Hematopoietic Progenitor Cell Function Research Articles

Age and Sex Divergence in Hematopoietic Radiosensitivity in Aged Mouse Models of the Hematopoietic Acute Radiation Syndrome.

The hematopoietic system is highly sensitive to stress from both aging and radiation exposure, and the hematopoietic acute radiation syndrome (H-ARS) should be modeled in the geriatric context separately from young for development of age-appropriate medical countermeasures (MCMs). Here we developed aging murine H-ARS models, defining radiation dose response relationships (DRRs) in 12-month-old middle-aged and 24-month-old geriatric male and female C57BL/6J mice, and characterized diverse factors affecting geriatric MCM testing. Groups of approximately 20 mice were exposed to ∼10 different doses of radiation to establish radiation DRRs for estimation of the LD50/30. Radioresistance increased with age and diverged dramatically between sexes. The LD50/30 in young adult mice averaged 853 cGy and was similar between sexes, but increased in middle age to 1,005 cGy in males and 920 cGy in females, with further sex divergence in geriatric mice to 1,008 cGy in males but 842 cGy in females. Correspondingly, neutrophils, platelets, and functional hematopoietic progenitor cells were all increased with age and rebounded faster after irradiation. These effects were higher in aged males, and neutrophil dysfunction was observed in aged females. Upstream of blood production, hematopoietic stem cell (HSC) markers associated with age and myeloid bias (CD61 and CD150) were higher in geriatric males vs. females, and sex-divergent gene signatures were found in HSCs relating to cholesterol metabolism, interferon signaling, and GIMAP family members. Fluid intake per gram body weight decreased with age in males, and decreased after irradiation in all mice. Geriatric mice of substrain C57BL/6JN sourced from the National Institute on Aging were significantly more radiosensitive than C57BL/6J mice from Jackson Labs aged at our institution, indicating mouse source and substrain should be considered in geriatric radiation studies. This work highlights the importance of sex, vendor, and other considerations in studies relating to hematopoiesis and aging, identifies novel sex-specific functional and molecular changes in aging hematopoietic cells at steady state and after irradiation, and presents well-characterized aging mouse models poised for MCM efficacy testing for treatment of acute radiation effects in the elderly.

P16INK4A-dependent senescence in the bone marrow niche drives age-related metabolic changes of hematopoietic progenitors

AbstractRapid and effective leukocyte response to infection is a fundamental function of the bone marrow (BM). However, with increasing age, this response becomes impaired, resulting in an increased burden of infectious diseases. Here, we investigate how aging changes the metabolism and function of hematopoietic progenitor cells (HPCs) and the impact of the BM niche on this phenotype. We found that, in response to lipopolysaccharide-induced stress, HPC mitochondrial function is impaired, and there is a failure to upregulate the TCA cycle in progenitor populations in aged animals compared with young animals. Furthermore, aged mesenchymal stromal cells (MSCs) of the BM niche, but not HPCs, exhibit a senescent phenotype, and selective depletion of senescent cells from the BM niche, as well as treatment with the senolytic drug ABT-263, improves mitochondrial function of HPCs when stressed with lipopolysaccharide. In summary, age-related HPC metabolic dysfunction occurs indirectly as a “bystander phenomenon” in the aging BM niche and can be restored by targeting senescent MSCs.

Ultrasound-assisted continuous-flow synthesis of PEGylated MIL-101(Cr) nanoparticles for hematopoietic radioprotection

Metal-organic frameworks (MOFs) are useful as drug delivery carriers with high loading capacity and excellent biocompatibility. We fabricated a new drug carrier based on MIL-101(Cr) environmentally and loaded it with 47.2 wt% WR-1065 (active metabolite of amifostine). Moreover, the permeability and stability of these nanoparticles increased after PEGylation by the N-hydroxysuccinimide active ester protocol. Then, a “green” continuous-flow system equipped with an ultrasound applicator was newly designed to prepare the nanoparticles under the effect of acoustic cavitation. Response surface methodology (RSM) was used to optimize the large-scale process conditions with Box-Behnken design to obtain high space-time yield (5785 kg m−3 day−1). These less toxic MOFs nanoparticles increased cell viability by scavenging the accumulated reactive oxygen species and resisting DNA damage after irradiation. They are capable of mitigating radiation injury, achieving a 30-d survival rate of 90% in mice after lethal total body irradiation (8.0 Gy). This countermeasure significantly improved the peripheral blood cell count, hematopoietic stem and progenitor cells frequency, and clonogenic function of hematopoietic progenitor cells. It probably prevents irradiation-induced hematopoietic damage through the p53-dependent apoptotic pathway. Therefore, ultrasound-assisted continuous-flow synthesis is a sustainable method to produce MOFs on a large scale for radioprotection.

Leukemic Stress Targets the mTOR Pathway to Suppress Residual HSC in the BM Microenvironment

The remodeling of the bone marrow (BM) microenvironment that occurs along with the progressive spread of acute myeloid leukemia (AML) cells can be considered a constitutive aspect of leukemogenesis. To date most studies have focused on the functional and in part inflammatory adaptation of stroma, and its potential role in extrinsic chemotherapy resistance. Much less is known about the impact of leukemic stress on residual hematopoietic cells. We previously identified the trafficking of select microRNAs (miRs-) in extracellular vesicles (EVs) between AML cells and hematopoietic progenitor cells (HPC). These studies revealed the mechanism underlying the suppression of HPC function in the AML niche (Hornick, Doron et. al., Science Signaling 2016). Several groups, including ours also noted the relative resistance of residual hematopoietic stem cells (HSC) to elimination from the BM of xenografted animals. In the current study we set out to understand how leukemic stress in the AML xenograft niche shapes HSC fate and function. Using AML cell lines (Molm14, U937, HL60) we established NSG xenografts, systematically tracking peripheral blood AML chimerism to recover murine hematopoietic cells at low or negative tumor burden, and replicating key assays using purified EV for intrafemoral injections. In immunofluorescent studies we initially confirmed the uptake of GFP labeled xenograft-derived EVs across the spectrum of HPC and HSC (KSL/CD150+/CD48-), as well as the successive loss and peripheral displacement of HPCs, and gains in HSC frequency in the leukemic niche. These HSC were found to be enriched for G0 cell cycle status with an increase in phospho- p53, but showed no evidence of apoptosis or senescence. To understand the mechanism underlying their apparent quiescence, we performed in vitro proteomics studies of AML EV exposed HPSC identified downregulation of ribosomal biogenesis pathways. We then confirmed in vivo that residual HSC from AML xenografts experienced a loss of protein synthesis (OPP assay). We next reasoned that deficits in ribosome dysfunction and protein synthesis may reflect deregulation by specific miRNAs highly abundant in AML EV. Here, we had an opportunity to profile EV miRNA from the plasma of 12 unselected AML patients at diagnosis versus 12 control samples, and we confirmed a significant enrichment for specific miRNAs, including miR-1246. Raptor is a component of the mTOR pathway and an annotated target of miR-1246. We demonstrated in a series of experiments that miR-1246 translationally suppresses Raptor and downregulates protein synthesis in residual HSC from AML xenografts. The transfection of synthetic anti-miR1246 sequences on the other hand reversed the effects of AML EV in murine HSC. In aggregate we show that direct crosstalk between AML and hematopoietic cells adds to the adaptive changes that occur in the AML niche. Our experiments suggest a functional significance for EV miRNA that can be detected in AML patient plasma in the regulation of residual BM HSC. More broadly, the mechanisms by which leukemic stress alters hematopoietic function remain underexplored, but our observations suggest that leukemia derived EV contribute to changes in competitive fitness of residual HSC. Disclosures No relevant conflicts of interest to declare.

Toll-like receptor signaling in hematopoietic stem and progenitor cells.

The innate immune system is essential in the protection against microbial infection and facilitating tissue repair mechanisms. During these stresses, the maintenance of innate immune cell numbers through stress-induced or emergency hematopoiesis is key for our survival. One major mechanism to recognize danger signals is through the activation of Toll-like receptors (TLRs) on the surface of hematopoietic cells, including hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC), and nonhematopoietic cells, which recognize pathogen-derived or damaged-induced compounds and can influence the emergency hematopoietic response. This review explores how direct pathogen-sensing by HSC/HPC regulates hematopoiesis, and the positive and negative consequences of these signals. Recent studies have highlighted new roles for TLRs in regulating HSC and HPC differentiation to innate immune cells of both myeloid and lymphoid origin and augmenting HSC and HPC migration capabilities. Most interestingly, new insights as to how acute versus chronic stimulation of TLR signaling regulates HSC and HPC function has been explored. Recent evidence suggests that TLRs may play an important role in many inflammation-associated diseases. This suggests a possible use for TLR agonists or antagonists as potential therapeutics. Understanding the direct effects of TLR signaling by HSC and HPC may help regulate inflammatory/danger signal-driven emergency hematopoiesis.

The role of PDK1 in the transition of endothelial to hematopoietic cells

目的研究磷酸肌醇依赖性激酶1（PDK1）在内皮细胞向造血细胞转化阶段对造血干细胞（HSC）发生的影响。方法应用Vec-Cre在内皮细胞中特异性敲除PDK1基因，取对照组PDK1fl/fl、PDK1fl/+小鼠及敲除组Vec-Cre；PDK1fl/fl小鼠胚胎的主动脉-性腺-中肾区（AGM区）细胞进行集落形成实验，检测PDK1基因对造血祖细胞功能的影响；取对照组和敲除组AGM区细胞行移植实验，检测PDK1对HSC功能的影响；取对照组和敲除组AGM区细胞，通过流式细胞术检测PDK1对能够向造血转化的CD31+c-Kithigh细胞群比例、细胞周期及细胞凋亡的影响；分选对照组和敲除组AGM区CD31+c-Kithigh细胞群，通过Real-time PCR检测PDK1对内皮向造血转换相关的转录因子（RUNX1、P2-RUNX1、GATA2）的影响。结果PDK1敲除后，造血祖细胞形成的克隆形态变小，数目减少[敲除组CFU-GM为（24±5）个/ee，对照组为（62±1）个/ee，P=0.001]；破坏了造血干细胞重建造血及多向分化的能力（敲除组移植5只，0只重建，对照组移植7只，5只重建，P=0.001）；AGM区CD31+c-Kithigh比例降低[敲除组CD31+c-Kithigh比例为（0.145±0.017）%，对照组比例为（0.385±0.04）%，P=0.001]；并且AGM区由内皮细胞向造血细胞转换的关键转录因子表达下降，但对CD31+c-Kithigh细胞的增殖和凋亡无明显影响。结论在内皮细胞中特异敲除PDK1基因，导致具有向造血转化的内皮细胞群比例降低，影响了HSC的发生，破坏了HSC重建造血的能力。

NOS2 deficiency has no influence on the radiosensitivity of the hematopoietic system

ObjectivePrevious studies have shown that inhibition of inducible NO synthase (NOS2 or iNOS) with an inhibitor can selectively protect several normal tissues against radiation during radiotherapy. However, the role of NOS2 in ionizing radiation (IR)-induced bone marrow (BM) suppression is unknown and thus was investigated in the present study using NOS2−/− and wild-type mice 14 days after they were exposed to a sublethal dose of total body irradiation (TBI).MethodsThe effects of different doses of IR (1, 2 and 4 Gy) on the apoptosis and colony-forming ability of bone marrow cells from wild-type (WT) and NOS2−/− mice were investigated in vitro. In addition, we exposed NOS2−/− mice and WT mice to 6-Gy TBI or sham irradiation. They were euthanized 14 days after TBI for analysis of peripheral blood cell counts and bone marrow cellularity. Colony-forming unit-granulocyte and macrophage, burst-forming unit-erythroid and CFU-granulocyte, erythroid, macrophage in bone marrow cells from the mice were determined to evaluate the function of hematopoietic progenitor cells (HPCs), and the ability of hematopoietic stem cells (HSCs) to self-renew was analysed by the cobblestone area forming cell assay. The cell cycling of HPCs and HSCs were measured by flow cytometry.ResultsExposure to 2 and 4 Gy IR induced bone marrow cell apoptosis and inhibited the proliferation of HPCs in vitro. However, there was no difference between the cells from WT mice and NOS2−/− mice in response to IR exposure in vitro. Exposure of WT mice and NOS2−/− mice to 6 Gy TBI decreased the white blood cell, red blood cell, and platelet counts in the peripheral blood and bone marrow mononuclear cells, and reduced the colony-forming ability of HPCs (P < 0.05), damaged the clonogenic function of HSCs. However, these changes were not significantly different in WT and NOS2−/− mice.ConclusionThese data suggest that IR induces BM suppression in a NOS2-independent manner.

Low doses of oxygen ion irradiation cause long-term damage to bone marrow hematopoietic progenitor and stem cells in mice.

During deep space missions, astronauts will be exposed to low doses of charged particle irradiation. The long-term health effects of these exposures are largely unknown. We previously showed that low doses of oxygen ion (16O) irradiation induced acute damage to the hematopoietic system, including hematopoietic progenitor and stem cells in a mouse model. However, the chronic effects of low dose 16O irradiation remain undefined. In the current study, we investigated the long-term effects of low dose 16O irradiation on the mouse hematopoietic system. Male C57BL/6J mice were exposed to 0.05 Gy, 0.1 Gy, 0.25 Gy and 1.0 Gy whole body 16O (600 MeV/n) irradiation. The effects of 16O irradiation on bone marrow (BM) hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) were examined three months after the exposure. The results showed that the frequencies and numbers of BM HPCs and HSCs were significantly reduced in 0.1 Gy, 0.25 Gy and 1.0 Gy irradiated mice compared to 0.05 Gy irradiated and non-irradiated mice. Exposure of mice to low dose 16O irradiation also significantly reduced the clongenic function of BM HPCs determined by the colony-forming unit assay. The functional defect of irradiated HSCs was detected by cobblestone area-forming cell assay after exposure of mice to 0.1 Gy, 0.25 Gy and 1.0 Gy of 16O irradiation, while it was not seen at three months after 0.5 Gy and 1.0 Gy of γ-ray irradiation. These adverse effects of 16O irradiation on HSCs coincided with an increased intracellular production of reactive oxygen species (ROS). However, there were comparable levels of cellular apoptosis and DNA damage between irradiated and non-irradiated HPCs and HSCs. These data suggest that exposure to low doses of 16O irradiation induces long-term hematopoietic injury, primarily via increased ROS production in HSCs.

Declined presentation the novel C/EBPalpha target gene EVI2B regulates myeloid differentiation and hematopoietic progenitor cell function

Declined presentation the novel C/EBPalpha target gene EVI2B regulates myeloid differentiation and hematopoietic progenitor cell function

Cytomegalovirus (CMV) Viral Load in Cord Blood (CB) Donors and Impact of Congenital Viremia on Markers of Hematopoietic Progenitor Cell (HPC) Potency

CMV infection is an established risk factor in patients undergoing hematopoietic stem cell transplantation (HSCT). Hence, the status of the donor is carefully considered, especially for high risk groups such as CMV seronegative recipients.The incidence of CMV neonatal infection is low thus, CB is an advantageous graft alternative to other HSC sources. Screening of CB donors is performed by testing the maternal sample for total Antibodies to CMV (CMV-Ab), while some CB banks, in addition, screen CB for CMV-DNA. Since CB units that have CMV viremia are not recommended for use in HSCT, their characteristics have not been fully evaluated. The aim of this study was to assess the impact of congenital CMV infection on CB cells, specifically on HPC concentration and their CFU functionality, in relation with the viral load in the CB and the donor's mother.Methods:To quantify CMV-DNA in CB a multiplex Real Time (RT) PCR assay was developed and validated using the Step One PlusTM RT PCR system (Applied Biosystems). The amplification target was located within the Glycoprotein B conserved sequence and a CMV standard control (SRM2366; National Institute of Standards and Technology) was used for validation experiments. To confirm the correct detection of different CMV genotypes and rare mutants, lyophilized synthetic oligonucleotides (SIGMA-ALDRICH) were used. Saliva culture and a validated qualitative PCR (PCR) were performed as described previously (Albano et al, Blood 2006;108). CB counts were measured in a Sysmex XE2100 analyzer, CD34+/CD45+ cells were evaluated using single platform, 3-color flow cytometry with 7-AAD, with ISHAGE gating strategy. The Colony Forming Unit (CFU) assay was performed using the National Cord Blood Program (NCBP) CFU strategy with High Resolution Digital Imaging (Albano et al, ASH 2008). CB was collected and all samples tested following NCBP approved procedures and donor consent.Results:We evaluated the incidence of CMV viremia in two groups of CB units donated to the NCBP. Initially, we screened prospectively 30,308 neonates for CMV infection by saliva culture (period 1/1995 - 3/2007, Group 1) and found 41 positive cases (0.14% incidence in CB donors), all from mothers with positive serum CMV antibodies at delivery.Based on this observation, subsequently, CMV-DNA was screened by PCR retrospectively in 4,712 newborns from CMV-Ab positive mothers (period 3/2007- 3/2016, Group 2). Eighteen cases had CMV-DNA detected (0.38%). Thus, a total of 59 newborns had CMV infection. Viremia was detected by RT-PCR in all of them (average viral load: 20.6 x104 vc/mL; range 110-2.3 x10^6 vc/mL). None of the neonates had clinical symptoms at birth. Screening by the Obstetric Hospital independently diagnosed CMV congenital infection in 2 of the infants.Only 7 of the 59 neonates had mothers who also carried CMV-DNA in blood at the time of delivery (range 300-5740 vc/mL) however, with no association to newborns' viral load.Potency evaluation, including HPC function (CFU), CD34+/CD45+ cells, and Total Nucleated Cells (TNC) was performed prospectively in a cohort of CMV positive (N=19) and control (N=76) CB samples collected and tested during the period 2006-2012. Control cases which were matched for birth date and hospital collection site; race, gender and delivery route were comparable to the CMV positive group. Birth weight and platelet counts were slightly lower in the group with CMV viremia, without reaching statistical significance. Interestingly, TNC and CD34+ cells were significantly higher in the CMV positive group, as well as HPC function (measured by Total CFU; Table). Across cellular progenitor lineages, CFUs showed a trend for high CFU-GM and CFU-GEMM with significantly elevated CFU-E (2843 +/-1949 x 10^3 vs 1389 +/- 1316 x 10^3; P= 0.029). The strong association seen between CD34+ and CFU content (R2=0.79; P<0.001) was consistent with all NCBP data. Notably, there was no association between these two parameters and CMV viral load at birth.Conclusions:The test developed, RT-PCR, is valid to detect and quantify CMV-DNA in CB.Maternal CMV-Ab status or maternal viremia at birth are poor predictors of CB CMV infection. The observations of this study suggestthatcongenital transmission of CMV as shown by CMV viremia at birth, impacts HPC, CD34+/CD45+ cells and erythrocytic precursors towards higher proliferation. Factors responsible for this effect need to be evaluated in future studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Re-Evaluation of Age-Related Dysregulation of Hematopoiesis Based on Oxygen Induced Changes Associated with Cell Collection/Processing in Ambient Air

Hematopoietic stem cell (HSC) numbers and function are reported to be dysregulated/compromised in old mice (e.g. ≥20 months of age: Cho, et. al., Blood 2008; Dykstra, et. al., J. Exp. Med. 2011; Sun, et. al., Cell Stem Cell 2014, amongst other papers). HSC numbers apparently increase with age, with decreased lymphoid production and compromised engrafting capability. However, what we know about aged HSCs is based on evaluation of these cells after they have first been collected and processed in ambient air. This is an hyperoxic environment, much increased in oxygen compared to the in vivo bone marrow (BM) hypoxic (~ 1-5 %) environment that the HSCs reside in. Moreover, there is little information on hematopoietic progenitor cells (HPCs) in terms of numbers and functional capacity in aged animals. Because of the need to better understand HSCs and HPCs in aging, where hematopoietic cell function appears to be compromised, and in context of blood disorders associated with aging, it is important to assess HSCs and HPCs removed from the body for analysis in situations as close as possible to that in which these cells find themselves in the body. It has recently become apparent that collection of BM cells from relatively young mice (e.g. 6-12 weeks of age), even for short periods (≤20 minutes) in ambient air, exposes these cells to the phenomenon of extra physiologic oxygen shock/stress (EPHOSS). EPHOSS triggers the opening of the mitochondrial permeability transition pore, resulting in enhanced production of mitochondrial reactive oxygen species that results in rapid cell differentiation, with decreased numbers of phenotypically- and functionally-defined long term (LT) repopulating, self-renewing HSCs and concomitant increases in rapidly cycling HPCs (Mantel, et. al. Cell 161:1553, 2015). By rigorous attention to detail in collection and processing mouse BM cells from young mice under constant hypoxic conditions of 3 % oxygen, it became clear that, in fact, there were on average 3-5 fold greater numbers of phenotyped- and functional LT HSCs. But, there were many fewer HPCs, and these HPCs were in a slow or non-cycling state when compared to cells collected and processed in ambient air, or when collected in 3% oxygen and then exposed to ambient air. We thus hypothesized that BM HSCs and HPCs from aged mice collected/processed in ambient air may not reflect their true numbers and functional characteristics in vivo. This led us to re-evalaute hematopoiesis in aged mice, compared to that in young mice, but in which BM cells were collected and processed for numbers and functions of HSCs and HPCs in a more physiological oxygen tension of 3 %, as reported (Mantel, et. al. Cell, 2015). We evaluated BM from CB6, Balb/c, and C57Bl/6 mice at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or 3% oxygen. Collection in air demonstrated that older mice had 2.6-2.8 and 1.5-1.9 fold more LT- and short term (ST)-HSCs, with 1.8-2.2 fold fewer phenotypically-defined common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMPs) and common lymphoid progenitors (CLPs), and 2-3 fold fewer functional myeloid HPCs (CFU-GM, BFU-E, CFU-GEMM) than younger mice. Moreover, HPCs of older mice, as assessed by colony assay, were in a slow cycling state. In contrast, BM of young and old mice collected/processed in hypoxia, demonstrated similar numbers of LT-HSC, ST-HSCs, CMPs, GMPs, and CLPs. Moreover, while CFU-GM, BFU-E, and CFU-GEMM in young mice were decreased after hypoxic collection/processing and were in slow cycle, those of older mice were greatly increased in numbers, and were in rapid cycle. Engrafting and other cell and intracellular studies are ongoing, but it is clear that hematopoietic cell studies previously reported in aged mice will have to be re-evaluated for better mechanistic understanding of their actual numbers and cell and intracellular characteristics as reflected in an in vivo hypoxic environment. DisclosuresBroxmeyer:CordUse: Other: SAB Member .

Chronic Trichuris muris infection alters hematopoiesis and causes IFN-γ-expressing T-cell accumulation in the mouse bone marrow.

Proinflammatory cytokines produced during immune responses to infectious stimuli are well-characterized to have secondary effects on the function of hematopoietic progenitor cells in the BM. However, these effects on the BM are poorly characterized during chronic infection with intestinal helminth parasites. In this study, we use the Trichuris muris model of infection and show that Th1 cell-associated, but not acute Th2 cell-associated, responses to chronic T. muris infection cause a major, transient expansion of CD48- CD150- multipotent progenitor cells in the BM that is dependent on the presence of adaptive immune cells and IFN-γ signaling. Chronic T. muris infection also broadly stimulated proliferation of BM progenitor cells including CD48- CD150+ hematopoietic stem cells. This shift in progenitor activity during chronic T. muris infection correlated with a functional increase in myeloid colony formation in vitro as well as neutrophilia in the BM and peripheral blood. In parallel, we observed an accumulation of CD4+ , CD8+ , and CD4- CD8- (double negative) T cells that expressed IFN-γ, displaying activated and central memory-type phenotypes in the bone marrow during chronic infection. Thus, these results demonstrate that Th1 cell-driven responses in the intestine during chronic helminth infection potently influence upstream hematopoietic processes in the BM via IFN-γ.

Ectopic expression of Hoxb4a in hemangioblasts promotes hematopoietic development in early embryogenesis of zebrafish.

Hemangioblast, including primitive hematopoietic progenitor cells, play an important role in hematopoietic development, however, the underlying mechanism for the propagation of hematopoietic progenitor cells remains elusive. A variety of regulatory molecules activated in early embryonic development play a critical role in the maintenance of function of hematopoietic progenitor cells. Homeobox transcription factors are an important class of early embryonic developmental regulators determining hematopoietic development. However, the effect of homeobox protein Hox-B4 (HOXB4) ectopic expression on the development of hemangioblasts has not been fully addressed. This study aimed to investigate the role of Hoxb4a, an ortholog gene of HOXB4 in zebrafish, in the hematopoietic development in zebrafish. A transgenic zebrafish line was established with Cre-loxP system that stably overexpressed enhanced green fluorescent protein (EGFP)-tagged Hoxb4a protein under the control of hemangioblast-specific lmo2 promoter. Overexpression of Hoxb4a in the development of hemangioblasts resulted in a considerable increase in the number of stem cell leukemia (scl) and lmo2-positive primitive hematopoietic progenitor cells occurring in the posterior intermediate cell mass (ICM). Interestingly, Hoxb4a overexpression also disrupted the development of myelomonocytes in the anterior yolk sac and the posterior ICM, without affecting erythropoiesis in the posterior ICM. Taken together, these results indicate that Hoxb4a favours the development of hematopoietic progenitor cells originated from hemangioblasts in vivo.

Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition

Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD), a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM) suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs) function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794) depletes mouse BM Lin−Sca-1+c-Kit+ cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU) assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs) in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs) but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs), respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.

Ultra-endurance exercise induces stress and inflammation and affects circulating hematopoietic progenitor cell function.

Although amateur sports have become increasingly competitive within recent decades, there are as yet few studies on the possible health risks for athletes. This study aims to determine the impact of ultra-endurance exercise-induced stress on the number and function of circulating hematopoietic progenitor cells (CPCs) and hematological, inflammatory, clinical, metabolic, and stress parameters in moderately trained amateur athletes. Following ultra-endurance exercise, there were significant increases in leukocytes, platelets, interleukin-6, fibrinogen, tissue enzymes, blood lactate, serum cortisol, and matrix metalloproteinase-9. Ultra-endurance exercise did not influence the number of CPCs but resulted in a highly significant decline of CPC functionality after the competition. Furthermore, Epstein-Barr virus was seen to be reactivated in one of seven athletes. The link between exercise-induced stress and decline of CPC functionality is supported by a negative correlation between cortisol and CPC function. We conclude that ultra-endurance exercise induces metabolic stress and an inflammatory response that affects not only mature hematopoietic cells but also the function of the immature hematopoietic stem and progenitor cell fraction, which make up the immune system and provide for regeneration.

Gfi-1 regulates the erythroid transcription factor network through Id2 repression in murine hematopoietic progenitor cells

AbstractGrowth factor independence 1 (Gfi-1) is a part of the transcriptional network that regulates the development of adult hematopoietic stem and progenitor cells. Gfi-1-null (Gfi-1−/−) mice have reduced numbers of hematopoietic stem cells (HSCs), impaired radioprotective function of hematopoietic progenitor cells (HPCs), and myeloid and erythroid hyperplasia. We found that the development of HPCs and erythropoiesis, but not HSC function, was rescued by reducing the expression of inhibitor of DNA-binding protein 2 (Id2) in Gfi-1−/− mice. Analysis of Gfi-1−/−;Id2+/− mice revealed that short-term HSCs, common myeloid progenitors (CMPs), erythroid burst-forming units, colony-forming units in spleen, and more differentiated red cells were partially restored by reducing Id2 levels in Gfi-1−/− mice. Moreover, short-term reconstituting cells, and, to a greater extent, CMP and megakaryocyte-erythroid progenitor development, and red blood cell production (anemia) were rescued in mice transplanted with Gfi-1−/−;Id2+/− bone marrow cells (BMCs) in comparison with Gfi-1−/− BMCs. Reduction of Id2 expression in Gfi-1−/− mice increased the expression of Gata1, Eklf, and EpoR, which are required for proper erythropoiesis. Reducing the levels of other Id family members (Id1 and Id3) in Gfi-1−/− mice did not rescue impaired HPC function or erythropoiesis. These data provide new evidence that Gfi-1 is linked to the erythroid gene regulatory network by repressing Id2 expression.

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

Resveratrol Rescues SIRT1-Dependent Adult Stem Cell Decline and Alleviates Progeroid Features in Laminopathy-Based Progeria

Abnormal splicing of LMNA gene or aberrant processing of prelamin A results in progeroid syndrome. Here we show that lamin A interacts with and activates SIRT1. SIRT1 exhibits reduced association with nuclear matrix (NM) and decreased deacetylase activity in the presence of progerin or prelamin A,leading to rapid depletion of adult stem cells (ASCs) in Zmpste24(-/-) mice. Resveratrol enhances the binding between SIRT1 and A-type lamins to increases its deacetylase activity. Resveratrol treatment rescues ASC decline, slows down body weight loss, improves trabecular bone structure and mineral density, and significantly extends the life span in Zmpste24(-/-) mice. Our data demonstrate lamin A as an activator of SIRT1 and provide a mechanistic explanation for the activation of SIRT1 by resveratrol. The link between conserved SIRT1 longevity pathway and progeria suggests a stem cell-based and SIRT1 pathway-dependent therapeutic strategy for progeria.

Abstract 18970: Angiotensin-(1-7) Enhances the Regenerative Capacity of Cardiac Progenitor Cells (CPCs, Islet-1 + ) in Ischemia-induced Cardiac Injury Model

Stem cell therapy has achieved limited success to provide sufficient exogenous reparative support in ischemic heart diseases (IHD). We evaluated the hypothesis that transduction of CPCs with Ang-(1-7) would improve their regenerative capacity, as this peptide has been shown to improve hematopoietic progenitor cell functions. First, we determined if Ang-(1-7) improves CPC functions in vitro . Angiogenesis and migratory potential of CPCs, lenti-Ang-(1-7) transduced CPCs and CPCs treated with 1µM Ang-(1-7) were evaluated. Ang-(1-7) enhanced the CPC capability to form tubes; protected CPCs from hypoxia (H) induced inhibition of migration (CPC 688.7 ± 52.8, H treated CPC 124.5 ± 187.7, H+lenti-Ang-(1-7) 617.2 ± 45.4, H+1µM Ang-(1-7) 878.8 ± 303.9; RFU); reduced ACE mRNA level in the CPCs and shifted the balance to the vasoprotective axis of the RAS. Next, in vivo studies were carried out by injecting CPCs or lenti-Ang-(1-7) transduced CPCs (4 × 10 6 ), into the jugular vein three days after permanent left anterior descending coronary artery ligation. Echocardiography, hemodynamic, histological parameters, and capillary vessel density were measured to assess cardiac function and the effects of cell transplantation four weeks thereafter. Results from the in vivo studies demonstrated that myocardial infarction (MI) significantly reduced fractional shortening (FS, 33.0% ± 1.5 vs 59.5% ± 1.9 for control), increased ventricular hypertrophy (VH, 3.6 ± 0.1 vs 2.7 ± 0.1for control, (g/kg)), and decreased dP/dt max (7235 ± 271.9 vs 10394 ± 326.3 for control, mmHg/sec). In comparison to MI, CPCs alone restored FS by 22%, reversed VH by 11%, and improved dP/dt max by 23%. Remarkably, Ang-(1-7) expressing CPCs further improved FS by 44%, decreased VH by 15%, and attenuated the reduction in dP/dt max by 42%. Finally, capillary density in the peri-infarct myocardium was increased by 59% with CPCs alone and this capillary density was further increased by 139% with Ang-(1-7) expressing CPCs. In conclusion, these observations, for the first time, demonstrate that Ang-(1-7) enhances the regenerative capacity of CPCs and promotes angiogenesis to improve cardiac hemodynamic following MI. They suggest that Ang-(1-7) transduced CPC could have therapeutic potential in IHD.

Hematopoietic Progenitor Cells and Restenosis After Carotid Endarterectomy

Hematopoietic progenitor cells (HPCs) may attenuate the response to vascular injury by maintaining endothelial integrity and function. Our aim was to determine whether circulating HPC number and function correlate with restenosis after carotid endarterectomy. HPC number (CD34(+)/CD133(+) cells), early colony-forming units, migratory capacity, and senescence were analyzed in blood collected preoperatively, 1 day, and 6 weeks postoperatively. Mobilizing cytokine levels were also measured. Stenosis was assessed by duplex scanning. HPC numbers (P<0.001) and early colony-forming unit count (P=0.001) fell rapidly 24 hours postoperatively. Restenosis at 6 months correlated negatively with the magnitude of postoperative falls in HPC numbers (R=-0.38, P=0.013) and early colony-forming unit counts (R=-0.42, P=0.008). The migratory capacity of preoperative HPCs correlated negatively with restenosis (R=-0.48, P=0.007). Preoperative SDF1 levels correlated with falls in HPC number (R=0.42, P=0.044) and early colony-forming unit counts (R=0.56, P=0.004). HPC function appears to be linked to the development of carotid artery restenosis after endarterectomy. These data support the concept that HPCs have a role in regulating remodeling of the injured arterial wall.