HIF-1α-stabilizing agent FG-4497 rescues human CD34+ cell mobilization in response to G-CSF in immunodeficient mice

Granulocyte colony-stimulating factor (G-CSF) is used routinely in the clinical setting to mobilize hematopoietic stem progenitor cells (HSPCs) into the patient's blood for collection and subsequent transplantation. However, a significant proportion of patients who have previously received chemotherapy or radiotherapy and require autologous HSPC transplantation cannot mobilize the minimal threshold of mobilized HSPCs to achieve rapid and successful hematopoietic reconstitution. Although several alternatives to the G-CSF regime have been tested, few are used in the clinical setting. We have shown previously in mice that administration of prolyl 4-hydroxylase domain enzyme (PHD) inhibitors, which stabilize hypoxiainducible factor (111F)-1 alpha, synergize with G-CSF in vivo to enhance mouse HSPC mobilization into blood, leading to enhanced engraftment via an HSPC-intrinsic mechanism. To evaluate whether PHD inhibitors could be used to enhance mobilization of human HSPCs, we humanized nonobese, diabetic severe combined immune-deficient Il2rg(-/-) mice by transplanting them with human umbilical cord blood CD34(+) HSPCs and then treating them with G-CSF with and without co-administration of the PHD inhibitor FG-4497. We observed that combination treatment with G-CSF and FG-4497 resulted in significant mobilization of human lineage-negative (Lin(-)) CD34(+) HSPCs and more primitive human Lin(-) CD34(+)CD38(-) HSPCs into blood and spleen, whereas mice treated with G-CSF alone did not mobilize human HSPCs significantly. These results suggest that the PHD inhibitor FG-4497 also increases human HSPC mobilization in a xenograft mouse model, suggesting the possibility of testing PHD inhibitors to boost HSPC mobilization in response to G-CSF in humans. Copyright (C) 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc

HIF prolyl hydroxylase inhibitor FG-4497 enhances mouse hematopoietic stem cell mobilization via VEGFR2/KDR

In normoxia, hypoxia-inducible transcription factors (HIFs) are rapidly degraded within the cytoplasm as a consequence of their prolyl hydroxylation by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes. We have previously shown that hematopoietic stem and progenitor cells (HSPCs) require HIF-1 for effective mobilization in response to granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist AMD3100/plerixafor. Conversely, HIF PHD inhibitors that stabilize HIF-1 protein in vivo enhance HSPC mobilization in response to G-CSF or AMD3100 in a cell-intrinsic manner. We now show that extrinsic mechanisms involving vascular endothelial growth factor receptor-2 (VEGFR2), via bone marrow (BM) endothelial cells, are also at play. PTK787/vatalanib, a tyrosine kinase inhibitor selective for VEGFR1 and VEGFR2, and neutralizing anti-VEGFR2 monoclonal antibody DC101 blocked enhancement of HSPC mobilization by FG-4497. VEGFR2 was absent on mesenchymal and hematopoietic cells and was detected only in Sca1 endothelial cells in the BM. We propose that HIF PHD inhibitor FG-4497 enhances HSPC mobilization by stabilizing HIF-1α in HSPCs as previously demonstrated, as well as by activating VEGFR2 signaling in BM endothelial cells, which facilitates HSPC egress from the BM into the circulation

NSG mice bone marrow cell characterisation and humanisation of the stromal compartment for the purpose of studying prostate cancer metastasis

Dr Nowlan’s PhD focused on efforts to better understand the biology of mouse bone marrow and to improve the utility of mouse model used to study prostate cancer bone marrow metastasis. She developed a new way of characterising the blood forming stem cells in the bone marrow mouse strains commonly used in the study of human cancers. Subsequently, she optimised methods to partially humanise mouse bone marrow with human bone marrow stromal cells, and then studied how the human stromal cells influenced the formation of prostate cancer lesions in mice

Direct bone marrow injection of human bone marrow-derived stromal cells into mouse femurs results in greater prostate cancer PC-3 cell proliferation, but not specifically proliferation within the injected femurs

Background: While prostate cancer (PCa) cells most often metastasize to bone in men, species-specific differences between human and mouse bone marrow mean that this pattern is not faithfully replicated in mice. Herein we evaluated the impact of partially humanizing mouse bone marrow with human bone marrow-derived stromal cells (BMSC, also known as "mesenchymal stem cells") on human PCa cell behaviour. Methods: BMSC are key cellular constituents of marrow. We used intrafemoral injection to transplant 5 × 105 luciferase (Luc) and green fluorescence protein (GFP) expressing human BMSC (hBMSC-Luc/GFP) into the right femur of non-obese diabetic (NOD)-severe combined immunodeficiency (scid) interleukin (IL)-2γ−/− (NSG) mice. Two weeks later, 2.5 × 106 PC-3 prostate cancer cells expressing DsRed (PC-3-DsRed) were delivered into the mice via intracardiac injection. PC-3-DsRed cells were tracked over time using an In Vivo Imaging System (IVIS) live animal imaging system, X-ray and IVIS imaging performed on harvested organs, and PC-3 cell numbers in femurs quantified using flow cytometry and histology. Results: Flow cytometry analysis revealed greater PC-3-DsRed cell numbers within femurs of the mice that received hBMSC-Luc/GFP. However, while there were overall greater PC-3-DsRed cell numbers in these animals, there were not more PC-3-DsRed in the femurs injected with hBMSC-Luc/GFP than in contralateral femurs. A similar proportion of mice in with or without hBMSC-Luc/GFP had bone lessions, but the absolute number of bone lesions was greater in mice that had received hBMSC-Luc/GFP. Conclusion: PC-3-DsRed cells preferentially populated bones in mice that had received hBMSC-Luc/GFP, although PC-3-DsRed cells not specifically localize in the bone marrow cavity where hBMSC-Luc/GFP had been transplanted. hBMSC-Luc/GFP appear to modify mouse biology in a manner that supports PC-3-DsRed tumor development, rather than specifically influencing PC-3-DsRed cell homing. This study provides useful insights into the role of humanizing murine bone marrow with hBMSC to study human PCa cell biology.</p

Human bone marrow-derived stromal cell behavior when injected directly into the bone marrow of NOD-scid-gamma mice pre-conditioned with sub-lethal irradiation

BackgroundDirect bone marrow injection of cells into murine marrow cavities is used in a range of cell characterization assays and to develop disease models. While human bone marrow-derived stromal cells (hBMSC, also known as mesenchymal stem cells (MSC)) are frequently described in therapeutic applications, or disease modeling, their behavior following direct injection into murine bone marrow is poorly characterized. Herein, we characterized hBMSC engraftment and persistence within the bone marrow of NOD-scid interleukin (IL)-2γ−/− (NSG) mice with or without prior 2 Gy total-body γ-irradiation of recipient mice.MethodsOne day after conditioning NSG mice with sublethal irradiation, 5 × 105 luciferase (Luc) and green fluorescent protein (GFP)-expressing hBMSC (hBMSC-Luc/GFP) were injected into the right femurs of animals. hBMSC-Luc/GFP were tracked in live animals using IVIS imaging, and histology was used to further characterize hBMSC location and behavior in tissues.ResultshBMSC-Luc/GFP number within injected marrow cavities declined rapidly over 4 weeks, but prior irradiation of animals delayed this decline. At 4 weeks, hBMSC-Luc/GFP colonized injected marrow cavities and distal marrow cavities at rates of 2.5 ± 2.2% and 1.7 ± 1.9% of total marrow nucleated cells, respectively in both irradiated and non-irradiated mice. In distal marrow cavities, hBMSC were not uniformly distributed and appeared to be co-localized in clusters, with the majority found in the endosteal region.ConclusionsWhile significant numbers of hBMSC-Luc/GFP could be deposited into the mouse bone marrow via direct bone marrow injection, IVIS imaging indicated that the number of hBMSC-Luc/GFP in that bone marrow cavity declined with time. Irradiation of mice prior to transplant only delayed the rate of hBMSC-Luc/GFP population decline in injected femurs. Clusters of hBMSC-Luc/GFP were observed in the histology of distal marrow cavities, suggesting that some transplanted cells actively homed to distal marrow cavities. Individual cell clusters may have arisen from discrete clones that homed to the marrow, and then underwent modest proliferation. The transient high-density population of hBMSC within the injected femur, or the longer-term low-density population of hBMSC in distal marrow cavities, offers useful models for studying disease or regenerative processes. Experimental designs should consider how relative hBMSC distribution and local hBMSC densities evolve over time

Flow cytometry analysis of cell cycling and proliferation in mouse hematopoietic stem and progenitor cells

The hematopoietic system is highly proliferative in the bone marrow (BM) due to the short half-life of granulocytes and platelets in the blood. Analysis of cell cycling and cell proliferation in vivo in specific populations of the mouse BM has highlighted some key properties of adult hematopoietic stem cells (HSCs). For instance, despite their enormous proliferation and repopulation potential, most true HSC are deeply quiescent in G(0) phase of the cell cycle and divide very infrequently, while less potent lineage-restricted progenitors divide rapidly to replace the daily consumption of blood leukocytes, erythrocytes, and platelets. In response to stress, e.g., following ablative chemotherapy or irradiation, HSC must enter the cell cycle to rapidly repopulate the BM with progenitors. Due to their extreme rarity in the BM, at least five color flow cytometry for cell surface antigens has to be combined with staining for DNA content and nuclear markers of proliferation to analyze cell cycle and proliferation of HSC in vivo. In this chapter, we describe two methods to stain mouse HSC to (1) distinguish all phases of the cell cycle (G(0), G(1), S, and G(2)/M) and (2) analyze the divisional history of HSC in vivo by incorporation of the thymidine analog 5-bromo-2-deoxyuridine

CD27, CD201, FLT3, CD48, and CD150 cell surface staining identifies long-term mouse hematopoietic stem cells in immunodeficient non-obese diabetic severe combined immune deficient derived strains

Staining for CD27 and CD201 (Endothelial protein C receptor) has been recently suggested as an alternative to stem cell antigen-1 (Sca1) to identify hematopoietic stem cells in inbred mouse strains with low or nil expression of SCA1. However, whether staining for CD27 and CD201 is compatible with low fms-like tyrosine kinase 3 (FLT3) expression and the 'SLAM' code defined by CD48 and CD150 to identify mouse long-term reconstituting hematopoietic stem cells has not been established. We compared the C57BL/6 strain, which expresses a high level of SCA1 on hematopoietic stem cells to non-obese diabetic severe combined immune deficient NOD.CB17-prkdcscid/Sz (NOD-scid) mice and NOD.CB17-prkdcscid il2rgtm1Wj1/Sz (NSG) mice which both express low to negative levels of SCA1 on hematopoietic stem cells. We demonstrate that hematopoietic stem cells are enriched within the linage-negative C-KIT+ CD27+ CD201+ FLT3- CD48- CD150+ population in serial dilution long-term competitive transplantation assays. We also make the novel observation that CD48 expression is up-regulated in Lin- KIT+ progenitors from NOD-scid and NSG strains, which otherwise have very few cells expressing the CD48 ligand CD244. Finally, we report that unlike hematopoietic stem cells, SCA1 expression is similar on bone marrow endothelial and mesenchymal progenitor cells in C57BL/6, NOD-scid and NSG mice. In conclusion, we propose that the combination of Lineage, KIT, CD27, CD201, FLT3, CD48, and CD150 antigens can be used to identify long-term reconstituting hematopoietic stem cells from mouse strains expressing low levels of SCA1 on hematopoietic cells

Bacillus okhensis sp. nov., a halotolerant and alkalitolerant bacterium from an Indian saltpan

A strictly aerobic, rod-shaped bacterium (0.6–0.8×2–3 μm), designated strain Kh10-101T, was isolated from a saltpan (22° 15′ N, 69° 1′ E) in the vicinity of Port Okha, India. The creamish pigmented colonies of strain Kh10-101T were round, flat and translucent with irregular margins and a smooth surface. The strain possessed up to three subpolar flagella, and was motile by a corkscrew motion. The strain grew optimally at 37 °C (temperature growth range 25–40 °C) in a complex glucose-containing medium with 5 % NaCl (NaCl growth range 0–10 %) at pH 9 (pH growth range pH 7–10), indicating that it was a mesophilic halotolerant alkaliphile. The strain was sensitive to lincomycin, meticillin, cefuroxime and cephalexin, but resistant to gentamicin, tetracycline and cotrimazine. Spores were not detected and cells were heat sensitive. The isolate metabolized a range of carbohydrates and hydrolysed casein, gelatin and starch. Growth was not observed on aromatic compounds, Tween 40 or Tween 80. Nitrate was not reduced and catalase was produced. Electron microscopic examination of thin sections revealed a single thick Gram-positive cell wall. The DNA G+C content was 41±1 mol%. Phylogenetic analyses of the 16S rRNA gene sequence revealed that strain Kh10-101T was a member of the sixth rRNA group of the genus Bacillus, which includes alkalitolerant, alkaliphilic and halotolerant species. The halotolerant obligate alkaliphile Bacillus krulwichiae is the closest relative of strain Kh10-101T (96 % similarity) but a number of phenotypic differences suggest that strain Kh10-101T (=JCM 13040T=ATCC BAA-1137T) should be designated the type strain of a new species, for which the name Bacillus okhensis sp. nov. is proposed

Mobilization of hematopoietic stem cells with highest self-renewal by G-CSF precedes clonogenic cell mobilization peak

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF. Copyright (C) 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc