Donor-derived Chimerism Research Articles

Predictors of growth patterns in children with mucopolysaccharidosis I after haematopoietic stem cell transplantation.

Mucopolysaccharidosis type I (MPS I) is an autosomal‐recessive metabolic disorder caused by an enzyme deficiency of lysosomal alpha‐l‐iduronidase (IDUA). Haematopoietic stem cell transplantation (HSCT) is the therapeutic option of choice in MPS I patients younger than 2.5 years, which has a positive impact on neurocognitive development. However, impaired growth remains a problem. In this monocentric study, 14 patients with MPS I (mean age 1.72 years, range 0.81–3.08) were monitored according to a standardised follow‐up program after successful allogeneic HSCT. A detailed anthropometric program was carried out to identify growth patterns and to determine predictors of growth in these children. All patients are alive and in outpatient care (mean follow‐up 8.1 years, range 0.1–16.0). Progressively lower standard deviation scores (SDS) were observed for body length (mean SDS −1.61; −4.58 – 3.29), weight (−0.56; −3.19 – 2.95), sitting height (−3.28; −7.37 – 0.26), leg length (−1.64; −3.88 – 1.49) and head circumference (0.91; −2.52 – 6.09). Already at the age of 24 months, significant disproportions were detected being associated with increasing deterioration in growth for age. Younger age at HSCT, lower counts for haemoglobin and platelets, lower potassium, higher donor‐derived chimerism, higher counts for leukocytes and recruitment of a matched unrelated donor (MUD) positively correlated with body length (p ≤ 0.05). In conclusion, this study characterised predictors and aspects of growth patterns in children with MPS I after HSCT, underlining that early HSCT of MUD is essential for slowing body disproportion.

Reverting to Quiescence Preserves Hematopoietic Stem Cells Following Genome Editing

Hematopoietic stem cells (HSCs) in steady-state are quiescent in cell cycle. CRISPR-Cas9 genome editing has revolutionized the HSC research and therapeutic application of HSCs for hematological diseases. Although these methods and clinical results are promising, keeping HSC function after highly efficient genome editing is still challenging because HSCs gradually lose their repopulation capacity following cell cycle activation. Preserving the function of HSCs after genome editing is an urgent issue. In this study, we interrogated the culture method following genome editing to reverse the cell cycle status of HSCs into a quiescent state to test how cell cycle status affects the genome editing efficiency and the HSC potential.To assess the relationship between genome editing efficiency and cell cycle status, we compared genome editing efficiency among the freshly-isolated or cultured HSCs and progenitors (HSPCs) at various time points. Ribonucleoprotein (RNP) complex was delivered into cells by electroporation. Genome-editing efficiency was evaluated by CD45 knockout rate comparing sgRNA for Rosa and CD45. All the HSPC fractions cultured displayed higher genome-editing efficiency following over-night preculture. Genome-editing efficiency of fresh HSC was lower than 20% while fresh granulocyte/monocyte progenitor (GMP) showed higher than 40%. Cell cycle analysis using EdU or Ki67 and Hoechst 33342 staining confirmed that genome-editing efficiency positively correlated with cell cycle activation. We further tested the effect of pre- and post-culture conditions for genome editing. While preculture with high cytokine concentration for a long period of time (> 16 hours) is required for the efficient genome editing, post-culture condition did not compromise the genome-editing efficiency.Given the quiescent nature of HSCs, we hypothesized that reverting activated HSCs back to quiescent state may improve the function of HSCs following genome editing. To test this, genome-edited HSCs were cultured in the quiescence-maintaining condition (SCF 1.5 ng/mL and TPO 1.0 ng/mL) under 1% O 2 atmosphere. After 7-day culture, more than 30% of cells kept the surface marker phenotype of CD150 + CD48 - LSK, and over 60% of cells were successfully underwent genome editing. Less than 10% of HSCs were EdU +, suggesting that HSCs reverted to cell cycle quiescence after genome editing. By contrast, HSCs cultured in a conventional high cytokine condition (proliferative condition) lost the surface marker phenotypes and highly incorporated EdU. To assess the long-term reconstitution potential of edited HSCs, the Evi1 expression level was evaluated using Evi1-IRES-GFP mice. The expression level of Evi1 was significantly higher in quiescent HSCs than proliferating HSCs after editing. These results suggest that, as post-electroporation culture, quiescence-maintaining condition reverts precultured HSCs back to a quiescent state in cell cycle. This protocol maintains phenotypic HSCs without compromising the genome-editing efficiency.To further determine the function of genome-edited HSCs, single cell colony assay was performed. Clonally sorted CD45 knockout HSCs cultured in the quiescence-maintaining condition after gene editing fully maintained colony-forming capacity, but HSCs cultured in the proliferating condition lost their capacity. We then performed transplantation assays using Ubc-GFP mice. GFP + HSCs were genome-edited for Rosa and transplanted into lethally-irradiated recipient mice with competitor cells. The donor-derived chimerism of edited HSCs with quiescence-maintaining condition in peripheral blood and bone marrow was generally superior to that of edited HSCs with proliferative condition. These data demonstrates that edited HSCs cultured in quiescence-maintaining condition maintain stem cell potential in vitro and in vivo.Altogether, we established an HSC-optimized, highly efficient genome-editing protocol. This study demonstrated that effectiveness of keeping HSC in a quiescent state even in the setting of genome editing. Our protocol is suitable for unveiling the function of genes distinguishing cycling and quiescent HSCs. DisclosuresKataoka: Celgene: Honoraria; Eisai: Honoraria; Astellas Pharma: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; MSD: Honoraria; Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Asahi Genomics: Current equity holder in publicly-traded company; Otsuka Pharmaceutical: Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding; Eisai: Other: Scholarship; Otsuka Pharmaceutical: Other: Scholarship; Ono Pharmaceutical: Other: Scholarship; Kyowa Kirin: Other: Scholarship; Shionogi: Other: Scholarship; Takeda Pharmaceutical: Other: Scholarship; Summitomo Dainippon Pharma: Other: Scholarship; Chugai Pharmaceutical: Other: Scholarship; Teijn Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Genetic Alterations: Patents & Royalties: PD-L1 abnormalties .

Prostaglandin E2 Promotes Recovery of Thymus in Survivors of the Hematopoietic Acute Radiation Syndrome (H-ARS)

We have previously developed a murine model of the hematopoietic acute radiation syndrome [H-ARS, total body irradiated (TBI)] and delayed effects of acute radiation exposure (DEARE) for efficacy testing of potential medical countermeasures (MCM) against these two syndromes. Using this model, we have previously reported that prophylactic use of 16,16 dimethyl-prostaglandin E2 (dmPGE2), a long-acting formulation of PGE, before irradiation significantly increases 30-day survival rate. The current study investigated the effects of the prophylactic use of dmPGE2 with or without therapeutic use of dmPGE2 in survivors on long-term thymus recovery in H-ARS survivors at 6 and 12-months post-LD70/30 (872cGy, 137Cs, 95.2±4.8 cGy/min). Mice were irradiated at 12 weeks of age and then injected subcutaneously with dmPGE2 (1 dose of 35 ug dmPGE2/mouse) or vehicle (Veh; ethanol/PBS solution) at 30“ or -1hr pre-TBI. Survivors were injected monthly with dmPGE2 or Veh and then analyzed for evidence of thymic reconstitution at 6 and 12 months.post-TBI. The three test groups were: prophylactic dmPGE2 + monthly dmPGE2 (PP), prophylactic dmPGE2 + monthly Veh (PV), and prophylactic Veh + monthly Veh (VV), n = 3-6 mice/group. Thymus weight and cellularity of mice in the dmPGE groups, PV and PP, were increased compared to Veh both at 6-months and 12-months post-TBI (p <0.05). The difference was not related to body weight. No difference was observed between PP and PV group. The number of thymocyte subgroups (CD4 single positive, CD8 single positive, double positive and double negative) was also increased in PV and PP mice. Complete blood count (CBC) was also analyzed in the same mice. The VV group showed myeloid skewing, evidenced as an increase in neutrophils and decrease in lymphocytes. The PV and PP groups both exhibited reversal of myeloid skewing, and once again dmPGE maintenance did not provide extra benefit. Thymic progenitors in bone marrow, known as the lymphoid-primed multipotent progenitors (LMPP, Lin-Kit+Sca1+Flt3hiCD34+) and common lymphoid progenitor (CLP, Lin-IL7R+KitloSca1lo Flt3+) were also increased in PV and PP groups compared with VV group (p <0.05). No difference was observed in common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and MEP (megakaryocyte-erythroid progenitor) between PP and PV group. Competitive bone marrow transplantation showed more than 10 times higher donor-derived chimerism in the thymus of PP and PV mice compared with the VV group. Again, no difference was observed between PP and PV group. In conclusion, these data show that dmPGE2, given before irradiation, protects thymic settling progenitors in bone marrow, resulting in reversal of thymic involution, increased peripheral lymphocytes, and partial alleviation of the myeloid skewing observed in radiation survivors. dmPGE maintenance does not bring extra benefit. F unded by Department of the Army PR140433P1 and NIAID 1U01AI107340-01. DisclosuresNo relevant conflicts of interest to declare.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Correction of murine sickle cell disease by allogeneic haematopoietic cell transplantation with anti-3rd party veto cells.

Despite advances in gene therapy allogeneic hematopoietic stem cell transplants (HSCT) remains the most effective way to cure sickle cell disease (SCD). However, there are substantial challenges including lack of suitable donors, therapy-related toxicity (TRM) and risk of graft-versus-host disease (GvHD). Perhaps the most critical question is when to do a transplant for SCD. Safer transplant protocols for HLA-disparate HSCT is needed before transplants are widely accepted for SCD. Although risk of GvHD and TRM are less with T-cell-deplete HSCT and reduced-intensity conditioning (RIC), transplant rejection is a challenge. We have reported graft rejection of T cell-depleted non-myeloablative HSCT can be overcome in wild type fully mis-matched recipient mice, using donor-derived anti-3rd party central memory CD8-positive veto cells combined with short-term low-dose rapamycin. Here, we report safety and efficacy of this approach in a murine model for SCD. Durable donor-derived chimerism was achieved using this strategy with reversal of pathological parameters of SCD, including complete conversion to normal donor-derived red cells, and correction of splenomegaly and the levels of circulating reticulocytes, hematocrit, and hemoglobin.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

Loss of Gpr68 Enhances Hematopoietic Stem Cell Function during Aging

Recent studies implicate metabolic plasticity in fine-tuning hematopoietic stem cell (HSC) homeostasis and function. HSC have been shown to rely on anaerobic glycolysis due to low energetic demand. In contrast, mitochondrial respiration is largely engaged to meet high energetic demand under certain pathophysiological processes, such as differentiation, aging and malignant transformation. The microenvironment, i.e. the HSC niche, has long been known as hypoxic. Glycolytic pathway is not only the metabolic adaptation to the hypoxic niche, but also essential for HSC function at least by providing prosurvival signals. However, glycolysis is reduced in HSC upon aging, which is associated with diminished regeneration ability of HSC. How glycolysis impacts HSC biology is unclear. G protein-coupled receptor 68 (GPR68, also known as OGR1) responds to extracellular acidosis, activating the phospholipase C (PLCb)/calcium pathway or the adenylyl cyclase (CA)/cAMP pathway. The end point product of glycolysis is lactate that leads to extracellular acidosis. This prompts us to examine the role of Gpr68 in HSC biology. Among the hematopoietic stem and progenitor cell (HSPC) subpopulations, we found that Gpr68 expression was most enriched in mouse long-term HSC (LT-HSC, Lin-Sca-1+c-Kit+CD34-CD135-). To understand the function of Gpr68 in HSC biology, we generated a genetic loss of function mouse model (i.e. the Gpr68fl/fl;VavCre+ mice) where Gpr68 was specifically deleted in hematopoietic cells, including the most primitive LT-HSC. We found that the number of LT-HSC was unaltered in bone marrow (BM) from Gpr68fl/fl;VavCre+ mice as compared to wild type (WT) control littermates (i.e. the Gpr68wt/wt;VavCre+ mice), indicating that Gpr68 was not required for HSC homeostasis. To examine the role of Gpr68 in HSC function, we performed competitive bone marrow transplantation (cBMT). The donor-derived chimerism (i.e. the frequency of CD45.2+ cells) was increased in recipients with BM cells from Gpr68fl/fl;VavCre+ mice (12-month old) compared to those from age-matched WT mice after primary cBMT, which was increased even more dramatically after secondary cBMT. This indicates enhanced HSC regeneration ability due to loss of Gpr68. Increased chimerism was observed in CD3+ T lymphocytes, B220+ B lymphocytes and CD11b+ myeloid cells, indicating that both lymphoid and myeloid lineages contributed to enhanced HSC function. Consistently, increased chimerism was also observed in BM HSPC subpopulations except the LT-HSC. Of note, increased chimerism was not observed in recipients that were transplanted with BM cells from young Gpr68fl/fl;VavCre+ mice (2-month old). In addition, BM cells from Gpr68fl/fl;VavCre+ mice produced slightly reduced colonies in methylcellulose, indicating that loss of Gpr68 did not lead to malignant transformation despite enhanced competitiveness of HSC. To further understand the mechanism of Gpr68 in regulating HSC function, we studied cellular features of HSPC subpopulations. The frequency of cells in cell cycle G0 phase was unchanged in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, indicating loss of quiescence may not contribute to enhanced HSC function. Intriguingly, the frequency of Annexin V+ cells was largely reduced in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, suggesting that loss of Gpr68 provides a pro-survival signal in HSC. We next examined two main signaling pathways downstream of Gpr68. Cytosolic calcium levels were reduced in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, indicating Gpr68 may activate a calcium-related proapoptotic pathway. In contrast, intracellular cAMP levels were increased in HSPC, possibly from unknown compensatory mechanism. In addition, Gpr68 expression was reduced in LT-HSC from older mice (12-month old) compared to younger mice (2-month old), indicating reduced Gpr68 function at 12-month of age. This is in line with reduced glycolysis in aged HSC. Our study suggests that Gpr68 mediates a proapoptotic pathway in LT-HSC, providing a negative regulation, possibly at downstream of glycolysis, that limits HSC function. Our study also suggests that inhibiting Gpr68 function would enhance HSC function, especially in aged people or during bone marrow transplantation. Disclosures No relevant conflicts of interest to declare.

A Phase 1 Dose-Escalation Study of Adding Venetoclax to a Reduced Intensity Conditioning (RIC) Regimen Prior to Allogeneic Hematopoietic Cell Transplantation for Patients with High Risk Myeloid Malignancies

A Phase 1 Dose-Escalation Study of Adding Venetoclax to a Reduced Intensity Conditioning (RIC) Regimen Prior to Allogeneic Hematopoietic Cell Transplantation for Patients with High Risk Myeloid Malignancies

Proof of Concept for Repair of Sickle Cell Disease By Non-Myeloablative MHC Disparate T Cell Depleted HSCT Combined with Donor-Derived Veto Cells

Although life extending medical treatments are available for sickle cell disease (SCD), allogenic hematopoietic stem cell transplantation (HSCT) is considered a treatment of choice (Ozdogu et al., Bone Marrow Transplant 2018; 53(7): 880-890). However, HSCT is associated with several limitations, including conditioning-related toxicity and graft-versus-host disease (GvHD), especially when using MHC disparate transplants. Thus, the development of safe transplantation protocols for MHC disparate HSCT in sickle cell disease is vital. While the risk of GvHD and conditioning toxicity can be effectively reduced by the use of T-cell-depleted HSCT (TD-HSCT) under reduced intensity conditioning (RIC), rejection of TD-HSCT remains a challenge. We previously demonstrated in wild type mice that this barrier can be overcome using donor-derived veto cells. Here, we demonstrate the safety and efficacy of this approach in a well-defined murine model for sickle cells disease. Veto activity is based on the ability of certain cells to attack host CTL-precursors (CTLp) which are directed against antigens expressed on the veto cells themselves, sparing cells that are not targeted against the veto cells including T cells needed for defense against pathogens. Central memory CD8 T cells exhibit the most robust veto activity upon transplantation; however, these cells are also endowed with marked GvH activity. We overcame this issue by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, respectively, under culture conditions favoring expression of central memory phenotype. Such anti-3rd party central memoryCD8 T cells (Tcm), which are endowed with marked veto activity, also exhibit reduced risk for GvHD in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin Hematol. 2019; 56(3): 173-182.) To generate Tcm veto cells, splenocytes obtained from Balb/c donors (H2d) were cultured against irradiated third-party splenocytes (FVB; H2q) under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators leads to selective 'death by neglect' of bystander anti-host T cell clones potentialy mediating GvHD, and these are further diluted out by subsequent expansion of anti-3rd party T clones during continued culture in the presence of IL-15. Apart from selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be important for attaining robust veto activity in vivo (Ophir et al., Blood. 2013; 121(7): 1220-8). We first calibrated the optimal irradiation dose for sickle mice (Berkeley model, H2b), comparing 4.5 Gy versus 5 Gy TBI in a conditioning protocol also including short term rapmycin treatment (Scheme 1). Higher levels of engraftment and chimerism were found in the group receiving 5Gy TBI (Fig. 1). All mice of both treatment groups survived &gt;140 days with no evidence of GvHD. To further evaluate this treatment approach, 8 week old sickle mice (N=7) were given bone marrow transplants using the protocol described in Scheme 1, including conditioning with 5 Gy TBI (day -1), rapamycin treatment (day -1 to day 4), and transplantation of NuBM (day 0) plus veto cells (day 7; Scheme 1). Notably, at 44 days post-transplant, 6 out of 7 mice receiving NuBM + TCM + Rapa (85.7%) showed donor chimerism in the peripheral blood, ranging between 77-94% (Fig.2A-B), while no chimerism was detected in mice receiving conditioning alone, or conditioning and transplantation with only NuBM or only veto cells . All mice in all groups survived (N=26) , and no GvHD was detected with a follow up of 77 days, even in the transplanted group which exhibited high donor-derived chimerism. Furthermore, reversal of sickle disease symptoms was observed, including reticulocyte levels (p=0.001;Fig.2C) and expression of wild type hemoglobin ( Fig.2D) in all engrafted mice. Our results offer a proof of concept for the treatment of sickle disease by MHC disparate non-myeloablative T cell depleted HSCT in conjunction with anti-3rd party central memory veto CD8 T cells. A clinical trial testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is currently ongoing at MD Anderson Cancer Center. If successful, our present results support further evaluation of this platform for sickle cell disease. Disclosures Lustig: Yeda Ltd.: Patents &amp; Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

Platelet Biogenesis in the Lung Circulation

Platelets are indispensable in hemostasis, thrombosis, and immune responses. In humans, billions of platelets are produced each day from megakaryocytes, however the mechanisms of mature platelet production are incompletely understood. Megakaryocytes are produced in the bone marrow and have been visualized to communicate with the bone marrow sinusoids to release proplatelet fragments. Megakaryocytes have also been found in other tissues, including the lung, but the function of megakaryocytes in these locations is unclear. Historical data indicate that the lung may be a site of platelet biogenesis. The concentration of megakaryocytes in the blood exiting the lung is much lower than the blood entering the lung (implying filtering) and conversely, platelet counts are higher in blood draining from the lungs. Additionally, when the lung circulation in entirely bypassed, megakaryocytes accumulate in the blood and there is a high incidence of thrombocytopenia. However, direct proof of platelet biogenesis in the lung is lacking. We used lung intravital microscopy combined with fluorescently labeled mouse strains and directly visualized intravascular megakaryocytes releasing platelets in the lung circulation. We also visualized megakaryocytes in the bone marrow and spleen releasing proplatelet fragments, and megakaryocyte migration in toto from the bone marrow, which are presumably the source material for lung platelet production. The megakaryocyte-releasing events in the lung were quantified and represent at least half of the total platelet production in mice, which can be increased by the application of thrombopoietin. We also observed a much larger extravascular pool of megakaryocytes in the lung that were not platelet generating as observed by lung intravital imaging. The function of these lung-resident megakaryocytes is unknown, but RNA-Seq data points to a potential role in lung immunity. Orthotopic, single-lung transplantation experiments into thrombocytopenic and hematopoietic progenitor-deficient animals (c-mpl-/-) revealed that peripheral blood platelet counts and bone marrow hematopoietic progenitors could be fully reconstituted by the lung transplant procedure implying the presence of hematopoietic progenitors in the mouse lung. Indeed, these progenitors were directly detected in the extravascular lung and purified populations of hematopoietic progenitors in the lung could correct thrombocytopenia in c-mpl-/-animals. Finally, the lung transplant procedure produced donor-derived chimerism of other hematopoietic lineages such as neutrophils and lymphocytes. We conclude that the lung has significant hematopoietic potential including being a major site of platelet biogenesis. DisclosuresNo relevant conflicts of interest to declare.

TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis

Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function.To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis.To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells).Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. DisclosuresNo relevant conflicts of interest to declare.

Thymus Transplantation Plus Allogeneic Bone Marrow Transplantation Can Induce Strong Graft-Versus Leukemia Effects with Low Risk of Graft-Versus-Host Disease

Abstract: [Objective] Allogeneic bone marrow transplantation (allo-BMT) has been used for the treatment of refractory leukemia and donor lymphocyte infusion (DLI) was used for the purpose of graft versus leukemia (GVL) effects. However, DLI is associated with a risk of graft versus host disease (GVHD). Thus, new cellular-based methods are desired. In the present study, we performed thymus transplantation (TT) plus allo-BMT to explore it’s anti leukemia effects. [Methods] Recipient B6 mice (H-2b) bearing leukemia (EL-4 cells, H-2b) were irradiated 8 Gy. The next day, bone marrow cells from BALB/c mice (H-2d) were transplanted into the B6 mice. Simultaneously, DLI and thymus transplantation from the same donor were carried out. The survival period of the recipient B6 mice were examined, histological studies were performed in the liver, intestine, and the engrafted thymus from the recipients 4 weeks after the BMT. Surface markers on lymphocytes from the spleen were analyzed by 3-color fluorescence staining using a FACScan system to determine chimerism. [Results]. All mice treated with BMT showed fully donor-derived chimerism(H-2d). The survival rate in mice treated with BMT plus TT was significantly prolonged compared with those treated with BMT alone or BMT plus DLI. Histologically, both the cortex and medullar areas of the engrafted thymus under the renal capsule were clearly shown. Normal T-cell differentiation was also observed in the engrafted thymus. Microscopic founding of small intestine and liver in the BMT plus TT group indicated mild GVHD, whereas those treated with BMT plus DLI showed moderated to serious GVHD. The number of the CD4+ cells was significantly greater in the mice treated with BMT+TT compared with those with BMT alone or those with BMT + DLI. The percentage of FoxP3+ regulatory T cells among CD4+ cells was higher in the mice treated with BMT + TT comparable to those treated with BMT + DLI. The results for CD8+ T cells were similar to those for CD4+ cells. [Conclusion]. Allo-BMT combined with TT induces high thymopoiesis, and can elicit strong GVL effects with mild GVHD reaction. We thus found that donor-derived T cells play an important role in the treatment of leukemia. Also the details of the mechanisms are still unknown, one possibilities may be the continuous supplementation of T cells from the allogeneic thymus. The results of the present study suggest this strategy will become a new way for the treatment of refractory or relapse leukemia in human. DisclosuresNo relevant conflicts of interest to declare.

Various Ages Thymus Transplantation Has Differential Ability of Anti Tumor Effects

[Objective] Our previously work has demonstrated that allogeneic bone marrow transplantation (allo-BMT) combined with thymus transplantation (TT) was effective in restoring donor-derived T cell function and was beneficial for enhancing graft versus tumor (GVT) effects. However, since the thymic cell functions differ with age, the most effective age of thymus should be explored. In the present study, we examined the effects of allo-BMT plus thymus transplantation (TT) from various ages (fetal, newborn, adult) to determine it’s anti tumor effects.[Methods] BALB/c mice (H-2d ) bearing Meth-A sarcoma (H-2d )were treated with allo-BMT combined with or without TT from various age B6 mice(H-2b), the tumor size and survival period of the recipient BALB/c mice were examined, histological studies were performed in the liver, intestine, and the engrafted thymus from the recipients 4 weeks after the BMT. Surface markers on lymphocytes from the spleen were analyzed by 3-color fluorescence staining using a FACScan system to determine chimerism. Cytokine production was examined for monitoring lymphocyte function.[Results]. All mice treated with BMT with or without TT showed fully donor-derived chimerism. The tumor size were significantly smaller in the mice treated with BMT plus TT than those treated with BMT alone. Interestingly, the mice treated with BMT plus newborn or fetal thymus showed the greatest degree of tumor regression. The survival rate in mice treated with BMT plus newborn thymus was significantly prolonged compared with those treated with BMT plus adult thymus or BMT plus fetal thymus. Histologically, both the cortex and medullar areas were clearly shown in each group. Normal T-cell differentiation was also observed in the engrafted thymus. The number of CD4+ T cells significantly increased in the mice treated with BMT plus TT compared with those treated BMT alone. The numbers were highest in the mice treated with BMT plus newborn thymus or BMT plus fetal thymus. Microscopic founding of small intestine and liver indicated no evidence of GVHD in all mice treated with BMT combined with or without TT. The production of IL-2 and IFN-γ was significantly elevated in the mice treated with BMT plus TT compared with those treated with BMT alone. However, the production of IL-2 has no significantly difference in all various age thymus transplantation groups. In contrast, the production of IFN-γ was the highest in the mice treated with BMT plus newborn thymus transplantation.[Conclusion]. The present study indicated that allo-BMT combined with TT induces high thymopoiesis, elicit strong GVT effects, and is effective for the host with cancer. And the combination of allo-BMT with newborn thymus is the most effect. We thus found that donor-derived T cells play an important role in the treatment of leukemia. As human thymus tissue can be obtained from patient with congenital heart disease or from aborted fetuses, so the results of the present study suggest this strategy will become a new way for the treatment of malignant tumors in human. DisclosuresNo relevant conflicts of interest to declare.

Generation and Analysis of a Novel Mouse Model for Chronic Myelomonocytic Leukemia (CMML) with Acquired Expression of c-CBLQ367P

Myelodysplastic syndromes (MDS) are disorders originated from hematopoietic stem cells (HSCs), which are characterized by ineffective hematopoiesis, dysplasia mainly in the myeloid lineage, and high progression ratio to acute myeloid leukemia (AML). Recently, we identified mutations of the c-CBL (Casitas B cell lymphoma, a cellular homologue of v-CBL) gene in patients with MDS and myeloproliferative neoplasms (MPN). The mutations are detected in about 8% of the patients with the highest frequency in chronic myelomonocytic leukemia (CMML) cases with acquired uniparental disomy (aUPD) at 11q. c-CBL encodes a RING finger-based E3 ubiquitin ligase that negatively regulates receptor-mediated intracellular signaling. c-CBL is highly expressed in HSCs, strongly suggesting that it functions as a fine regulator of hematopoietic homeostasis. In fact, c-CBL knockout (KO) mice showed a myeloproliferative phenotype, owing to the hyper-responsiveness of HSCs to cytokine stimulation and subsequent augmented hematopoietic progenitor pool. In addition, c-CBL knockin (KI) mice harboring a mutation in the RING finger domain in one allele and a null mutation in the other allele exhibit an MPD-like disease and eventually progress to AML. These findings indicate that dysfunction of c-CBL perturbs normal hematopoietic development and contributes to hematopoietic abnormalities, but the precise leukemogenic mechanism(s) remains elusive. To gain insights into this issue and to create a novel animal model for mutated c-CBL-harboring leukemia, we generated conditional knock-in (cKI) mice that express wild-type c-CBL at steady state and inducibly express c-CBLQ367P, which was identified in patients with chronic myelomonocytic leukemia (CMML). After induced expression of c-CBLQ367P, the cKI mice exhibited a rapid and sustained increase in myelomonocytic cells with dysplasia in the peripheral blood and splenic enlargement with proliferation of myeloid cells, which closely resemble to the phenotype of human CMML. The bone marrow (BM) was hypercellular with predominance of myeloid cells, and increased number of HSC subpopulations and early myeloid progenitors were observed. In addition, phosphorylation of AKT, STAT3 and STAT5 was detected in long-term hematopoietic stem cells (LT-HSCs) of c-CBLQ367P cKI mice, indicating that PI3K/AKT and JAK/STAT signaling pathways are activated in c-CBLQ367P LT-HSCs. Moreover, competitive repopulation assays revealed that mice transplanted with c-CBLQ367P LT-HSCs showed significantly higher donor-derived chimerism than those transplanted with control LT-HSCs and displayed expansion of myelomonocytic cells as observed in c-CBLQ367P cKI mice, indicating that c-CBLQ367P conferred a proliferative advantage to LT-HSCs and that the phenotypes observed in c-CBLQ367P cKI mice were hematopoietic cell-intrinsic. CMML is known to progress to AML, possibly with additional genetic aberrations. To investigate the mechanism(s) underlying the disease evolution, we performed retroviral insertional mutagenesis using MOL4070A, a derivative of Moloney murine leukemia virus capable of inducing myeloid diseases. Almost all MOL4070A-infected c-CBLQ367P cKI mice developed AML, while no disease was observed in virus-injected control mice. Inverse PCR method identified Evi1 gene as a common integration site in the diseased mice and high Evi1 expression was detected in Evi1-integrated tumors. Mice transplanted with Evi1-transduced c-CBLQ367P cKI c-kit-positive BM cells developed AML at a high frequency and in a shortened period as compared to those transplanted with Evi1-transduced control cells. Taken together, we demonstrated that acquired expression of c-CBLQ367P plays a causative role in the development of CMML by activating PI3K/AKT and JAK/STAT pathways in HSCs and found that Evi1 overexpression cooperates with c-CBLQ367P to develop AML. Our mouse model provides a powerful tool for understanding of the pathogenesis of CMML and for developing novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

The orally bioavailable MDM2 antagonist RG7112 and pegylated interferon α 2a target JAK2V617F-positive progenitor and stem cells

The Philadelphia chromosomal-negative chronic myeloproliferative neoplasms (MPNs) originate at the level of the hematopoietic stem cell (HSC). The protracted clinical course of the MPNs has limited the use of potentially toxic treatment modalities, which may eliminate the responsible malignant clone. Treatment with low doses of RG7112, an orally available small-molecule inhibitor of p53-MDM2, both alone and combined with pegylated interferon α 2a (Peg-IFNα 2a), significantly decreased MPN colony-forming unit-granulocyte macrophage and burst-forming unit-erythroid numbers and preferentially eliminated the total number of JAKV617F(+) MPN hematopoietic progenitor cells. The effects of RG7112 and Peg-IFNα 2a on MPN progenitor cells were dependent on blocking p53-MDM2 interactions and activating the p53 pathway, thereby increasing MPN CD34(+) cell apoptosis. Treatment of polycythemia vera (PV) and primary myelofibrosis (PMF) CD34(+) cells with low doses of RG7112 and Peg-IFNα 2a before their transplantation into immune-deficient mice decreased the degree of donor-derived chimerism as well as the JAK2V617F allele burden, indicating that these drugs can each alone or in combination deplete MPN HSCs. These results provide a rationale for the use of combinations of low doses of RG7112 and Peg-IFNα 2a for the treatment of PV or PMF patients with the intent of altering their natural history.

Passenger Lymphocyte Syndrome Following Solid Organ Transplantation: Graft Source, Incidence, Specificity, Duration, and Severity Of Hemolysis

BackgroundPassenger Lymphocyte Syndrome (PLS) is a rare complication of solid organ transplantation (SOT) and is marked by production of donor-derived antibodies towards host red blood cell (RBC) antigens. At Canada's largest SOT program, >400 transplants are conducted annually. The affiliated Transfusion Laboratory detects possible cases of PLS when previously seronegative hosts develop (delayed, non-transfusion-attributable) post-transplant antibodies (Ab) bearing unexpected, autoreactive (rather than alloreactive) RBC specificities. Donor attributability is established by discovery of the same specificity (on serologic lookback) and/or by the antithetical (seroconversion risk) phenotype. The incidence, duration, and severity of PLS remain unknown due to the infrequency of reported cases and the general absence of active surveillance. MethodBetween 1/10/2006 – 30/06/2013, PLS cases were discovered by the Blood Bank through pre-transfusion specimens. The specificities, phase (direct or indirect), correlative markers of hemolysis, and temporal profile of the Ab were subsequently followed according to patient and clinician interest. The severity of hemolysis (grade 0 to 5) at the time of nadir hemoglobin was judged according to the tiers defined by the Canadian Blood Services/IVIG Hemolysis Pharmacovigilance Group (CL#2011-34). Implicated organ and donor review (for relevant serology, and outcomes in any parallel organ distributions) were also attempted. ResultsOver 88 months, 2772 transplants occurred (992 kidney, 187 pancreas [with/after kidney in 113/74 respectively], 909 liver, 529 lung [437 double, 83 single, 9 +heart], 151 heart, 4 bowel). PLS was found in 14 or 0.5% (sex: 5F: 9M; mean age 56 years), with the implicated organ being lung or liver (9 [2%] and 5 [0.6%] respectively). All had a negative pre-transplant DAT with a pre-sensitized donor. PLS Ab specificities included ABO (8: 5A, 3B), RH (5 with ≥1 target [C:3, D:5, E:2, V:1]), and others (KIDD: Jka in 2; MNS: N in 1; unidentified in 1). Multiple Ab occurred in 5 (36%), usually within the same system (3 RH cases: C,D,E in 2; C,D,V in 1), but otherwise towards ABO + ≥1 target (B,Jka,N; B, unidentified). The time to the first detectable expression of an Ab (as signaled by a +DAT and/or screen) was 4-120 days (mean 24, median 15, n=14). Clinically significant hemolysis occurred in 11 (79%), with six grade 3 and five grade 4; the duration of hemolysis was 0-776 days (mean 148, median 38, n=11). The duration of Ab detectability (or the point of “last positivity”) was 12-851 days (mean 196, median 78, n=10), while the point of disappearance (“subsequent sustained negativity”) occurred between day 12-1288 (mean 298, median 45, n=10). Lookbacks were feasible with 5 implicated donors, and in only 1 case (of a contralateral lung's transplantation) was PLS detected in another recipient. PLS did not account directly for any deaths in the cohort, although hemolytic morbidity required treatment in 2 cases. Transfusion support with antigen-negative blood was provided to 11 patients for anemia that may have been due to hemolysis and/or other causes while the Ab was still evident. In one case, severe hemolysis provoked rate-uncontrolled atrial fibrillation with a supply and demand ischemia which culminated in irreversible congestive heart failure and cardiac death 2 months after PLS resolution. [Display omitted] Discussion/ConclusionsThis is the largest series of PLS reported to date. Clinician-driven submissions of blood for transfusion compatibility testing, and the discovery of discrepant serologies, represented an unforced but prospective means by which to identify PLS in roughly 1 in 200 transplants. Several cases transcended the limits of previously described temporal profiles (with the most delayed onset at 120 days, and the most prolonged duration at 2.3 years). The duration of hemolysis was consistently shorter than the duration of seropositivity, and was rarely life-threatening, while the most severe grades associated with RH rather than ABO or other targets. The surveillance achieved in this cohort revealed a longer-than-previously-appreciated duration of Ab productivity, and raised the question of whether or not such prolongation is a function of observation bias or the current-day armamentarium of anti-rejection therapeutics, which may be more permissive to donor-derived chimerism. Disclosures:No relevant conflicts of interest to declare.

CD166 (ALCAM): A Functional Marker of Primitive Murine and Human Hematopoietic Stem Cells and Cellular Elements of Their Niche

AbstractAbstract 640Phenotypic definition of murine and human hematopoietic stem cells (HSC) relies on a large number of markers. Few surface antigens with functional importance have been identified as key common markers on adult murine and human HSC. Intimate interactions between HSC and elements of the hematopoietic niche (HN) depend on ligand-counter ligand molecules co-expressed independently on niche components or putative HSC. To date, not a single surface marker has been identified as a common marker expressed on murine and human HSC and on cells of the HN. We previously demonstrated that CD166 is expressed on osteoblasts (OB) that mediate a hematopoiesis enhancing activity (HEA). Given that CD166 is a member of the immunoglobulin superfamily that can mediate homophilic cell-cell interactions, we investigated the role of CD166 in identifying HSC and the impact of CD166 on hematopoiesis, stem cell engraftment, and the HN. Interestingly, CD166+, but not CD166- fractions of murine and human repopulating HSC identified by a rigorous hierarchical classification for each species mediated robust long-term engraftment. In the murine system, 25 sorted Lineage- Sca1+ ckit+ (LSK) CD48-CD150+CD166+ cells mediated 69.5 ± 7.3% chimerism 4 months post-transplantation (PT) while donor-derived chimerism supported by 25 sorted LSKCD48-CD150+CD166- cells was only 13.6 ± 11.6% (p<0.01) suggesting that CD166 identifies long-term repopulating cells beyond what is possible with SLAM markers. In the human system, 1000 cord blood-derived Lin-CD34+CD38-CD49f-CD166+ cells and Lin-CD34+CD38-CD49f+CD166+ cells engrafted at 44.5 ± 9.7% and 38.4 ± 8.9%, respectively 16 weeks PT in conditioned NSG mice. More importantly, chimerism derived from Lin-CD34+CD38-CD49f+CD166- cells was 1.6 ± 0.1% (p<0.01 vs both fractions) demonstrating that the CD166+ but not the CD166- fraction of CD34+Lin-CD38- cells (regardless of the status of CD49f expression) contains long-term engrafting human HSC. In CD166 knockout (KO) mice, numbers of LSKCD48-CD150+ cells in the bone marrow and Lin-CD48- cells in the peripheral blood were significantly reduced relative to wild-type (WT) controls although other hematopoietic parameters in KO mice were within normal ranges. Phenotypically defined HSC from CD166−/− mice failed to engraft in lethally irradiated WT recipients. Levels of engraftment 4mo PT of 1,000 LSK cells from WT donors into WT recipients was 71.8% ± 8.3% while that obtained from a similar number of KO cells was 5.8% ± 2.8% (p<0.01). To permit direct comparison of KO and WT mice as recipients, both genotypes were transplanted with purified LSK cells from GFP C57BL/6 mice. While short-term repopulating GFP cells engrafted efficiently in KO mice 1mo PT (66.0% ± 6.5%), reconstitution declined substantially 2mo PT and was 10.3% ± 2.7% at 3mo PT (compared to 52.6% ± 10.4% in WT hosts, p<0.01) and less than 5% at 4mo PT, demonstrating that the CD166−/− hematopoietic niche can not support long-term repopulating cells. We used our previously described co-culture system to assess the impact of homophilic CD166 interactions on the HEA of OB. The highest HEA was reached when both OB and LSK cells expressed CD166. However, when either or both cell types lacked CD166 expression, the degree of HEA was significantly lower demonstrating that homophilic CD166 interactions are critical to maintaining HSC function. Since Stat3 has 3 binding sites on the CD166 promoter, we examined the relationship between expression of Stat3 and CD166. HSC from Stat3−/− mice which do not engraft efficiently in WT recipients expressed very low levels of CD166. In addition, pharmacologic inhibition of Stat3 expression led to a simultaneous inhibition of CD166 expression. Reconstitution kinetics data and survival of KO mice under hematopoietic stress conditions suggested that CD166−/− HSC have an intrinsic self-renewal capacity precluding them from both rapid proliferation and expansion and maintenance of the stem cell pool in the HN. Our data illustrate for the first time, that CD166 is a universal marker of both murine and human HSC and OB within the HN and suggest that CD166 may modulate HSC-niche interactions and impact stem cell fate. The conserved homology between murine and human CD166 provides an excellent bridge between human and murine studies for efficient translational investigations and interventions for enhancing stem cell engraftment and clinical utility. Disclosures:Broxmeyer:CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

Paracrine Proangiopoietic Effects of Human Umbilical Cord Blood-Derived Purified CD133+Cells—Implications for Stem Cell Therapies in Regenerative Medicine

CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance