Number Of Hematopoietic Stem Cells Research Articles

Extracellular DEK Regulates Hematopoiesis through CXCR2-Mediated Intracellular Signaling That Does Not Require Its Translocation into the Nucleus or Binding to DNA

DEK is a highly conserved, ubiquitously expressed and structurally unique DNA-binding nuclear protein involved in transcription, DNA replication, and DNA repair implicating a role for DEK in gene regulation. Interestingly, hematopoietic cells, following stimulation with inflammatory mediators, can actively secrete DEK, which can then be taken up by cells via binding to heparan sulfate proteoglycans (HSPGs), translocating to the nucleus, and mediating its normal function of gene regulation. Previous work from our group indicates that both endogenous, intracellular DEK and secreted, extracellular DEK regulates normal hematopoiesis by decreasing numbers of hematopoietic progenitor cells (HPCs) and increasing numbers of hematopoietic stem cells (HSCs) suggesting that DEK plays a role in regulating HSC/HPC function and/or cell fate. Extracellular DEK-mediated regulation of hematopoiesis occurs in a HSPG-, CXCR2-, Gai protein-dependent manner. Neutralizing these pathways inhibits extracellular DEK regulation of HSCs and HPCs. However, what was not known is whether the HSPG-dependent internalization of DEK is required for the extracellular DEK-mediated regulation of hematopoiesis as HSPGs are also required for maximum binding efficiency of ELR-CXC chemokines to CXCR2. To address this unknown, two mutant forms of recombinant (r)DEK were utilized: DEK that lacks its nuclear translocation sequence (NTS) and DEK that lacks its DNA-binding motif (DBM). First we examined whether NTS- or DMB-rDEK could alter HPC or HSC numbers in vitro / ex vivo . We had previously shown that C57Bl/6 bone marrow (BM) cells when in culture with rDEK inhibited HPC colony formation in a dose-dependent manner, with the minimum dose of WT rDEK required for inhibition being 12.5 nM (p=0.002). Both NTS- and DMB-rDEK also inhibited HPC colony formation in a dose-dependent manner starting at 12.5 nM (p=0.03 and p=0.02 respectively) suggesting that neither the ability of DEK to get into the nucleus or bind to DNA is required to regulate HPC numbers in BM. To examine whether NTS- or DMB-rDEK can still enhance HSC numbers, ex vivo HSC expansion assays were performed. C57Bl/6 lineage negative BM cells were cultured in SCF, TPO, and Flt3L with vehicle control, WT-rDEK, NTS-rDEK, or DMB-rDEK then examined for HSC expansion on days 4 and 7. On both days, WT-, NTS-rDEK and DMB-rDEK significantly expanded long-term HSC numbers when compared to vehicle control, suggesting that like DEK-mediated suppression of HPCs, DEK-mediated expansion of HSCs does not require DEK to translocate to the nucleus or bind to DNA. To examine whether extracellular NTS- and DMB-rDEK can still mediate one of DEK's known nuclear functions, heterochromatin stability, DEK-/- lineage negative BM cells were incubated for 12 hours with WT rDEK, NTS-rDEK or DMB-rDEK then examined for H3K9 trimethylation in HSCs and HPCs by ImageStream flow cytometry. Neither NTS- or DMB-rDEK were able to enhance the trimethylation of H3K9 in DEK-/- HSCs or HPCs. However, incubating DEK-/- lineage negative BM with WT rDEK, increased H3K9me3 in the nucleus of the cells by ~26%. When utilizing WT BM, incubating cells for 12 hours with WT, NTS- or DMB-rDEK did not result in changes in H3K9Me3 that was detectable by ImageStream technology suggesting that extracellular DEK-mediated changes in chromatin remodeling in HSCs and HPCs only occurs when the cells lack endogenous DEK. Co-culturing C57Bl/6 BM cells with WT rDEK mediated AKT and ERK1/2 phosphorylation in HSCs and ERK1/2 in HPCs which is blocked when BM cells are pretreated with a neutralizing antibody against CXCR2 or when preteated with heparin neutralizing HSPGs. Both NTS- and DMB-rDEK were able to mediate similar activation of AKT and ERK1/2 in HSCs and HPCs as WT rDEK but not when the BM was pretreated with anti-CXCR2 antibody or heparin. Taken together, these data suggest that extracellular DEK functions to regulate HSC and HPC numbers/function by binding to CXCR2 to mediate intracellular signaling events in C57Bl/6 BM cells. The fact that HSPGs are required for extracellular DEK's effect, but DEK does not have to get into the nucleus and bind DNA, also suggests that internalization of DEK is not required to regulate hematopoiesis in cells with normal levels of endogenous DEK. Therefore, like the ELR-CXC chemokines, DEK requires HSPGs for its CXCR2-mediated signaling processes in both HSCs and HPCs. DisclosuresNo relevant conflicts of interest to declare.

A Novel View of Umbilical Cord Blood Versus Maternal Peripheral Blood — Evidence for an Increase in the Number of Circulating Stem Cells on Both Sides of the Fetal–Maternal Circulation Barrier.

Background. Umbilical cord blood (UCB) is a rich source of stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitors cells (EPCs), and very small embryonic-like stem cells (VSELs). We envision that these cells are mobilized into UCB in response to hypoxia and increases in the levels of pro-mobilizing factors released in response to delivery stress. We have hypothesized that these stem cells may play a role in repairing tissue/organ injuries that occur in the newborn child after delivery and that mobilization of these cells into newborn peripheral blood could be a kind of autologous, physiological stem cell therapy that all newborns experience after delivery. Aim of the study. Interestingly, in contrast to the intensive research on stem cells present in UCB, parallel research on stem cells released into maternal blood has been lacking. This is despite the fact that, like the newborn child, the mother is exposed to several types of injuries related to delivery, such as damage due to passage of the fetus through the reproductive tract, hypoxia, and the release of pro-mobilizing cytokines and bioactive lipids. Hypothesis. We hypothesized that the number of circulating stem cells also increases in the maternal circulation due to stress related to delivery as part of an autologous regeneration mechanism.Materials and Methods. To evaluate the possibility of a parallel increase in the number of circulating stem cells in the maternal circulation, we analyzed the number of circulating HSCs, MSCs, EPCs, and VSELs in maternal peripheral blood 12-24 hours before and 24 hours after normal delivery (n=17). Specifically, we analyzed CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic SCs (HSCs), the CD34+/CD133+/KDR+ population of endothelial progenitor cells (EPCs), the CD45-/CD105+/CD90+/CD29+ population of mesenchymal SCs (MSCs), and the CD34+ and CD133+ populations of Lin−CD45− very small embryonic-like stem cells (VSELs). We also employed standard clonogenic assays to enumerate the number of hematopoietic progenitors circulating in maternal blood. Results. We observed that the number of HSCs, MSCs, EPCs, VSELs and clonogenic progenitors increases significantly in maternal blood at 24 hours after normal delivery (n=17). Conclusions. Based on this observation, we propose that delivery stress is associated with an increase in the number of circulating stem cells, not only on the fetal side but also on the maternal side of the fetal-maternal circulatory barrier. We propose that, most likely, the increase in the number of circulating stem cells in maternal blood indicates that these cells play a role in repairing small tissue/organ injuries that occur in the mother's body due to delivery. Currently, we are determining whether these changes correspond with other clinical parameters, such as the extent of the mother's internal injuries or the Apgar status of the newborn child, and whether there are changes in the number of circulating stem cells in maternal blood after planned C-sections. We propose that this phenomenon of maternally circulating stem cells could potentially be harnessed to accelerate the recovery of the mother following delivery. DisclosuresNo relevant conflicts of interest to declare.

Comparison of Peripheral Blood Stem Cell Mobilization with Etoposide or Cyclophosphamide in Patients with Multiple Myeloma

▪For successful autologous stem cell transplantation, collection of the sufficient number of hematopoietic stem cells is essential after induction therapy for transplant candidates in multiple myeloma (MM). In this study, we compared the efficacy and safety of stem cell mobilization with etoposide (VP-16; 375 mg/m2 on days 1 and 2) or cyclophosphamide (CY; 3.0 g/m2 on day 1) in patients with MM. Granulocyte-colony stimulating factor (G-CSF, 10 mg/kg/day, subcutaneously) was administered from the development of neutropenia to the final collecting day. Mobilization failure was defined as total collected CD34+ cell count < 4.0 x 106 cells/kg in three apheresis procedures. Seventy-six patients were mobilized with CY and G-CSF, and 46 patients were mobilized with VP-16 and G-CSF between February 2008 and July 2017. Median total collected number of CD34+ cells was significantly higher in patients who were mobilized with VP-16 compared with those who were mobilized with CY (27.6 x 106 cells/kg vs. 9.5 x 106 cells/kg, P < 0.001). In addition, the rate of mobilization failure was significantly lower in VP-16 group compared with CY group (2.2% vs. 23.7%, HR 0.072, 95% CI 0.009-0.557, P = 0.001). One patients who were mobilized with VP-16 showed mobilization failure and did not achieve the sufficient collection of stem cells in subsequent mobilization process with plerixafor. Severe infections during mobilization period were frequently developed in CY group (15.8% in CY group vs. 8.6% in VP-16 group, P = 0.407). The median time to platelet engraftment (> 20 × 109/L) and neutrophil recovery (> 0.5 × 109/L) did not different between two group after autologous transplantation. In conclusion, VP-16 and G-CSF was a very effective and safe method for peripheral blood stem cell mobilization and is considered as a more suitable chemo-mobilization regimen compared to CY and G-CSF in patients with MM. DisclosuresNo relevant conflicts of interest to declare.

Zebrafish gata2 Orthologs Are Necessary but Not Sufficient for Developmental Hematopoiesis

Germline mutations in the GATA2 gene have been identified in patients with a variety of hematological disorders unified by their with predisposition to myelodysplastic syndromes (MDS) and defective immunity. The GATA2 gene encodes a transcription factor that plays a major role in hematopoiesis. However, the precise effects of GATA2 haploinsufficiency in developmental hematopoiesis and the impact of this in MDS predisposition remain unknown. We have developed zebrafish mutants of gata2 to model this and demonstrate that heterozygosity results in defective primitive and definitive granulopoiesis.Zebrafish carry two orthologues to the mammalian GATA2 gene: gata2a and gata2b . gata2a and gata2b demonstrate distinct expression patterns. By 32 hours post fertilization (hpf), expression of gata2a is seen throughout the dorsal aorta (DA) and cardinal vein, primitive erythrocytes and the nervous system. In contrast, gata2b expression is restricted to the ventral DA (VDA) and branchiomotor neurons. We hypothesised that this division of expression may translate into a requirement for both ohnologs as necessary but not sufficient for normal developmental hematopoiesis.Homozygous gata2a mutants carrying an 11bp deletion (gata2aum27 ) have been previously described. These mutants have abnormal vascular development in the aorta resulting in defective circulation. We analysed developmental hematopoiesis in these mutants. gata2aum27/um27 and gata2aum27 /+ showed normal hemangioblasts, and primitive hematopoiesis before 18 somites. At 22hpf however myeloid transcription factor cebpa showed a dose dependent decrease in expression resulting from gata2a loss, while pu.1 expression remained normal. This suggests that Gata2a may be a direct regulator of cebpa in primitive myeloid cells, or that cebpa expression defines a sub-population of myeloid cells. We then assessed hematopoietic stem cells (HSCs) in gata2aum27 mutants. gata2aum27/um27 showed severely decreased or absent HSCs in the caudal hematopoietic tissue (CHT, fetal liver equivalent) at 52hpf, likely due to absent blood flow in the aorta. gata2aum27/+ mutants also showed a decrease in HSCs at this time point, however this decrease was transient, resolving by 72hpf (A). Further assessment of definitive hematopoiesis showed that gata2um27/um27 lacked mature granulocytes and thymocytes, in keeping with the observed HSC defect. By contrast myeloid cells in gata2aum27/+ at 4dpf (assessed by Sudan Black (SB) stain) were unaffected (B,C) . Interestingly lck-expressing thymocytes showed a range of phenotypes, from normal, through reduced and some with a complete absence of thymocytes in one of their thymii.We next used CRISPR to generate a gata2b mutant line. This line carries a 4bp deletion leading to a truncated protein lacking both zinc fingers (gata2bU5008 ). In contrast to gata2aum27/um27 , gata2bu5008/5008 have normal circulation and were viable beyond 5dpf. Loss of gata2b by morpholino knockdown results in absence of HSC, however gata2b5008/+ showed no difference in HSC numbers in the CHT at 60hpf. Myeloid cells were unaffected in gata2b5008 mutants by SB at 48hpf, however, by 4dpf gata2b5008 show a dose dependent reduction in myeloid cells (D). This suggests that Gata2b regulates myeloid cell proliferation or differentiation after the emergence of HSCs.We next studied haematopoiesis in crosses of gata2aum27/+ to gata2bu5008/+ . Quantification of granulocytes showed normal numbers at 48hpf in all resulting genotypes. By 4dpf gata2b5008/+ have a reduced number of SB expressing granulocytes regardless of their gata2a genotype, suggesting that Gata2a is dispensable for definitive myelopoiesis (E). Quantification of HSCs show HSC numbers are unaffected in gata2aum27/+ ; gata2bu5008/+ at 48hpf. Assessment of thymocytes by contrast shows a greater than additive effect on thymocyte numbers than in single mutants alone, suggesting that both Gata2a and Gata2b contribute to lymphoid development in an additive manner. Further characterisation of the effects of gata2aum27/+ ; gata2bu5008/+ in combination is ongoing.In conclusion our results support the notion at that haploinsufficient levels of Gata2a and Gata2b are necessary but not sufficient for normal developmental hematopoiesis and that combined heterozygous loss of these genes models the haploinsufficieny observed in patients in GATA2 haploinsufficient diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hmgb2 Is a Novel Transcriptional Regulator of Latexin in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) provide life-long production of blood cells and undergo self-renewal division in order to sustain the stem cell pool. Precisely regulated HSC pool maintenance and blood cell production is vital for organismal survival. We previously have identified a protein, latexin (Lxn), as a novel regulator of HSCs in mice, whose natural variation in the expression is inversely correlated with HSC population size. However, the molecular mechanisms involved in transcriptional regulation of Lxn in HSCs have not been clearly defined. By using DNA pull down and mass spectrometry, we identified several proteins that bind to the promoter region of Lxn. One top candidate is Hmgb2(High mobility group protein B2). ChIP Q-PCR assay and luciferase assay confirmed the binding of Hmgb2 to Lxn promoter with the potential role of transcriptional repressor. We also found that knock-down of Hmgb2 increases the endogenous Lxn expression at both mRNA and protein levels in hematopoietic cell line EML cells and primary HSC cells. As a result, HSC number was demonstrated decreased in vitro and in vivo due to decreased self-renewal and proliferation and increased apoptosis. This study, for the first time, revealed the transcriptional regulation of Lxn by Hmgb2. Disclosures No relevant conflicts of interest to declare.

YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biophysical Forces of Blood Flow

Hematopoietic stem cells (HSCs) are first specified during embryonic development from hemogenic endothelial cells (HEC) in the ventral dorsal aorta (VDA). Prior studies from our lab and others demonstrated that HSC formation is coordinated with the onset of rigorous circulatory flow, which exposes the vascular endothelium to both wall shear stress (WSS) and circumferential stretch (CS). To better understand how these biomechanical forces drive HEC specification and HSC production, we engineered a microfluidics-based dorsal aorta-on-a-chip that recapitulates the biophysical features present in the vasculature. Using this biomimetic microsystem, human iPS-derived HEC exposed to WSS and CS through continuous media flow and cyclic stretching of the adherent cell layer showed an upregulation of RUNX1 (WSS, p<0.01; CS, p<0.05), the main transcription factor involved in HSC specification, by qPCR compared to cells in the static control. Interestingly, the YAP (Yes-associated protein) target genes CYR61, ANKRD1 and CTGF were also upregulated in human iPS-derived HEC after exposure to CS, suggesting a possible downstream mechanism for the effects of blood flow on HEC. YAP is the most downstream effector protein in the Hippo signaling pathway and is known to regulate stem cell dynamics and organ size. Prior work identified YAP as a mechanosensor, where it is activated in response to Rho GTPase activity and actomyosin filament rearrangement independent of the core Hippo pathway. To test whether YAP functions in response to mechanical force-activated Rho, iPS-derived HEC were exposed to circumferential stretch in the presence of a Rho inhibitor, which abolished CS-induced activation of YAP targets and RUNX1, indicating that Rho proteins are involved in mediating the effects of blood flow on HECs. Furthermore, treatment of iPS-derived HEC cultured under static conditions with either direct or indirect activators of the Rho GTPase family increased YAP activity and RUNX1 levels, demonstrating that biophysical forces from blood flow can be mimicked in vitro with Rho modulation. To investigate the implication of these findings in vivo, we utilized the zebrafish model: as in mammalian systems, HSCs are born in the VDA (24-36 hours post fertilization (hpf)) and migrate to the secondary sites of hematopoiesis to expand and differentiate before colonizing adult tissues. Heat-shock mediated overexpression of Yap at 12 hpf significantly increased runx1 + HEC specification at 36 hpf by both in situ hybridization and qPCR (p<0.01). This impact on HSC number was sustained throughout development, as Yap overexpression also significantly increased the number of Cd41+ HSCs present in the caudal hematopoietic tissue (CHT) at 72 hpf (p<0.05). In contrast, Yap knockout mutants exhibited a significant reduction in runx1 at 36 hpf (p<0.0001) despite normal Flk1+ vascular structure, indicative of a role for Yap in HSC specification. In line with the human in vitro data, activation of Rho GTPase increased expression of Yap target genes and runx1 at 36 hpf (p<0.05), suggesting a pathway in which Yap induces Runx1 downstream of Rho stimulation. Prior analysis of the silent heart mutant line as well as morpholino knockdown of cardiac troponin T type A (tnnt2a) revealed a significant deficiency in HEC specification and HSC production in the absence of heartbeat and blood flow. Importantly, compound-mediated activation of Rho GTPase (12-36 hpf) significantly increased runx1 expression in tnnt2a morphant embryos by in situ hybridization and qPCR (p<0.01), demonstrating that Rho activation can mimic the effects of blood flow on HSC production in vivo . Taken together, our findings point to a functional intersection between blood flow, Rho GTPase function and YAP activation in RUNX1-dependent HSC production; this mechanochemical mechanism may be exploited to improve in vitro human HSC differentiation protocols for the treatment for blood disorders. DisclosuresIngber:Emulate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

MLL3 Deletion Enhances HSC Self-Renewal

Mixed lineage leukemia 3 (MLL3 ) is one of the most commonly mutated genes in human cancer, and it is believed to function as a tumor suppressor. It is one of many genes affected as a result of mono-allelic 7q deletions that occur commonly in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MLL3 encodes a histone methyltransferase that catalyzes the monomethylation of Histone 3 lysine 4 (H3K4), a known feature of enhancer elements. MLL3 also nucleates a COMPASS-like complex that removes repressive H3K27me3 marks, recruits the histone acetyltransferase p300/CBP to enhancers and promotes transcript elongation at promoters. For these reasons, MLL3 binding at cis-regulatory elements is thought to potentiate enhancer activity and transcription. Previous studies have shown that Mll3 deficiency causes a mild myeloproliferative disorder in mice (PLoS ONE 11:e0162515), and shRNA knockdown of Mll3 has been shown to cooperate with loss of Nf1 and p53 to promote AML (Cancer Cell 25:652). In the latter study, Mll3 silencing was associated with a hematopoietic stem cell (HSC)-like gene expression signature within the AML cells. These studies implicate MLL3 as a hematopoietic tumor suppressor, though its precise function in HSC self-renewal and lineage commitment has not been described.To better understand how Mll3 regulates HSC self-renewal, we developed a novel loss-of-function mouse allele in which a 5-base pair deletion and frameshift mutation was created near the N-terminus of the protein. Homozygous Mll3-/- mice are viable through most of gestation, but they die just prior to birth. Heterozygous Mll3+/- mice are born at normal Mendelian ratios, their development is normal after birth and they are fertile. To test whether Mll3 regulates hematopoiesis, we determined the frequencies and absolute numbers of HSCs, lineage restricted hematopoietic progenitors (HPCs), and granulocyte-monocyte progenitors (GMPs) in control, Mll3+/- and Mll3-/- fetal livers (E18.5) and control and Mll3+/- adult bone marrow. Heterozygous and homozygous Mll3 deletion caused a modest increase in HSC numbers and a modest decrease in HPC frequency in fetal livers. Mll3 heterozygosity resulted in a significant expansion of the HSC population in adult bone marrow. To test whether these increases in HSC numbers reflected enhanced self-renewal, we performed serial transplantation assays with fetal and adult HSCs. While primary transplants showed only a modest increase in repopulating activity in Mll3+/- and Mll3-/- HSCs, this effect was greatly enhanced in secondary transplants. Thus, Mll3 deficiency enhances HSC self-renewal capacity. Despite this phenotype, RNAseq showed no differences in the transcriptomes of wild type and Mll3+/- HSCs or HPCs. This suggests that Mll3 loss may reduce the probability of lineage commitment during successive HSC divisions without altering the commitment pathways themselves. Ongoing experiments will test this hypothesis. Enhanced self-renewal, particularly under conditions of hematopoietic stress, may convey a selective advantage to Mll3 -heterozygous HSCs and enhance progression to MDS. DisclosuresNo relevant conflicts of interest to declare.

CXCL12-Expressing Bone Marrow Niches Differentially Regulate CML Compared to Normal Stem Cells

CXCL12 plays a major role in hematopoietic stem cells (HSC) localization to regulatory niches in the bone marrow microenvironment (BMM). Previous studies indicate that CXCL12 deletion from Prx+ mesenchymal stem cells (MSC) reduces murine HSC numbers, and from Tek+ endothelial cells (EC) modestly reduces their repopulating activity. In contrast, CXCL12 deletion from Ocn+ osteoblasts or Osx+ osteoprogenitors does not affect HSC function (Greenbaum et al. Nature. 2013 Mar 14;495(7440):227-30; Ding et al. Nature. 2013 Mar 14;495(7440):231-5.). These studies identified MSC and endothelial cells as important HSC niche components. However, niche cells for CML leukemic stem cells (LSC) remain poorly characterized. To evaluate the contribution of CXCL12-expressing populations to LSC regulation, we transplanted normal and CML BM cells (CD45.1/2+; 2*106/mouse) into CXCL12f/f (loxP sites flanking exon 2) mice crossed with Tek-Cre and Prx-Cre mice ( reported in ASH 2016, Blood, Abstract: 26) . We showed that CXCL12 deletion from Prx+ MSC resulted in reduction in normal HSC (LSK Flt3- CD150+ CD48- cells) numbers and function compared to controls, but did not observe significant reduction of HSC with CXCL12 deletion from Tek+ EC. Interestingly, transplantation of CML cells into mice with CXCL12 deleted from Prx+ MSC led to reduced survival compared to control mice associated with increased WBC counts, BM cellularity, LSC numbers, and secondary repopulation capacity. In contrast, CXCL12 deletion from Tek+ cells led to enhanced survival, associated with reduced BM cellularity and reduced LSC numbers. In further studies, we transplanted mixtures of equal numbers of normal and CML cells into Prx-Cre and Tek-Cre mice, and found a marked growth advantage of LSC compared with HSC in Prx-Cre mice, and a growth advantage of HSC over LSC in Tek-Cre knockout mice. We also show that both HSC and LSC demonstrate increased cell cycling via EdU labeling. Consistent with this, we found that Nilotinib treatment leads to significantly greater reduction in BM cellularity, LSC numbers and secondary repopulation potential in Prx-Cre mice, compared to Tek-Cre or WT mice. To evaluate the effects of CML on CXCL12-expressing BMM cells, we crossed CXCL12GFP (GFP reporter knocked into the CXCL12 locus) and SCL-tTA-BCR-ABL mice to generate CXCL12GFP-BCR-ABL mice. CML development lead to significantly reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) but increased GFP+ endothelial cells (CD45-Ter119-CD31+), and within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased compared to WT mice. To further understand niche mediated interactions that regulate LSC fate, we studied gene expression profiles of LSC from mice following Cre-mediated targeting of CXCL12, compared to LSC from Cre-negative mice. Our preliminary analysis indicates significant enrichment of genes associated with cell cycle progression and Myc activation, and negative enrichment of genes related to TGF-b, STAT3, and Polycomb Repressive Complex 2 (PRC2) activity in LSC from Prx1+Cre CXCL12f/f mice. Reduced TGF-b, STAT3 and PRC2/EZH2 activity represent potential mechanisms for regulation of CML LSC maintenance and/or TKI resistance, and may potentially contribute to Prx1+ MSC-mediated LSC regulation. We conclude that CXCL12+ BM MSC inhibit CML development, maintain LSC quiescence, and enhance TKI resistance, while CXCL12+ EC enhance LSC maintenance and disease development. Leukemia associated reduction in CXCL12+MSC and increase in CXCL12+EC may enhance CML development. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM MSC and EC for CML LSC compared with normal HSC. DisclosuresNo relevant conflicts of interest to declare.

Prostaglandin E2 Prevents Apoptosis and Promotes Homeostasis of Hematopoietic Stem Cells after Lethal Irradiation through Alteration of p53 and TNF Signaling

Background: The hematopoietic system is the most sensitive organ to therapeutic or unintended radiation. In a mouse model of the hematopoietic acute radiation syndrome (H-ARS) a single dose of 16,16 dimethyl prostaglandin E2 (dmPGE2) given 15 min to 3 h before LD50/30-90/30 total body irradiation (TBI) provides 100% survival and accelerates hematopoietic recovery. The mechanisms responsible, however, are not known.Methods: C57BL/6 or FGD5-ZsGreen (JAX) mice received 35 µg dmPGE2 or vehicle 30 min before LD50/30 (853 cGy) γ-irradiation, along with non-irradiated dmPGE2 and vehicle controls. Bone marrow (BM) cells were harvested at multiple time points post-TBI and analyzed by flow cytometry for hematopoietic stem cell (HSC) markers as well as cleaved-caspase 3, cleaved-PARP, and p21. HSC were sorted for RNA-seq 1 h post-TBI based on SLAM-LS (Sca-1, CD150, CD48, Lineage-neg) and ZsGreen fluorescence. FGD5-ZsGreen mice express ZsGreen specifically in HSC, and were used to maximize HSC purity as the standard cKit marker is affected by irradiation. RNA-seq data were analyzed using Ingenuity Pathway Analysis.Results: The number of marker-defined HSC in dmPGE2-treated mice was preserved at 24 h post-TBI, but then declined to levels of vehicle-treated mice by day 3, implying that a qualitative rather than quantitative difference in HSC may be facilitating animal survival. To investigate gene expression changes in HSC after dmPGE2 treatment and irradiation, BM cells were harvested 1 h post-TBI and HSC sorted for RNA-seq. We first reasoned that changes observed with dmPGE2 alone may be involved in priming HSC to respond differently to irradiation, perhaps preserving HSC properties and function. The most activated upstream regulator by dmPGE2 alone was TNF, based on the expression patterns of known TNF-regulated genes, with an activation z-score of +5.26 (p = 1.39 x 10-23). Interestingly, TNF was also the upstream regulator most activated by irradiation alone (z-score +8.26, p = 5.88 x 10-37), to a greater degree than dmPGE2 alone, and different sets of TNF regulated genes were increased or decreased in each case. Significantly, with dmPGE2 plus irradiation, TNF-regulated genes in HSC adopted an expression pattern similar to dmPGE2-only, and generally opposite to irradiation alone. The direct comparison of dmPGE2-irradiation with vehicle-irradiation resulted in a significant net inhibition of TNF activation. Approximately one third of these altered genes are also NF-kB-regulated, suggesting that NF-kB-mediated TNF signaling induced by irradiation is prevented by dmPGE2 treatment. TNF mRNA itself was highly upregulated in both irradiated groups, but while TNF receptor 1 expression was not affected, dmPGE2 (with and without irradiation) significantly increased expression of TNF receptor 2, likely influencing TNF downstream effects.Strong changes were also seen in the p53-mediated apoptosis pathway. This pathway was not increased by dmPGE2 alone, but was greatly activated by irradiation (p = 1.47 x 10-27) and was the top inhibited regulator by dmPGE2-irradiation compared to irradiation alone (p = 1.86 x 10-18). At the protein level, a corresponding decrease in HSC production of cleaved caspase-3 and cleaved-PARP was detected from 1 h to 9 d post-TBI in PGE2-treated animals after irradiation, supporting its protection of HSC from irradiation-induced apoptosis. One dramatically affected gene involved in both the TNF and p53 pathways was the cdk inhibitor p21 (Cdkn1a). Confirming the observed gene expression pattern, p21 protein was substantially increased in HSC by 24 h post-TBI, and this was prevented by dmPGE2.Finally, irradiation stimulated a widespread increase in HSC transcriptional activity that was generally prevented by dmPGE2, suggesting preservation of a more quiescent state. In addition, transcription factors implicated in HSC maintenance/function were significantly decreased with irradiation and protected by dmPGE2, including Gata2, Meis1, Hoxa9, Tal1, and Gfi1.Conclusions: DmPGE2 induces rapid gene expression changes in HSC that dampen transcriptional activation induced by high-dose irradiation. This includes inhibiting activation of p53 and NFKB-mediated TNF activity, reducing apoptosis and maintaining essential HSC properties. This work provides insight into the mechanism of how dmPGE2 facilitates survival and hematopoietic recovery from lethal irradiation. DisclosuresNo relevant conflicts of interest to declare.

BET-Inhibition By JQ1 Promotes Proliferation and Self-Renewal Capacity of Hematopoietic Stem Cells

Background/significance: Bromodomain and extra terminal domain (BET) proteins have been shown to be critical for maintaining the function of leukemia stem cells. Although BET-inhibitors are in clinical development and show promising activity in different hematologic malignancies, a systematic analysis of the consequences of pharmacological BET-inhibition on healthy hematopoietic (stem) cells is outstanding.Methods: C57BL/6J (WT) or B6.SJL-Ptprca Pepcb/BoyJ mice, which carry the congenic pan-leukocyte marker CD45.1 were treated for 3 weeks with the BET-Inhibitor JQ1 or a placebo. For competitive repopulation transplantations, bone marrow from treated CD45.1 mice was transplanted together with WT competitor cells (CD45.2) into lethally irradiated WT mice (9 Gy) in different ratios. Engraftment of all major hematopoietic lineages was monitored in peripheral blood (PB) and BM using differential blood counting and flow cytometry. For the determination of HSC frequency in vivo, extreme limiting dilution analysis (ELDA) was carried out by transplanting 2x103, 2x104 or 2x105 JQ1- or placebo-treated bone marrow cells from WT mice together with 2x105 bone marrow cells from CD45.2 mice. Engraftment was assumed when CD45.1+ myeloid cells were present in the peripheral blood in frequencies > 1 %.Colony formation assays were used to measure the abundance of colony forming progenitor cells (M3434) as well as megakaryocytic progenitors (MegaCult-C). For analysis of recovery after myelosuppression, JQ1- or placebo-treated WT mice received sublethal irradiation (5 Gy) and hematopoietic repopulation was monitored. For analysis of HSC proliferation, 1 mg BrdU was injected 24 h, 12 h, and 6 h before sacrifice.Results: As a first step, we analyzed the mRNA expression patterns of Brd2-4 in all major hematopoietic subpopulations, because BET-family members represent the main molecular targets of JQ1. Expression of Brd4 mRNA is most abundant in megakaryocytes (MK), followed by B cells, T cells and hematopoietic stem cells (HSC) and expression levels of Brd2+3 show similar distribution.JQ1-treatment decreases the numbers of pre-, immature and mature B cells while numbers of early pro-B cells remain constant (pre-B cells: 125 ± 7 vs. 52 ± 5 and immature B cells: 121 ± 9 vs. 26 ± 5 x104 cells/tibia; n=4; *p<0.05). Furthermore, it increases apoptosis in all T cell subsets, all together leading to reduced cellularity in thymus, BM and spleen. Intriguingly, JQ1 induces a 3-fold increase in the number of hematopoietic stem and progenitor cells (25 ± 2 vs. 70 ± 5 x103 LSK/femur; n=5; *p<0.05) and mobilizes HSC into the peripheral blood (4 ± 1 vs. 28 ± 2 x106 CFU/ml blood; n=5; *p<0.05). Consistently, extreme limiting dilution analysis (ELDA) showed a 3-fold increase in stem cell frequencies in the bone marrow upon JQ1-treatment (1/19480 vs. 1/6284; n=8-12; *p<0.05).We found that the elevated HSC numbers arise from increased cycling of LT-HSC, as these cells incorporate more BrdU upon JQ1-treatment (29 ± 2 vs. 41 ± 3 % BrdU+ LT-HSC in bone marrow; n=5; *p<0.05). Moreover and in contrast to placebo, JQ1-treated bone marrow cells preserve their colony-forming properties during several rounds of replating in vitro, indicating sustained stemness (18 ± 4 vs. 77 ± 10 CFU/1x104 bone marrow cells after third replating; n=3-8; *p<0.05). Due to increased numbers of HSC, JQ1-treated bone marrow engrafts better after competitive stem cell transplantation and repopulates the hematopoietic system significantly faster after sublethal myeloablation. Six weeks after irradiation, total leukocyte counts of JQ1-treated mice fully recover to levels before irradiation (94 ± 7 % recovery; n=5; *p<0.05), whereas recovery is slower in the control group (47 ± 5 % recovery; n=5; *p<0.05).Conclusions: JQ1 induces proliferation and mobilization of HSC, thereby increasing their number in bone marrow and peripheral blood. Therefore, BET-inhibition might benefit patients after myelosuppression or stem cell transplantation. DisclosuresLoges:BerGenBio ASA: Research Funding.

Setd2 Is Critical for Maintaining Hematopoietic Homeostasis

The histone H3 lysine 36 methyltransferase SETD2 is frequently mutated in various cancers, including leukemia. However, there has not been any functional model to show the contribution of SETD2 in hematopoiesis or the causal role of SETD2 mutation in tumorigenesis. In this study, using a conditional Setd2 knock-out mouse model, we show that Setd2 deficiency skews hematopoietic differentiation and reduces multipotent progenitors; although the number of phenotypic hematopoietic stem cells (HSCs) in Setd2 -deleted mice is unchanged, functional assays, including serial BM transplantation, reveal that the self-renewal and competitiveness of HSCs are impaired. Intriguingly, Setd2 -deleted HSCs, through a latency period, can acquire abilities to overcome the growth disadvantage and eventually develop into hematopoietic malignancy characteristic of myelodysplastic syndrome. Gene expression profile of Setd2 -deleted hematopoietic stem/progenitor cells partially resemblesthe Dnmt3a/Tet2 double knock-out, involving activation of the erythroid transcription factor Klf1 related pathway, which plays an important role in hematopoietic malignant transformation. Setd2 deficiency also induces DNA replication stress in HSCs, as reflected by activated E2F gene regulatory network and repressed ribonucleotide reductase subunit Rrm2b, which results in proliferation and cell cycle abnormalities and DNA instability of HSCs, allowing accumulation of secondary mutation(s) that synergistically contribute to tumorigenesis. Thus, our results demonstrate that Setd2 is required for HSC self-renewal, and provide evidence supporting the causal role of Setd2 deficiency in tumorigenesis. The underlying mechanism shall advance our understanding of epigenetic regulation of cancer and provide potential new therapeutic targets. DisclosuresNo relevant conflicts of interest to declare.

Prostaglandin E2 Protects HSC from DNA Damage Induced By Ionizing Radiation By Preventing Generation of Reactive Oxygen Species and Histone Acetylation

The hematopoietic system is highly radiosensitive; thus therapeutic and unwanted irradiation can produce significant/lethal acute and delayed bone marrow injury. In a murine hematopoietic-acute radiation syndrome (H-ARS) model, a single dose of 16,16 dimethyl Prostaglandin E2 (dmPGE2) given 15 min to 3 hrs before lethal (LD50/30-90/30) total body irradiation (TBI) results in 100% survival of mice at 30 days, with accelerated recovery of WBC, and hematopoietic stem and progenitor cell number and function.To explore mechanisms responsible for the radio-protective effect(s) of dmPGE2 we evaluated hematopoietic cells at early points post-TBI, focusing on oxidative pathways and epigenetic changes. Hematopoietic cells were isolated and evaluated at 1 hr and 1, 3 and 9 days post LD50 [853 cGy] TBI. Mitochondrial ROS and membrane potential were measured by flow cytometry in SLAM-SKL cells. Protein levels of gH2AX, LC3b, BNIP3, P16, P53, P62 acetylated H3K9 and H4K16 histones and SIRT1 were measured by flow cytometry. Acetylation of H3K9 and H4K16 at the KU70, Ku80, LC3b, Atg5, Atg7, Atg12, BNIP3, NF-kB and p53 promoters was evaluated by ChIP.Lethal radiation significantly increased HSC mitochondrial ROS and reduced membrane potential at 1,3 and 9 days post-TBI. Significant DNA damage was observed at 1 hour as measured by gH2AX. In contrast, mitochondrial ROS and DNA damage (gH2AX) were significantly lower in mice that received dmPGE2 before irradiation. In vehicle treated irradiated mice, we observed hyper-acetylated H3K9 and H4K16 at the Ku70/80 promoter, suggesting diminished capacity of Ku70/80 to repair DNA damage. In contrast, in mice treated with dmPGE2, H3K9 and H4K16 acetylation was significantly lower. Reduced mitochondrial membrane potential seen in vehicle treated mice was also prevented. The senescence markers SA-beta-gal and P16 in HSC were significantly reduced in PGE2 treated mice.The sirtuin family NAD-dependent deacetylase, SIRT1, has broad functions including gene silencing and DNA repair. In control irradiated mice SIRT1 levels dramatically decreased over 9 days post-irradiation, but remained significantly higher in dmPGE treated mice, which may account for the low acetylation levels of H3K9 and H4K16 in PGE treated mice. Consistent with elevated SIRT1 in PGE2 treated mice, we also observed global deacetylation of H3K9 and H4K16 in lineage negative and SLAM LSK cells at 1-3 days post-TBI. P53 is a key transcriptional regulator of genes involved in cell cycle, apoptosis and DNA repair and is acetylated after DNA damage. In control mice, irradiation resulted in robust acetylation of histone proteins at the p53 promoter, whereas dmPGE2 administration prevented acetylation of H4K16, but not H3K9. In addition, dmPGE2 blocked p53-mediated apoptosis via repression of p53 transcription.Since SIRT1 promotes autophagy, we evaluated autophagy related genes. CHIP analyses revealed low levels of acetylated-H3K9 at the LC3B, ATG7 and NF-KB promoters and deacetylated H4K16 at the LC3B, ATG7, BNIP3 and NF-kB promoters in PGE treated mice compared to vehicle controls. No effects on histone deacetylation of the Atg5 and Atg12 genes. BNIP3 is a mitophagy marker that when elevated downregulates mitochondrial mass and respiration. In irradiated control mice, BNIP3 levels were decreased at 1through 9 days, but remained elevated in mice treated with dmPGE2. The autophagy substrate P62 was significantly increased at 1 and 3 days post-irradiation in control mice, but was significantly lower in PGE2 treated mice. SIRT1 activation also leads to decreased binding of NFkB-p65 to DNA and attenuated oxidative stress through reduced transcription. CHIP data showed that TBI increased acetylation of H3K9 and H4K16 at the NF-kB p65 promoter in vehicle treated mice, whereas hyperacetylation of H3K9 and H4K16 and NF-kB transcription did not occur in mice treated with dmPGE2 before irradiation.These studies suggest that the radio protective effect of dmPGE2 and accelerated recovery of hematopoiesis following radiation exposure is mediated in part through reduction in oxidative stress and DNA damage induced in HSC and epigenetically, through prevention of histone acetylation of genes associated with DNA repair, autophagy and mitophagy, likely mediated through elevation of SIRT1. DisclosuresNo relevant conflicts of interest to declare.

Identification of T Cell Receptors Specific to Antigens Presented By HLA-B5401 on IPS Cell-Derived Hematopoietic Stem Cells in a Patient with Acquired Aplastic Anemia Carrying B5401-Lacking Leukocytes

[Background] Cytotoxic T lymphocytes (CTLs) specific to hematopoietic stem cells (HSCs) are thought to play an important role in the development of acquired aplastic anemia (AA). The presence of HLA class I allele-lacking leukocytes as a result of 6pLOH or allelic mutations in about 25% of AA patients strongly suggests that CTLs specific to antigens presented by the specific HLA alleles are involved in the destruction of normal HSCs as well as in the escape of HLA-lacking HSCs. Establishing CTLs from these patients by culturing the patients' CD8+ cells with targeted HSCs may be useful for identifying autoantigens of AA. However, it is generally impossible to obtain sufficient numbers of HSCs from the patients for functional assays and in vitro stimulation of patient's T cells with HSCs may induce clonal expansion of CTLs irrelevant to pathogenesis of AA. To solve these problems, we generated iPS cells (iPSCs) from an untreated AA patient who possessed HLA-B5401-lacking leukocytes for inducing HSCs and attempted to identify cDNAs of T-cell receptor (TCR) α and β chains of predominant T cells in the patient's bone marrow (BM) using a single T-cell analysis, which may specifically recognize autologous HSCs. [Methods] iPSCs were generated from the peripheral blood mononuclear cells of an AA patient (56-year-old man), whose moderate AA later responded to cyclosporine therapy, using Yamanaka's method. The patient's monocytes accounted for 20% wild-type, 20% 6pLOH(+) and 60% B5401-lacking cells at the time of sampling. CD34+ cells were induced from these iPSCs and injected into the BM of C57BL/6.Rag2 mice harboring NOD-Sirpa (BRGS). CD8+ T cells in the BM of the patient were subjected to a single cell sorting followed by cDNA amplification of TCRVβ chains. TCRα chains of five T cells that showed the five most recurrent TCRVβs were determined, and the TCRβ cDNAs were retrovirally transfected to Jurkat cells that lacked TCRβ chain and were transfected with IFN-γ cDNA. Specific responses of each TCR transfectant to iPSC-derived CD34+ cells were determined using ELISA for detecting IFN-γ. [Results] Five iPSC clones, including wild type (n=2), 6pLOH(+) (n=1), and B5401-lacking (B5401[-]) (n=2) clones, that had different mutations in B*54:01 were generated. Three-week culture of the iPSCs in the presence of various growth factors produced hematopoietic cells consisting of 50% CD34+ cells of each phenotype, which were able to reconstitute of BRGS mice with 3.5% to 8.9% human CD45+ cells in the peripheral blood at 9-12 weeks after injection. When the four TCR transfectants were tested for their response to wild-type and B5401-lacking CD34+ cells, all four transfectants secreted higher amounts of IFN-γ in response to wild-type cells than to B5401(-) CD34+ cells. [Conclusions] HSCs with original genotypes capable of reconstituting hematopoiesis in immunodeficient mice were successfully induced from iPSCs that had been generated from an AA patient's monocytes. The TCR transfectants showing a specific response to B5401(+) wild-type CD34+ cells will be useful for identifying the ligand peptides presented by B5401. DisclosuresNo relevant conflicts of interest to declare.

Bioactive SL-13R Peptide Expands Human Umbilical Cord Blood Hematopoietic Stem Cells and Maintains Long-Term Reconstitution Ability

Hematopoietic stem cell (HSC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSCs, the presence of an insufficient number of HSCs in these preparations limits their use, prompting need for ex vivo HSC amplification. To establish a more efficient method to expand UCB HSCs, we developed the SL-13R peptide and cultured UCB CD34+ cells with peptide in serum-free medium containing a cytokine cocktail. Following 9 days of culture with peptide, the number of total cells, CD34+CD38- cells and hematopoietic colonies significantly increased relative to control cells grown without peptide. Transplantation of these cells into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution ability. To understand how the peptide promotes HSC expansion, we identified PLEC and ERLIN2 proteins as peptide interactors and undertook loss-of-function analysis to determine whether either was required for peptide function in our culture system. PLEC knockdown UCB CD34+ cells cultured with peptide showed a decreased number of hematopoietic colonies relative to peptide-treated, non-knockdown controls. By contrast, ERLIN2 knockdown had little effect in the presence of peptide. This work suggests that PLEC functions in HSC expansion promoted by SL-13R. In summary, we have identified a novel peptide promoting expansion of UCB CD34+ cells with long-term reconstitution ability. Its use may facilitate clinical use of UCB HSCs. DisclosuresSugiyama:Science Lustre, Ltd: Equity Ownership.

Unique Roles for Grb2-Associated Binding (Gab) Adaptors in Hematopoietic Stem Cell Homeostasis and Repopulation

Homeostasis of hematopoietic stem cells (HSCs) is essential for the continuous replenishment of the hematopoietic system throughout the entire lifespan of an organism and is a tightly regulated process, controlled by intrinsic and extrinsic signals. Although a variety of molecules controlling HSC homeostasis are known, it remains unclear how signaling is regulated. The growth factor receptor-bound protein 2 (Grb2)-associated binding (Gab) proteins are a family of Pleckstrin homology domain containing adaptor proteins that dock with receptor tyrosine kinases (RTKs) and non-RTKs to control cell activation of ERK and PI3K pathways. The murine Gab family includes Gab1, Gab2, and Gab3. Gab1 and Gab2 are expressed throughout hematopoiesis including HSCs but Gab3 is limited mostly to macrophages, dendritic cells, and T-cells. Previously we reported that Gab2 is intrinsically required for hematopoietic cell responses to early-acting cytokines, with defective hematopoiesis in Gab2-/- mice (obtained from Toshio Hirano, Osaka, Japan; Nishida K et al, Blood, 99, 2002). Little is known about redundancy among Gabs in hematopoiesis and it is possible that roles for Gab1 and Gab3 have been obscured by the prominence of Gab2. Using Gab1flox/flox (Bard-Chapeau EA et al, Nat. Med. 11, 2005) and Gab3-/- mice (obtained from Pam Hankey-Giblin, Penn. State; Seiffert M et al, Mol. Cell. Biol. 23, 2003) we set out to make compound mutant mice. Gab3 knockout mice do not have reported hematopoietic phenotypes. First, we generated Gab2/3 double knockout mice. In the peripheral blood, we observed greater B-lymphocyte and erythroid deficiency compared to wild-type or Gab2-/- mice. In non-competitive bone marrow transplantation, Gab2/3-/- bone marrow was sufficient to reconstitute relatively normal myeloid hematology in irradiated recipients but resulted in significantly reduced donor derived B- and T-lymphocytes relative to wild type or Gab2-/- donor bone marrow cells, which were the same. In competitive bone marrow transplantation against Boy J (CD45.1) we observed sharp reductions in multilineage engraftment of Gab2/3-/- (13-15 fold) compared to Gab2-/- (4-6 fold) mice while Gab3-/- mice were normal. These data combined with the limited expression pattern for Gab3 suggest a novel critical extrinsic role in hematopoiesis. Despite severe multilineage engraftment defects, we surprisingly observed that Gab2/3-/- mice had significantly increased numbers of KLS cells (~2-fold) as well as LT-HSCs (KLSCD150+CD48-, ~3-fold) compared to the wild-type controls. Associated with the increased HSC numbers were cell cycle changes observed by staining with Pyronin Y/Hoechst 33342. During steady-state, Gab2/3-/- KLS cells were less quiescent with a lower percentage of cells in G0 (Gab2/3-/- vs WT: 40.5 ± 6.1 vs 57.4 ± 11.4, N=4, p=0.04) and more cells in G1 (Gab2/3-/- vs WT: 19.5 ± 0.7 vs 13.5 ± 3.5, N=4, p=0.01) phase of the cell cycle. Interestingly, KLS cells from Gab2/3-/- mice were less apoptotic than wild-type control (1.05 ± 0.49% in Gab2/3-/- (N=7) vs 5.97 ± 1.40% in WT KLS (N=10), p<0.001). Together these indicate that the HSCs were expanded but functionally defective. To further explore the mechanisms for Gab2/3-/- defects, we found that Gab1 expression levels were increased 2-fold. Since germline Gab1 deficiency results in embryonic lethality, Gab1 floxed alleles were deleted conditionally using Vav1-Cre to generate viable adult Gab1/2/3-/- mice. Real-time PCR confirmed the average of 94% Gab1 deletion efficiency (N=10) and Western analysis showed no detectable level of Gab1 in splenocytes. Interestingly, Gab1 deletion reversed the KLS expansion observed in the Gab2/3-/- mice indicating that Gab1 has effects that oppose Gab2/3 at the KLS level. However, Gab1/2/3-/- mice had severe defects in multilineage engraftment in competitive repopulation (39-52 fold). Altogether, our data indicate that Gab2/3 play positive roles while Gab1 plays a negative role in HSC homeostasis but all three Gabs are essential for multilineage repopulation. Further studies of Gab1 and Gab3 function in intrinsic and extrinsic regulation of HSC biology and repopulation are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Oral Squamous Cell Carcinomas in Patient with Graft Versus-Host Disease Following Allogenic Bone Marrow Transplantation: A Case Report during Mutant COVID-19 Pandemic

Aplastic anaemia is a severe haematological disorder characterized by an inadequate number of hematopoietic stem cells, resulting in pancytopenia, formed by a hypocellular bone marrow. Disorders of this nature are widely treated with haematopoietic stem cell transplantation (HSCT). A potential chronic complication following (HSCT) is the growth of secondary malignancies. Notably, patients suffering from chronic graft versus host disease (cGvHD) secondary to HSCT have been shown to be more susceptible to oral squamous cell carcinoma (OSCC). Here, we present a rare case of a 30-year-old Libyan woman treated with HSCT for aplastic anaemia, with subsequent complications of cGvHD and OSCC after few months of HSCT. These carcinomatous lesions were detected in the buccal gingiva and retromolar pad area at the age of 31. The present case report emphasizes the connection between oral cGvHD and OSCC, and the potential appearance of OSCC after HSCT at any time of patient life. Thus, closer follow-up is mandatory for all patients treated with HSCT who developed cGvHD, and efficient cGvHD prevention and therapeutic approaches are needed.

C/EBP homologous protein deficiency enhances hematopoietic stem cell function via reducing ATF3/ROS-induced cell apoptosis.

Hematopoietic stem cells (HSCs) reside in a quiescent niche to reserve their capacity of self‐renewal. Upon hematopoietic injuries, HSCs enter the cell cycle and encounter protein homeostasis problems caused by accumulation of misfolded proteins. However, the mechanism by which protein homeostasis influences HSC function and maintenance remains poorly understood. Here, we show that C/EBP homologous protein (CHOP), demonstrated previously to induces cell death upon unfolded protein response (UPR), plays an important role in HSCs regeneration. CHOP−/− mice showed normal hematopoietic stem and progenitor cell frequencies in steady state. However, when treated with 5‐FU, CHOP deficiency resulted in higher survival rates, associated with an increased number of HSCs and reduced level of apoptosis. In serial competitive transplantation experiments, CHOP−/− HSCs showed a dramatic enhancement of repopulation ability and a reduction of protein aggresomes. Mechanistically, CHOP deletion causes reduced ATF3 expression and further leads to decreased protein aggregation and ROS. In addition, CHOP−/− HSCs exhibited an increased resistance to IR‐induced DNA damage and improved HSCs homeostasis and function in telomere dysfunctional (G3Terc −/−) mice. In summary, these findings disclose a new role of CHOP in the regulation of the HSCs function and homeostasis through reducing ATF3 and ROS signaling.

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.

Evaluation of a clinical-grade, cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34+ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP). MethodscGMP VPA-mediated expansion was initiated with CD34+ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts. ResultsThe authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products. ConclusionsThe authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34+ cells for clinical utilization.

Usefulness of hematopoietic progenitor cell monitoring to predict autologous peripheral blood stem cell harvest timing: A single-center retrospective study

IntroductionIn autologous peripheral blood stem cell harvest (APBSCH), CD34-positive cells have been measured to assess the numbers of hematopoietic stem cells, but measurement requires specialized equipment. Recently, there was a report that peripheral blood hematopoietic progenitor cells (HPCs) are useful indicators of the presence of hematopoietic stem cells. We examined the usefulness of HPC monitoring to predict APBSCH timing. MethodsWe retrospectively analyzed the relationship between HPC and collected CD34-positive cells in 84 consecutive patients who underwent APBSCH. ResultsAccording to the receiver operating characteristics curve for the collection of ≥2 × 106 CD34-positive cells/kg, the HPC cut-off value on the day before collection was 21/μL, while that on the day of collection was 41/μL. No significant factors were found in the univariate analysis except for the HPC count on the day before collection (p < 0.001) and the day of collection (p < 0.001). According to the multivariate analysis, the HPC count on the day before collection (p < 0.001) and the day of collection (p < 0.001) were also factors that strongly influenced the quantity of CD34-positive cells collected. ConclusionOur results suggest that the HPC count on not only the day of collection but also the day before collection is a good indicator for appropriate APBSCH timing.