Long-term Bone Marrow Cultures Research Articles

Regenerative potential of multinucleated cells: bone marrow adiponectin-positive multinucleated cells take the lead

BackgroundPolyploid cells can be found in a wide evolutionary spectrum of organisms. These cells are assumed to be involved in tissue regeneration and resistance to stressors. Although the appearance of large multinucleated cells (LMCs) in long-term culture of bone marrow (BM) mesenchymal cells has been reported, the presence and characteristics of such cells in native BM and their putative role in BM reconstitution following injury have not been fully investigated.MethodsBM-derived LMCs were explored by time-lapse microscopy from the first hours post-isolation to assess their colony formation and plasticity. In addition, sub-lethally irradiated mice were killed every other day for four weeks to investigate the histopathological processes during BM regeneration. Moreover, LMCs from GFP transgenic mice were transplanted to BM-ablated recipients to evaluate their contribution to tissue reconstruction.ResultsBM-isolated LMCs produced mononucleated cells with characteristics of mesenchymal stromal cells. Time-series inspections of BM sections following irradiation revealed that LMCs are highly resistant to injury and originate mononucleated cells which reconstitute the tissue. The regeneration process was synchronized with a transient augmentation of adipocytes suggesting their contribution to tissue repair. Additionally, LMCs were found to be adiponectin positive linking the observations on multinucleation and adipogenesis to BM regeneration. Notably, transplantation of LMCs to myeloablated recipients could reconstitute both the hematopoietic system and BM stroma.ConclusionsA population of resistant multinucleated cells reside in the BM that serves as the common origin of stromal and hematopoietic lineages with a key role in tissue regeneration. Furthermore, this study underscores the contribution of adipocytes in BM reconstruction.Graphical

Targeted Radiation Evokes Catecholamine Production Triggering Systemic Inflammatory Responses

▪Targeted irradiation (TR) is widely used for tumor treatment in the clinic. TR benefits tumor therapy through direct effects as well as poorly understood systemic (abscopal) effects. Recent studies suggest that the systemic innate and acquired immune responses to TR contribute to elimination of tumor cells, but also cause systemic inflammation with prolonged tissue injury that may result in secondary malignancies. To elucidate and eventually target the mechanisms underlying these systemic effects of TR, we utilized a murine model using the small animal radiation research platform (SARRP).To define the dynamics of cytokine production and immune responses after TR, we administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. We analyzed bone marrow (BM) and BM extracellular fluid (BMEF) from both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. As a tumor-bearing model, we utilized 3-4 weeks old C57BL/6 mice injected with Rhabdomyosarcoma (RMS) in one hind limb, and treated with (1) one dose i.p injection of 1mg/Kg Vincristine (Vin) as chemotherapy model, (2) 4.8GyX5times fractionated TR to the tumor area and (3) combination (TR+Vin) therapy. Analysis of peripheral blood (PB), BM, BMEF was performed 3 weeks after the final TR dose (n = 5-13 mice/time point).We found that multiple inflammatory cytokines and chemokines, such as IL-1b, IL-18, CCL2, CCL3, CXCL2, CXCL9, CXCL10 were upregulated from very early phase (2hrs) up to 48hrs in BMEF of the radiated tibiae. Consistent with the dynamics of these cytokines, we observed influx of myeloid cells in both TR and CONT side and expansion of T cells peaking at 6hrs in BM. At the same time of these immune responses, Norepinephrine (NE) was elevated in BMEF even in CONT side. In the tumor-bearing model of RMS, fractionated TR eliminated the tumor while systemically expanding CD8+ cytotoxic T cells and reducing neutrophils. Vin alone did not eliminate the tumor and was associated with systemic decrease of lymphoid cells and expansion of neutrophils. In Vin+TR, tumor control and CD8+ cell expansion were restored, with normalization of neutrophils. These data suggest that TR in the setting of tumor differentially activates lymphoid and myeloid cells. Since recent studies showed catecholamine production from myeloid cells may augment cytokine production in the setting of infection, we hypothesized that BM myeloid cells respond to radiation-induced cell damage by producing catecholamines that trigger a systemic inflammatory response after TR. To test this hypothesis, we utilized standard long-term bone marrow cultures (LT-BM) that reproduce three-dimensional BM structures with myeloid-skewing in vitro, and irradiated them to look at inflammatory changes induced by radiation at 2, 6 and 24hrs. In this experimental model, 5Gy of radiation led to the elevation of NE along with the production of chemokines CCL2, CCL3, CXCL2, CXCL9 mostly peaking at 6hrs in the cell culture supernatants. In contrast, these responses could not be reproduced in spleen cultures, which also had a much lower baseline NE production compared to LT-BMs. These data indicate that radiation induced-chemokine elevations might come from myeloid cells stimulated by NE, independent of systemic innervation. To define the contribution of catecholamines to cytokine production in LT-BM, we directly stimulated culture-LT-BM with NE and Isoproterenol, a pan beta stimulant. While both agents showed similar effect and increased CXCL2, CXCL9, CCL2 and CCL3 at 6hrs, they decreased CXCL10 level, suggesting that catecholamine mostly stimulate myeloid cells but rather inhibit lymphoid activation through chemokine production.Together, these data show that local irradiation initiates global immune responses, and identify local BM production of NE as its potential trigger. Blocking local catecholamine production in the bone marrow could therefore be a positive adjuvant to TR in tumor treatment by inhibiting unfavorable effects of radiation, such as chronic inflammation with systemic increases of neutrophils, while facilitating expansion and recruitment of the cytotoxic T cells which play an essential beneficial role in tumor immunity. DisclosuresNo relevant conflicts of interest to declare.

Radiation Resistance of C57Bl/6 Mouse Bone Marrow Stromal Cell Lines Induced By Apoptosis Inhibitor JP4-039 or Necroptosis Inhibitor Necrostatin-1, but Not By Simultaneous Administration of Both Mitigators

Introduction: Necroptosis is a TNFa activated, caspase-independent cell death pathway that involves active disintegration of mitochondrial, lysosomal and plasma membranes (Vandenabeele, et al, Nat Rev Mol Cell Biol., 2010 Oct; 11(10):700-14). We measured clonogenic radiation survival of C57/Bl6 mouse bone marrow stromal cell lines in the presence of a mitochondrial targeted inhibitor of apoptosis (JP4-039), an inhibitor of necroptosis (Necrostatin-1) or both drugs.Materials and Methods: C57BL/6 mouse long term bone marrow cultures (LTBMCs) were established from adult female C57Bl/6 mice. Bone marrow stromal cell lines were derived from the adherent layers of LTBMCs. Radiosensitivity of C57Bl/6 bone marrow stromal cell lines and fresh bone marrow CFU-GEMM (semisolid medium with added hemopoietic growth factors) was measured in clonogenic radiation survival curves. Stromal Cells were irradiated to doses of 0 to 8 Gy, plated in T-75 tissue culture flasks, incubated for 8 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted at day 7. CFU-GEMMs from fresh bone marrow were scored at day 14. Both cultured cell populations were tested with addition of radiation mitigators: JP4-039 (10µM), Necrostatin-1 (40µM), or both drugs added to culture medium 30 minutes post irradiation. C57BL/6 mouse LTBMCs generated hemopoietic cells for 20 weeks. Marrow stromal cell lines were derived from 20 week old hemopoieticly-inactive culture adherent layer cells. Statistical analysis was performed using Student's t-test, with a two-tail p-value < 0.05 considered significant.Results: Radiation survival curves showed that C57Bl/6 fresh bone marrow CFU-GEMM and stromal cell lines were comparable to those for other mouse strains (CFU-GEMM Do = 1.67 ± 0.02, stromal cells Do=1.43 ± 0.14). Both fresh marrow CFU-GEMM and stromal cells (ň = 5.26 ± 0.72) were radioresistant when grown in presence of JP4-039 (ň = 8.85 ± 1.23, p = 0.012) or necrostatin-1 (n=7.97 ± 1.12, p = 0.024). Addition of both JP4-039 and Necrostatin-1 (ň = 5.79 ± 1.44) at the same time after irradiation (30 min) did not improve clonogenic survival of either fresh marrow CFU-GEMM or stromal cell lines.Conclusions: Both freshly explanted CFU-GEMM marrow hematopoietic progenitor cells and LTBMC derived marrow stromal cell lines display radioresistance when treated with each of two radiation mitigators that target different cell death pathways. While each drug delivered independently induced radioresistance, combined delivery of JP4-039 and Necrostatin-1 at 30 min after irradiation did not. Thus, the timing of delivery of drugs that target different cell death pathways is likely critical in designing effective protocols for radiation mitigation. DisclosuresNo relevant conflicts of interest to declare.

Radiation-Induced Senescence in p16+/LUC Mouse Lung Compared to Bone Marrow Multilineage Hematopoietic Progenitor Cells.

We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-β. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and β-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.

Functionalized Silica Star-Shaped Nanoparticles and Human Mesenchymal Stem Cells: An In Vitro Model.

The biomedical translational applications of functionalized nanoparticles require comprehensive studies on their effect on human stem cells. Here, we have tested neat star-shaped mesoporous silica nanoparticles (s-MSN) and their chemically functionalized derivates; we examined nanoparticles (NPs) with similar dimensions but different surface chemistry, due to the amino groups grafted on silica nanoparticles (s-MSN-NH2), and gold nanoseeds chemically adsorbed on silica nanoparticles (s-MSN-Au). The different samples were dropped on glass coverslips to obtain a homogeneous deposition differing only for NPs’ chemical functionalization and suitable for long-term culture of human Bone Marrow–Mesenchymal stem cells (hBM-MSCs) and Adipose stem cells (hASCs). Our model allowed us to demonstrate that hBM-MSCs and hASCs have comparable growth curves, viability, and canonical Vinculin Focal adhesion spots on functionalized s-MSN-NH2 and s-MSN-Au as on neat s-MSN and control systems, but also to show morphological changes on all NP types compared to the control counterparts. The new shape was stem-cell-specific and was maintained on all types of NPs. Compared to the other NPs, s-MSN-Au exerted a small genotoxic effect on both stem cell types, which, however, did not affect the stem cell behavior, likely due to a peculiar stem cell metabolic restoration response.

Suppression of Hematopoietic Activity in Tenascin-C–Deficient Mice

AbstractTenascin-C (TN-C), a member of the extracellular matrix (ECM) glycoprotein family, is expressed on the surface of stromal cells in the hematopoietic system or lymphoid organs. Recently, TN-C–deficient mutant mice produced by TN-C gene targeting through homologous recombination were shown to develop normally, although TNs have been reported to play important roles in organogenesis and carcinogenesis. In the present study, we found that colony-forming capacity of bone marrow (BM) cells was considerably lower in TN-C–deficient mice (a decrease of ∼35% from control), although their mononuclear cell count and BM architecture showed no significant difference from those of normal mice. Furthermore, in long-term BM culture in vitro, hematopoietic cell production (a decrease of ∼40% in Dexter's condition and of ∼65% in Whitlock-Witte's condition from control), colony-forming capacity of the produced cells (a decrease of ∼60% from control), and longevity of the cultures were markedly lower in the TN-C–deficient mice than in control mice, whereas hematopoiesis in the TN-C–deficient mutant mice was sustained. The addition of TN-C glycoprotein to long-term BM cultures of TN-C–deficient mice clearly induced the recovery of hematopoietic cell production and colony-forming capacity of hematopoietic progenitor cells. Thus, these results provide direct evidence that an ECM glycoprotein component, TN-C, plays a relevant role in hematopoiesis through interactions between stromal cells and hematopoietic progenitor cells.

Surface modifications to polydimethylsiloxane substrate for stabilizing prolonged bone marrow stromal cell culture

Polydimethylsiloxane (PDMS) has been extensively used as a supporting material for studies of cell mechanobiology, cell micropatterning and microscale-cell analysis in microfluidic chips due to its numerous advantages, such as low cytotoxicity, ease of modification, inexpensive costs and biocompatibility. However, the innate hydrophobicity of PDMS often poses a problem for stable cell adhesion, seriously limiting its applicability for prolonged cell culture. UV exposure and protein coating are suboptimal solutions, while chemical surface functionalization is often associated with laborious procedures and producing environmental toxics. Plasma treatment can render a hydrophilic substrate by altering the surface chemistry, but such effect is often short-lived due to its tendency to hydrophobic recovery. Variation of physical properties of the substratum are known to influence cell behaviour. Nevertheless, the combination of varying PDMS substratum properties via base:curing agent ratio and plasma treatment to stabilize the long-term culture of bone marrow derived stromal cells (BMSCs) still remain poorly understood. In this study, we developed a protocol to maintain the hydrophilicity of the plasma-treated PDMS over a range of substratum properties. This study demonstrated that varying the substratum properties of PDMS can enhance the stability of BMSC culture for at least three weeks, while plasma treatment with or without additional collagen coating further enhanced such effect. The changes in the physical properties of PDMS have rendered difference in BMSCs adhesion, proliferation and in-vitro plasticity, thereby offering a simple and effective strategy for PDMS surface modification to enable long term cell analysis in PDMS-based culture platform.

Extracellular annexin-A1 promotes myeloid/granulocytic differentiation of hematopoietic stem/progenitor cells via the Ca2+/MAPK signalling transduction pathway

Annexin A1 (AnxA1) modulates neutrophil life span and bone marrow/blood cell trafficking thorough activation of formyl-peptide receptors (FPRs). Here, we investigated the effect of exogenous AnxA1 on haematopoiesis in the mouse. Treatment of C57BL/6 mice with recombinant AnxA1 (rAnxA1) reduced the granulocyte–macrophage progenitor (GMP) population in the bone marrow, enhanced the number of mature granulocytes Gr-1+Mac-1+ in the bone marrow as well as peripheral granulocytic neutrophils and increased expression of mitotic cyclin B1 on hematopoietic stem cells (HSCs)/progenitor cells (Lin−Sca-1+c-Kit+: LSK). These effects were abolished by simultaneous treatment with Boc-2, an FPR pan-antagonist. In in vitro studies, rAnxA1 reduced both HSC (LSKCD90lowFLK-2−) and GMP populations while enhancing mature cells (Gr1+Mac1+). Moreover, rAnxA1 induced LSK cell proliferation (Ki67+), increasing the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCLγ2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways.

Radioresistance of Serpinb3a-/- Mice and Derived Hematopoietic and Marrow Stromal Cell Lines.

Serpins are a group of serine-proteases involved in multiple signal transduction pathways in mammalian cells. In particular, Serpinb3a is involved in the lysosomal necrosis cell death pathway with components that overlap with radiation-induced apoptosis. We investigated the radiation response of Serpinb3a-/- mice compared to Serpinb3a+/+ mice on the Balb/c background. Serpinb3a-/- mice showed significant radioresistance to a dose of 8.0 Gy total-body irradiation, compared to Serpinb3a+/+ Balb/c mice. Long-term bone marrow cultures from Serpinb3a-/- mice showed increased longevity. In clonogenic survival assays, fresh bone marrow hematopoietic progenitors, as well as clonal interleukin-3 (IL-3)-dependent hematopoietic progenitor and bone marrow stromal cell lines from Serpinb3a-/- mice were radioresistant. Serpinb3a-/- mouse bone marrow-derived stromal cell lines had increased baseline and postirradiation antioxidant capacity. Serpinb3a-/- bone marrow stromal cells showed increased radiation-induced RNA transcripts for MnSOD and p21, and decreased levels of p53 and TGF-b. Both irradiated Serpinb3a-/- mouse bone marrow stromal cell lines and plasma removed from total-body irradiated mice had decreased levels of expression of stress response and inflammation-associated proteins. Abrogation of Serpinb3a may be a potential new target for mitigation of radiation effects.

Abstract 2913: Serpinb3a-/- mice are radioresistant

Abstract Serpins are a superfamily of serine protease inhibitors that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit their target peptidases. Currently 36 human serpins have been identified, but the biological roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). Serpinb3a (the mouse orthologue of human SERPINB3 and -B4) is an intracellular serpin and inhibits both serine and cysteine peptidases invitro. We now report radioresistance of Serpinb3a-/- mice and their hematopoietic progenitor cells. Serpinb3a-/- and control Balb/c mice were irradiated to a dose of 8.0 Gy total body irradiation and followed for the development of the hematopoietic syndrome. Long Term Bone Marrow Cultures (LTBMCs) were established from the bone marrow flushed from the femurs and tibias of Serpinb3a-/- and Balb/c mice. Irradiation survival curves were performed on bone marrow stromal cell lines established from the LTBMCs and on fresh bone marrow isolated from the femurs of sacrificed Serpinb3a-/- and Balb/c mice. Colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 3 experiments were analyzed using linear quadratic and single-hit, multi-target models. Serpinb3a-/- mice showed increased survival following 8.0 Gy TBI compared to Balb/c mice (p &amp;lt; 0.0001). CFU-GM from Serpinb3a-/- were radioresistant compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). Serpinb3a-/- bone marrow stromal cells were radioresistant with an increased Do (1.97 + 0.11 Gy) compared to bone marrow stromal cells from Balb/c mice (Do = 1.45 + 0.03 Gy, p = 0.0113). Loss of Serpinb3a provides a radiation survival advantage, increases radioresistance of both hematopoietic progenitor cells and marrow stromal cells, and may lead to identification of new targets for design of radiation mitigator drugs. This research was supported by grant NIAID/NIH U19-A168021. Citation Format: Stephanie Thermozier, Michael Epperly, Darcy Franicola, Xichen Zhang, Renee Fisher, Donna Shields, Hong Wang, John Willis, Cliff Luke, Gary Silverman, Joel Greenberger. Serpinb3a-/- mice are radioresistant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2913.

Abstract 5155: Increased longevity of continuous bone marrow cultures and radioresistance of bone marrow stromal cells from SOD193A ALS (Amyotrophic Lateral Sclerosis) mice

Abstract Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss. Materials and Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis. Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p &amp;lt; 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466). Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748). Conclusions: The results of hematopoiesis in LTBMCs in the absence of functional SOD1 showed improved LTBMC longevity and radioresistance of marrow stromal cells both unexpected pleiotropic effects of the SOD1G93A genotype. Marrow transplant after TBI prolonged the paralysis free interval in ALS mice. Citation Format: Andrew Henderson, Michael W. Epperly, Renee Fisher, Donna Shields, Lora Rigatti, Christopher Donnelly, Simon Watkins, Joel S. Greenberger. Increased longevity of continuous bone marrow cultures and radioresistance of bone marrow stromal cells from SOD193A ALS (Amyotrophic Lateral Sclerosis) mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5155.

TGF-B Inhibition Rescues Hematopoietic Defects in Fanconi Anemia

Four different combinations of double knock-out mouse strains have been created (SMAD3-/- Fancd2-/-) consisting of combinations of the individual knock-out strains on either the C57BL/6 or 129/Sv background. Sets of breeding experiments were carried out and the frequency of detection of double knock-out mice was significantly below the expected frequency of detecting DKO mice by breeding double heterozygotes (1 out of 16 or 6.25%). In fact, DKO mice were detected at a frequency ranging from 3.4% to 0.54%. Mice of all four DKO genotypes showed resistance of bone marrow hematopoietic progenitor cells to canonical TGF-β signaling, consistent with the SMAD3-/- parent. However, the predominant phenotype was that of the Fancd2-/- parent including reduced duration of hematopoiesis in long-term bone marrow cultures, reduced marrow stem cell competitive repopulation capacity, and retained mitomycin C and radiation sensitivity of bone marrow stromal cells. These mice should be a valuable resource for elucidating the bypass pathways involved in the reduced, but successful gestation of SMAD3-/- Fancd2-/- mice, and the interactions of the FA and TGF-β signaling pathways.Supported by the NIAID/NIH U19-A168021 and the Fanconi Anemia Research Fund. DisclosuresNo relevant conflicts of interest to declare.

Increased Longevity of Continuous Bone Marrow Cultures and Radioresistance of Bone Marrow Stromal Cells from SOD193A ALS (Amyotrophic Lateral Sclerosis) Mice

Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss.Materials & Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis.Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p < 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466).Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748).Conclusions: The results showing increased longevity of hematopoiesis in LTBMCs of marrow from mice displaying an absence of SOD1 and the radioresistance of derived bone marrow stromal cell lines represent two unexpected pleiotrophic effects of the SOD1 G93A genotype. Further studies will be required to determine how marrow transplant after TBI prolonged the paralysis free interval in these ALS mice. DisclosuresNo relevant conflicts of interest to declare.

Amelioration of Head and Neck Radiation-Induced Mucositis and Distant Marrow Suppression in Fanca-/- and Fancg-/- Mice by Intraoral Administration of GS-Nitroxide (JP4-039).

Squamous cell carcinomas of the head and neck are appearing with increased frequency in both marrow transplanted and non-transplanted Fanconi anemia (FA) patients. FA patients commonly display radiosensitivity of epithelial tissues, complicating effective radiotherapy. Fancd2-/- mice (C57BL/6J and 129/Sv background) demonstrate epithelial tissue sensitivity to single-fraction or fractionated irradiation to the head and neck and distant marrow suppression (abscopal effect), both ameliorated by intraoral administration of the mitochondrial-targeted antioxidant, GS-nitroxide, JP4-039. We now report that mice of two other FA genotypes, Fancg-/- (B6) and the most prevalent human genotype Fanca-/- (129/Sv), also demonstrate: 1. reduced longevity of hematopoiesis in long-term bone marrow cultures; 2. radiosensitivity of bone marrow stromal cell lines; and 3. head and neck radiation-induced severe mucositis and abscopal suppression of distant marrow hematopoiesis. Intraoral administration of JP4-039/F15, but not non-mitochondrial-targeted 4-amino-Tempo/F15 or F15 alone, prior to each radiation treatment ameliorated both local and abscopal radiation effects. Head and neck irradiated TGF-β-resistant SMAD3-/- (129/Sv) mice and double-knockout SMAD3-/- Fancd2-/- (129/Sv) mice treated daily with TGF-β receptor antagonist, LY364947, still displayed abscopal bone marrow suppression, implicating a non-TGF-β mechanism. Thus, amelioration of both local normal tissue radiosensitivity and distant marrow suppression by intraoral administration of JP4-039 in Fancg-/- and Fanca-/- mice supports a clinical trial of this locally administered normal tissue radioprotector and mitigator during head and neck irradiation in FA patients.

Scribble Controls HSC Self-Renewal through Polarity-Dependent Activation of the Hippo Signaling Pathway

Search of molecular targets to expand functional hematopoietic stem cells (HSC) for transplantation and hematological cancer therapy remains paramount, particularly when manipulation of cellular quiescence is required. HSC are highly quiescent cells with the ability to rapidly enter the cell cycle in response to microenvironment cues and divide through changes in their polarity. Despite cellular polarity being one of the most basic properties of all living cells, the role that polarity regulators have on polarization and function of HSC remains controversial. Scribble, of the Scribble Polarity Complex, controls the spatial organization of intracellular proteins and negatively regulates the cell cycle. By using a combination of constitutive and inducible hematopoietic-specific Scribble-deficient animal models, hematopoietic reconstitution assays, structure-function mutants of Scribble and intracellular protein trafficking analysis, we identified the functional relevance of Scribble in HSC activity. We observed that Scribble is polarized in HSC and deletion of Scribble leads to a ~ 50% reduction in the bone marrow (BM) HSC population (p<0.001). Scribble-deficient HSC are less quiescent (G0: 93 vs 88%, p<0.05; S/G2/M 4 vs 10%, for WT and KO, respectively, p<0.01) and contain ~70% reduction of dormant, long-term HSC (p<0.01). This data indicates that Scribble is required to maintain HSC quiescence. Functionally, stroma-dependent long-term BM cultures revealed that Scribble-deficient BM contains a ~ 60% reduction in the number of early cobblestone area-forming cells (CAFC) while their content of late (28-35 day) CAFC (primitive progenitors/HSC) is 3-5 fold increased. Serial transplantation experiments demonstrated that Scribble-deficient BM HSC have increased competitive repopulating potential (46% vs 90% BM chimerism for WT and KO BM from tertiary recipient mice, respectively, p<0.001). Moreover, deletion of hematopoietic Scribble protects mice from 5-fluorouracil (5-FU)-mediated hematopoietic exhaustion and death by BM aplasia (>30 days median survival vs 16 days for KO mice, p<0.001). This set of functional data indicates that Scribble deficient HSC show enhanced long-term self-renewal capacity in stroma-dependent cultures and in vivo following transplantation or chemotherapeutic stress without the development of stem cell exhaustion or leukemia. Comparative transcriptional analysis of Scribble-deficient and WT HSC identified transcriptional regulation of the Hippo-dependent signaling pathway. We observed that Yap1 is polarized in a Scribble-dependent manner within WT HSC and co-localizes in the cytoplasm with the active form of the upstream inhibitory kinase, Lats1. Notably, deletion of Scribble in HSC leads to a disruption of the phosphorylated Lats1/Yap1 complex resulting in Yap1 translocation to the nucleus. Subsequently, cytoplasmic polarization of Yap1 can be restored when Scribble-deficient HSC are rescued with full length or PDZ domain containing mutants of Scribble. Expression of the N-terminus leucine-rich repeat (LRR) domain of Scribble successfully restored activation of Lats1 as well as phosphorylated Lats1 co-localization with Scribble, however, was not sufficient to prevent Yap1 translocation. Combined deletion of Yap1 and Taz using an inducible hematopoietic-specific model generates hematopoietic stem and progenitor cells (HSC/P) with enhanced cellular proliferation (HSC G0: 88 vs. 58%, p<0.0001; G1: 6 vs. 25%, p<0.0001; S/G2/M 6 vs. 18%, p<0.0001 for WT controls and Yap1/Taz double deficient BM, respectively) leading to an increase in colony forming units and early CAFC potential (2 and 3 fold respectively; p<0.0001). Yap1/Taz double KO HSC are prone to exhaustion evidenced by their inability to regenerate murine hematopoiesis after serial administration of 5-FU (12 days median survival vs 20 days for WT control mice, p<0.001). Triple deficiency of Scribble, Yap1 and Taz restores the effect of both Scribble and Yap1/Taz deficiencies to a normal hematopoietic response after myeloablation. Altogether, this data identifies Scribble as a negative regulator of cell cycle entry and self-renewal of HSC, and provides compelling evidence of key polarity and signaling programs used to maintain HSC quiescence that can be utilized as novel targets to improve HSC based transplantations and hematological cancer therapies. DisclosuresNo relevant conflicts of interest to declare.

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.

SVP-B5 peptide from Buthus martensii Karsch scorpion venom exerts hyperproliferative effects on irradiated hematopoietic cells.

Previous studies have demonstrated the radioprotective efficacy of scorpion venom peptide, fraction II (SVPII) from the venom of Buthus martensii Karsch. In the present study, the SVP-B5 polypeptide, which is one of the active components of SVPII, was purified using a two-step chromatographic process. SVP-B5 significantly promoted the proliferation of irradiated M-NFS-60 mouse-derived myelocytic leukemia cells. In addition, SVP-B5 effectively and persistently promoted hematopoietic recovery and expansion of hematopoietic cells after irradiation as demonstrated by cobblestone area forming cell and long-term bone marrow culture assays. Treatment of M-NFS-60 cells with SVP-B5 upregulated the expression of interleukin 3 receptor and activated the Janus kinase-2/signal transducer and activator of transcription 5 signaling pathway. In conclusion, the present study demonstrated that SVP-B5 has growth factor-like properties and may be used as a therapeutic modality in the recovery of severe myelosuppression, which is a common side effect of radiotherapy.

The effects of interleukin-1 beta and gamma-quantum braking radiation on mesenchymal progenitor cells

In murine bone-marrow stromal microenvironment cells and in human multipotent mesenchymal stromal cells (MMSCs), proinflammatory cytokine interleukin-1 beta (IL-1β) serves as a growth factor. In murine bone tissue, IL-1β expression increases in vivo after irradiation. Here, we have presented our evaluation of the effects of exogenous IL-1β on the expression of NF-kB transcription factors in human MMSCs and stromal layer cells of murine long-term bone marrow cultures (LTBMCs). The cytokine signaling pathway was also activated in murine LTBMC by braking electron radiation in doses of 3-12 Gy. The level of expression of genes that code for IL-1β, IL-1β type-I receptor and NF-kB and IKK protein families have been studied at different time points post exposure. In both human and murine stromal cells, exogenous IL-1β led to an increase in the level of expression of its own gene, while levels of expression of NF-kB and IKK gene families were not substantially changed. Nevertheless, in human cells, a significant correlation between levels of expression of IL-1β and all NF-kB family genes was detected. It points to a similarity in IL-1β signal pathways in mesenchymal and hematopoietic cells, where the posttranslational modifications of NF-kB transcription factors play a major role. The irradiation of murine LTBMC resulted in a transient increase in the expression of genes that code NF-kB transcription factors and IL-1β. These results indicate an important role of Rel, Rela, Relb, and Nfkb2 genes in the induction of IL-1β signal pathway in murine stromal cells. An increase in IL-1β expression after the irradiation of stromal cells may be related to both the induction of inflammation due to massive cell death and to a profound stimulation of the expression of this proinflammatory cytokine expression.

Radiation Resistance of Double Knockout (DKO) Smad3-/- Fancd2-/- (129/Sv) Mouse Bone Marrow Stromal Cell Lines

Introduction: Bone marrow progenitor cells from Fanconi Anemia (FA) patients have a hyperactive TGF-β signaling pathway which may explain hematopoietic stem cell depletion leading to bone marrow failure (Zhang, et al, Cell Stem Cell, 18:668-681, 2016). To determine whether blockade of TGF-B signaling by knockout of Smad3 normalized hematopoiesis in Fancd2-/- mice, we bred two double knockout mouse strains. The first (DKO#1) was derived by crossing C57BL/6 Fancd2 +/- mice with 129/Sv Smad3 +/- mice. The second (DKO#2) was derived by mating 129/Sv Fancd2 +/- mice with the same 129/Sv Smad3 +/- mice.Materials and Methods: Long term bone marrow cultures (LTBMCs) were established from DKO#1, DKO#2, parental mouse strains and F1 (129/Sv X C57Bl/6) control mice for DKO#1, 129/Sv Smad3 -/-, and C57BL/6 Smad3 -/- mice . The cultures were scored for weekly numbers of cobblestone islands as an indicator of stem cells in the adherent layer, weekly production of nonadherent cells, and cells producing Day 7 and Day 14 CFU-GEMM .Bone marrow stromal cell lines were derived from the adherent layer of LTBMCs and radiosensitivity measured in clonogenic radiation survival curves. Cells were irradiated to doses of 0 to 8 Gy, plated in 4 well linbro plates, incubated for 7 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted. Western analysis of the cell lines quantitated expression levels of proteins involved in the TGF-β signaling pathway, and DNA double strand break repair by homologous recombination and nonhomologous end-joining.Results: Both DKO1 and DKO2 LTBMCs showed decreased duration and magnitude of hematopoiesis, thus being similar to that observed with both C57BL/6 Fancd2-/- and 129/Sv Fancd2-/- mouse marrow cultures. Both were significantly lower than that for control mouse or Smad3-/- (129/Sv) marrow cultures. As expected, radiation survival curves showed that both C57Bl/6 Fancd2-/- and 129/Sv Fancd2-/- marrow stromal cell lines were radiosensitive compared to control cell lines including: F1 control, C57BL/6 control, and 129/Sv control (Berhane, et al, Rad Res. 181:76-89, 2014, Berhane et.al, Rad Res 182:35-49, 2014). In contrast marrow stromal cell lines from Smad3-/- (129/Sv) marrow cultures were radioresistant (Epperly, et al, Rad Res 165:671-677, 2006). Fresh marrow CFU-GEMM from both DKO#1 and DKO#2 mice showed resistance to abrogation of colony formation by increasing concentrations of TGF-B, (similar to the 129/Sv Smad3-/- cell line). In contrast, cell lines from all controls and both Fancd2-/- mice showed clear TGF-B mediated inhibition of hemopoietic colony formation. In striking contrast to the above similarities between DKO#1 and DKO#2 mice, marrow stromal cell lines from DKO#1 were radiosensitive (like theirFancd2-/- parent) while those from DKO#2 were radioresistant (like their Smad3-/- parent). Thus, DKO#1 retained the C57Bl/6 Fancd2-/- genotype cell line radiosensitivity: Do of 1.41 ± 0.03 Gy and 1.45 ± 0.05 Gy respectively, and were more radiosensitive than the control F1 bone marrow stromal cell line (p = 0.0230 and 0.0418, respectively) and DKO#2 retained the 129/Sv Smad3-/- cell line genotype radioresistance (Do = 2.15 ± 0.13 Gy) compared to control 129/Sv stromal cells (Do = 1.86 ± 0.04, p = 0.0054). Western analysis revealed that p21 was elevated in DKO#2 but not DKO#1 marrow stromal cell lines.Conclusions: While marrow from both DKO#1 and DKO#2 mice showed resistance to TGF-B signaling consistent with their smad3-/- genotype, only DKO#1 stromal cells retained the radiosensitivity of their Fancd2-/- genotype. Reduced p21 in irradiated DKO#1 marrow stromal cells may have allowed procession through G to S phase causing reduced time for DNA repair and radiosensitivity. The irradiated DKO#2 cells may have been blocked by p21 from passing through the G1 checkpoint and may have allowed DNA strand break repair and radioresistance. These DKO mice and derived cell lines should be valuable for analysis of the interaction of TGF-B signaling and FA pathways.Supported by NIAID/NIH U19-AI068021 DisclosuresNo relevant conflicts of interest to declare.

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2-/- mouse long-term bone marrow cultures.

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2−/− (129/Sv), K14E7 Fancd2+/+, Fancd2−/−, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2−/− and Fancd2−/− LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2−/− and Fancd2−/− cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2−/− cultures. In contrast, Fancd2−/− mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2−/− mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2−/−, but not control Fancd2+/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2−/− genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.