and dysregulated Th1 immune response contributes to pathophysiology of marrow failure (Solomou EE et al., Blood . 2006; 107:3983). In the present study, we studied the role of T-bet in inducing bone marrow failure in a mouse model of immune-mediated BM failure, employing mice engineered with a germline T-bet deletion as lymphocyte donors. Compared with T-bet +/+ wild-type controls, T-bet−/− mice have similar cellular composition in various lymphohematopietic tissues including peripheral blood, spleen, thymus, lymph nodes (LN), and BM. Incubation of effector T-bet−/− LN cells with MHC-mismatched target CByB6F1 (F1) BM cells in an in vitro cytotoxicity assay resulted in a significantly lower proportion of apoptotic target cells than did wild-type T-bet+/+ LN effector cells, suggesting that T-bet−/− effector LN cells are functionally defective. While infusion of 5×106 wild-type T-bet+/+ LN cells into sublethally-irradiated F1 mice led to severe pancytopenia and aplastic bone marrow in recipient mice, infusion of the same number of T-bet−/− LN cells failed to result in marrow failure, and recipients had relatively normal blood counts and bone marrow cellularity. By flow cytometry, both expansion of CD4+ and CD8+ T cells and elevation in intracellular Th1 cytokine gamma interferon (IFN-γ), which are characteristic of marrow cells in recipients received B6 LN cells, were absent in recipients receiving T-bet −/− LN cells. Serum IFN-γ concentration in F1 mice infused with T-bet −/− LN cells was similar to the level in F1 control mice received TBI alone, and both were significantly lower than serum IFN-γ in recipients of wild-type B6 LN cells. In contrast, serum TGF-γ concentration was higher in F1 mice that received TBI alone or TBI plus T-bet −/− LN cell infusion, compared with mice that received TBI plus B6 LN cells. An increase of T-bet −/− LN cell infusion to 10×106 cells per recipient led to very mild BM failure. Contrary to the markedly increased number of CD4+ and CD8+ T cells and elevated IFN-γ level in the BM of F1 mice which have received wild type B6 LN cells, F1 mice infused with T-bet −/− LN have low CD4+ and CD8+ cells and low IFN-γ level in the BM similar to F1 mice received TBI alone, but they show increased IL4 and IL17 levels within bone marrow T cells, indicating that the diminished Th1 immune response due to T-bet deficiency was partially compensated by up-regulated Th2 and Th17 responses. Our data demonstrated that T-bet plays a critical role in immune mediated bone marrow failure. Approaches targeting to T-bet signal pathway may lead to novel treatment for bone marrow failure and other autoimmune diseases.

B- and T-cell Development Both Involve Activity of the Unfolded Protein Response Pathway

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.

Pbx1 Regulates Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence

Self-renewal is a defining characteristic of stem cells; however, the molecular pathways underlying its regulation are poorly understood. Here, we demonstrate that conditional inactivation of the Pbx1 proto-oncogene in the hematopoietic compartment results in a progressive loss of long-term hematopoietic stem cells (LT-HSCs) that is associated with concomitant reduction in their quiescence, leading to a defect in the maintenance of self-renewal as assessed by serial transplantation. Transcriptional profiling revealed that multiple stem cell maintenance factors are perturbed in Pbx1-deficient LT-HSCs, which prematurely express a large subset of genes, including cell-cycle regulators, normally expressed in non-self-renewing multipotent progenitors. A significant proportion of Pbx1-dependent genes is associated with the TGF-beta pathway, which serves a major role in maintaining HSC quiescence. Prospectively isolated, Pbx1-deficient LT-HSCs display altered transcriptional responses to TGF-beta stimulation in vitro, suggesting a possible mechanism through which Pbx1 maintenance of stem cell quiescence may in part be achieved.