Cobblestone Area-forming Cells Research Articles

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p<0.0001 and p<0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values <0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

Targeting S100A9 in the Inflammatory Myelodysplastic Hematopoietic Niche Reprograms the Functional Properties of CD271+ Mesenchymal Stromal Cells

Myelodysplastic syndromes (MDS) belong to the most common hematological malignancies in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An involvement of the bone marrow microenvironment (BMME) in disease initiation and progression is indisputable. The profound immune dysregulation and sustained inflammation, essentially mediated by mesenchymal stromal cells (MSCs), are further factors contributing to ineffective hematopoiesis and driving disease progression. The alarmin S100A9 is a TLR4 and CD33 ligand that acts as key player for the inflammasome activation in the MDS BM. The novel small molecular drug Tasquinimod (TASQ, Active Biotech) is a S100A9 inhibitor and has demonstrated antitumor and immunomodulatory properties in a broad range of solid tumors; however, little is known about its effects in myeloid malignancies. Here, we aim to decipher the effects of TASQ on MDS MSC subpopulations in more detail. Therefore, MSCs were isolated from BM aspirates ('asp-MSCs', n=12) or biopsies ('bp-MSCs', n=5) of patients with either low-risk MDS, CMML or age-adjusted healthy donors and were exposed to TASQ (10 µM) in vitro. Flow cytometry analysis of the MSC markers CD44, CD73, CD90, CD105, and CD146 did not show significant differences in neither asp- nor bp-MSCs after treatment. Interestingly, CD271, which is hard to detect in asp-MSCs, was decreased between 40% and 70% in bp-MSCs after TASQ treatment. This was accompanied by an inhibited expression of the checkpoint inhibitor PD-L1. The MDS BM has a higher potency for adipogenic differentiation which further supports the inflammatory conditions and the suppression of normal hematopoiesis. Treatment with TASQ resulted in about 50% inhibition of adipogenesis as demonstrated by significantly lower expression of adipocyte protein 2 in differentiated asp- and bp-MSCs. To investigate possible metabolic modulations of MSCs by TASQ, the oxygen consumption rate was measured as a readout of mitochondrial function by using the Cell Mito Stress Test (Agilent Technologies) in a XF96 analyzer. The basal and the maximal respiratory rate as well as ATP production were increased in TASQ-treated bp-MSC cultures suggesting a positive effect for their bioenergetic capacity. This was supported by detection of higher levels of glutamate dehydrogenase 1, a key enzyme in glutaminolysis converting glutamate to α-ketoglutarate for entry into the TCA cycle, and of the glutamine transporter Slc1a3 in TASQ-treated cells. NMR analyses revealed increased levels of glutamate and citrate as well as of several lipids, such as free cholesterol LDL, phospholipids LDL-6 and Apo-B LDL6 in supernatants of bp-MSCs after TASQ treatment. To investigate the impact of TASQ on CD271 expression in more detail, we sorted CD45-/CD73+/CD90+/CD271+/- bp-MSCs. Gene expression analysis by RNA sequencing of sorted MSCs revealed differential expression of several pathways, e.g. an enrichment of inflammation related molecules in the CD271+ samples, whereby TASQ could abrogate this effect. Moreover, a cytokine array analysis of culture supernatants of sorted cells showed enhanced IFNg, IL1b and IL-6 secretion of CD271+ MSCs compared to CD271- and TASQ-treated cells. To evaluate the impact on the hematopoietic support of MSCs, we analyzed CD271-sorted bp-MSC co-cultures with CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed a significantly decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay for CD271+ MSCs, nominating this MSC subpopulation as responsible for the diminished hematopoietic support. Interestingly, the number of CAF-C and CFU-E/CFU-GM could be increased by TASQ pre-treatment of the stromal layer. In conclusion, CD271+ MSCs play a crucial role in the inflammatory MDS BMME. The inhibition by TASQ efficiently blocks adipogenic differentiation and secretion of pro-inflammatory cytokines, thereby modulating the MSC energetic profile and enhancing their potential to support hematopoiesis in vitro.

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions.We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes.The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied.MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA.Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun.Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression.The work is supported by the RFBR, project 19-015-00280. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

IntroductionMyelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species.Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies.AimWe investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro.MethodsImmunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system.MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs.ResultsImmunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro.Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation.Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%.To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM.ConclusionIn summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. DisclosuresBalaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells

BackgroundUmbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo.MethodsUCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed.ResultsMSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells.ConclusionsAT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

Pharmacological Inhibition of Luteinizing Hormone Signaling Promotes HSC Quiescence and Hematopoietic Regeneration

During hematopoiesis there is a balance between divergent functions such as self-renewal, differentiation, and proliferation. This process allows for continued blood formation throughout life as well as hematopoietic regeneration after periods of stress. It was recently shown that suppression of sex steroids, in addition to promoting T and B cell lymphopoiesis, promotes hematopoietic rejuvenation by mediating functional changes to HSC pool (Khong et al., 2015).Although those changes suggested a direct impact of testosterone on HSC function, when we examined the expression of hormone receptors on the hematopoietic compartment, we found almost no detectable expression of the androgen receptor. In contrast, expression of luteinizing hormone (LHR) was highly enriched on HSCs with nearly absent expression in downstream hematopoietic progenitor cells as well as in the stromal cell compartment. Consistent with the mouse data, high expression of LHR was also found in human HSCs (Fig. 1A). Given the peculiar expression of the LHR on HSCs, we sought to examine the direct effect of LH on HSC function. LH significantly enhanced mouse HSC colony formation in cobblestone area-forming cell and colony-forming cell assays. In addition, LH significantly expand human HSCs in a stroma-free culture system, without impacting on their colony-forming potential (Fig. 1B).Given the direct effects of LH on HSCs, we investigated if blocking LH could attenuate HSC proliferation in two different models of hematopoietic stress that force HSCs out of their quiescent status. Pharmacological suppression of LH using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) inhibits HSCs from entry into cell cycle after Poly I:C and sub-lethal dose of total body irradiation (SL-TBI) exposure. Importantly, LH inhibition also significantly improved the proportion of live HSCs after SL-TBI (Fig. 1C). To gain some insights into the direct impact of LH on HSCs, we generated a hematopoietic chimera using KiLHRD582G mice, which have a constitutively-active D582G mutation in the Lhr gene. While there was no difference in primary reconstitution in mice receiving WT or KiLHRD582G HSCs, we found that mice reconstituted with KiLHRD582G and subsequently exposed to SL-TBI showed significantly higher proportion of apoptotic HSC compared to mice reconstituted with WT HSCs (Fig. 1D). To elucidate the molecular changes induced by LH inhibition, we assessed the expression of genes associated with quiescence, proliferation, DNA damage and apoptosis in HSC after SL-TBI. We found that LH suppression promoted higher levels of Hes1, Cdkn1a (p21), Bcl2 and Mcl1, genes previously identified to enforce HSC quiescence and survival (Fig. 1E). Consistent with these data, mice treated with the LHRH-Ant showed improved survival when exposed to successive doses of 5-fluorouracil, a well-established method to challenge HSC quiescence and self-renewal potential. It has been previously showing that promoting quiescence early after hematopoietic insults can mitigate HSC exhaustion and, ultimately, promote hematopoietic recovery and mouse survival. Mice receiving LHRH-Ant 24h after lethal TBI (840cGy) exposure showed a significant increase in survival compared to control animals. In fact, LH suppression spared LT-HSCs from radiation toxicity thus promoting hematopoietic recovery (Fig. 1F). Consistent with a negative role of LH on HSC recovery after radiation, mice deficient for LHR had a statistically significant increase in survival when exposed to L-TBI compared to control littermate.In conclusion our studies demonstrate that LH can directly regulate HSC quiescence, and that LH inhibition with a LHRH-Ant can be used as a radiation mitigation strategy. [Display omitted] DisclosuresJenq:Seres: Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. van den Brink:Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; Therakos Institute: Other: Speaking engagement; PureTech Health: Consultancy.

Functional and Phenotypic Characterization of Cord Blood and Bone Marrow Subsets Expressing FLT3 (CD135) Receptor Tyrosine Kinase

AbstractThe class III receptor tyrosine kinase FLT3/FLK2 (FLT3; CD135) represents an important molecule involved in early steps of hematopoiesis. Here we compare cell-surface expression of FLT3 on bone marrow (BM) and cord blood (CB) cells using monoclonal antibodies (MoAbs) specific for the extracellular domain of human FLT3. Flow cytometric analysis of MACS-purified BM and CB cells showed that 63% to 82% of BM CD34+ and 88% to 95% of the CB CD34+ cells coexpress FLT3. Clonogenic assays and morphological characterization of FACS-sorted BM CD34+ cells demonstrate that colony-forming unit–granulocyte-macrophage (CFU-GM) and immature myelo-monocytic precursor cells are enriched in the subpopulation staining most brightly with the FLT3 MoAb whereas the majority of the burst-forming units-erythroid (BTU-E) and small cells with lymphoid morphology are found in the FLT3− population. In contrast, statistically indistinguishable proportions of CFU-granulocyte-erythrocyte-megakaryocyte-macrophage (CFU-GEMM) and more primitive cobblestone area forming cells (CAFC) were detected in both fractions, albeit the FLT3+ fraction consistently showed more CAFC activity than the FLT3− fraction. Although in both, BM and CB the majority of CD34+CD117+ (KIT+), CD34+CD90+ (Thy-1+), and CD34+CD109+ cells coexpress FLT3, three-color phenotypic analyses are consistent with the functional findings and suggest that the most primitive cells defined as CD34+CD38−, CD34+CD71low, CD34+HLA-DR−, CD34+CD117low, CD34+CD90+, and CD34+CD109+ express low levels of cell-surface FLT3 and were therefore not enriched to a statistically significant extent with the bright versus negative sorting scheme. Thus, clear segregation of the most primitive progenitors from BM CD34+ cells was confounded by low apparent levels of FLT3 cell-surface expression on these cells, whereas myeloid progenitors unambiguously segregated with the FLT3 brightest cells and erythroid progenitors with the FLT3 dimmest. Additional phenotypic analyses using MoAbs against progenitor/stem cell markers including the mucinlike molecule MGC-24v (CD164), the receptor tyrosine kinases TIE, FMS (CD115), and KIT (CD117) further illustrate the differences in surface antigen expression profiles of BM and CB CD34+cells. Notably, CD115 is rarely detected on CB CD34+cells, whereas 20% to 25% of the BM CD34+FLT3+ cells are CD115+. Furthermore, 80% to 95% of the CB CD34+CD117+ but only 60% to 75% of the BM CD34+CD117+ cells coexpress FLT3. Only a negligible amount of CD34+CD19+ are detected in CB, while in BM 20% to 30% of CD34+CD19+ presumed pro/pre-B cells coexpress FLT3. In contrast, the majority of the CD34+CD164+ and CD34+TIE+ subsets in both CB and BM coexpress FLT3. Analysis of unseparated cells showed that FLT3 expression is not restricted to CD34+ subsets. About 65% to 70% of lymphocyte-gated BM CD34−FLT3+ cells are positive for the monocytic marker CD115 whereas 25% to 30% of these cells consist of CD10 expressing B-cell precursors. Finally, CD34−monocytes in BM, CB, and PB express FLT3 whereas granulocytes are FLT3−. Our data show that detectable FLT3 appears first at low levels on the surface of primitive multilineage progenitor cells and disappears during defined stages of B-cell development, but is upregulated and maintained during monocytic maturation. © 1997 by The American Society of Hematology.

PKC-Epsilon Overexpression Is Associated with Poor Outcomes in AML and Promotes Chemoresistance and Hematopoietic Stem Cell Quiescence

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies which are characterized by the accumulation of clonal, abnormally differentiated blasts in the bone marrow. For most patients, prognosis remains poor as a large proportion relapse following a period of complete remission. Protein kinase c epsilon (PKCε) is a pleiotropic regulator of cell proliferation, differentiation, apoptotic and survival signaling. Uniquely in the PKC family, PKCε also has demonstrated oncogenic properties and high expression of PKCε has been associated with poor patient outcomes, disease aggressiveness, and chemoresistance in solid cancers. A role for PKCε has also been implicated in AML; however, its clinical significance has not been elucidated. To address this, we first determined whether PKCε was aberrantly expressed in AML. Patient samples were collected from the MRC AML14 and AML15 clinical trials and quantitative estimation of PKCε at the protein and mRNA level was conducted. PKCε overexpression (&gt;2SD of mean of controls) occurred in 32% (43/132) of patients based on mRNA expression and 37% (26/70) of samples based on protein expression, compared to normal human CD34+ cells. In addition, a positive correlation between PKCε mRNA and protein expression was established (R=0.64, p&lt;0.01). We next evaluated the clinical impact of high PKCε expression in AML using the TCGA RNAseq dataset. High PKCε expression (&gt;median), correlated with significantly worse overall survival (95% CI (34-54); p&lt;0.05), with the median survival for being 11 months, compared with 24 months for patients with low PKCε expression. High PKCε expression did not correlate with age, molecular abnormality, gender, or WBC, and is therefore likely to be an independent prognostic factor. To study the mechanisms through which PKCε could contribute to poor outcomes in AML, the influence of PKCε overexpression on the chemosensitivity of AML cell lines was explored. In the AML cell lines investigated (U937, HEL, TF-1 and NOMO-1), PKCε overexpression was associated with significantly increased daunorubicin (DNR) resistance in U937 and HEL cells (2.6-fold, n=4; p&lt;0.05 for both lines) without affecting their sensitivity to cytarabine, suggesting that PKCε can selectively promote resistance to DNR. The therapeutic mechanism of DNR is, in part, accounted for by the generation of reactive oxygen species (ROS) and we are currently investigating whether PKCε expression protects against ROS-generating agents as has previously been suggested. We next examined the effect of PKCε on normal human hematopoiesis. RNAseq data from both human and murine hematopoietic cells show that PKCε is endogenously expressed in normal hematopoietic cells, with the highest levels of expression occurring in the HSC compartment and monocytes. Overexpression of PKCε in human CD34+ progenitor cells promoted myeloid differentiation of unilineage monocyte and granulocyte progenitors which showed an increase in expression CD14 and CD15, with CD11b being significantly upregulated on monocytes and granulocytes by 1.4-fold±0.3 (n=4, p&lt;0.05) and 1.6-fold±0.2 (n=3, p&lt;0.05), respectively. Conversely, shRNA mediated PKCε knockdown in these cells reduced the expression of these myeloid markers, supporting a role for PKCε in developmental regulation of the myeloid lineage. We next examined the effect of PKCε overexpression on the self-renewal and hematopoietic activity of human HSC (CD34+CD38-). Transduced HSC were scored for colony formation, in addition to being seeded onto MS5 stroma and assessed for their capacity to generate cobblestone area forming cells (CAFC). PKCε had no significant impact on colony forming efficiency but ablated CAFC formation suggesting that PKCε selectively promotes quiescence of HSC (n=2). Since quiescence of leukemic stem cells (LSC) is widely thought to contribute to relapse in AML, this may represent a further mechanism through which PKCε expression confers a poor outcome in this disease and we are currently investigating this. In summary, we show that elevated expression of PKCε occurs frequently in AML and our data indicate it is an independent prognostic marker of poor outcome in this disease. Experimental models suggest that overexpression of PKCε may contribute to treatment resistance through promoting DNR resistance and potentially by promoting LSC quiescence. Disclosures Knapper: Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Honoraria; Tolero: Membership on an entity's Board of Directors or advisory committees.

Tet2 regulates the function of mesenchymal stem cells

Tet2 (member 2 of the Tet family) plays an important role in DNA demethylation modification, epigenetic regulation, and hematopoiesis. In our previous study, we found that Tet2 knockout mice progressively developed lymphocytic leukemia and myeloid leukemia with aging. However，the role of Tet2 in bone marrow microenvironment is unclear. Here in this study, we found that more Tet2-/- mesenchymal stem cells (MSCs) from bone marrow were in the G2/M cell cycle stages. The division time of Tet2-/- MSCs was shorter than that of the control cells. The growth rate of Tet2-/- MSCs was accelerated. The cobblestone area-forming cells assay (CAFC) showed that Tet2 knockout MSCs supported the expansion of hematopoietic stem cells (HSCs) and the differentiation of HSCs was skewed towards myeloid cells. Through the dot blotting experiment, we found that the total methylation level was increased in Tet2-/- bone marrow cells (BM). We used the methylation-chip to analyze the methylation level of Tet2-/- bone marrow cells and found that the level of methylation was increased in the transcriptional starting area (TSS), exons (EXONS) and 3' untranslated region (3' UTR). Moreover, we found that the cytokines secreted by Tet2-/- MSCs, such as IL-8 and IL-18, were decreased. While the expressions of GM-CSF and CCL-3, which supported hematopoietic stem cells to differentiate to myeloid cells, were increased in Tet2-/- MSCs. Our results demonstrated that Tet2 regulates MSCs to support hematopoiesis.

Hematopoiesis-supportive function of growth-arrested human adipose-tissue stromal cells under physiological hypoxia

Exvivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of exvivo expansion is strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O2. Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell-cell (ICAM1, HCAM/CD44) and cell-matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O2 (20%). The number of ICAM-1 positive ASCs was increased under low O2 as well. These alterations contributed into the exvivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p<0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration invivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion.

TGFBI Expressed by Bone Marrow Niche Cells and Hematopoietic Stem and Progenitor Cells Regulates Hematopoiesis.

The interactions of hematopoietic stem and progenitor cells (HSPCs) with extracellular matrix (ECM) components and cells from the bone marrow (BM) microenvironment control their homeostasis. Regenerative BM conditions can induce expression of the ECM protein transforming growth factor beta-induced gene H3 (TGFBI or BIGH3) in murine HSPCs. In this study, we examined how increased or reduced TGFBI expression in human HSPCs and BM mesenchymal stromal cells (MSCs) affects HSPC maintenance, differentiation, and migration. HSPCs that overexpressed TGFBI showed accelerated megakaryopoiesis, whereas granulocyte differentiation and proliferation of granulocyte, erythrocyte, and monocyte cultures were reduced. In addition, both upregulation and downregulation of TGFBI expression impaired HSPC colony-forming capacity of HSPCs. Interestingly, the colony-forming capacity of HSPCs with reduced TGFBI levels was increased after long-term co-culture with MSCs, as measured by long-term culture-colony forming cell (LTC-CFC) formation. Moreover, TGFBI downregulation in HSPCs resulted in increased cobblestone area-forming cell (CAFC) frequency, a measure for hematopoietic stem cell (HSC) capacity. Concordantly, TGFBI upregulation in HSPCs resulted in a decrease of CAFC and LTC-CFC frequency. These results indicate that reduced TGFBI levels in HSPCs enhanced HSC maintenance, but only in the presence of MSCs. In addition, reduced levels of TGFBI in MSCs affected MSC/HSPC interaction, as observed by an increased migration of HSPCs under the stromal layer. In conclusion, tight regulation of TGFBI expression in the BM niche is essential for balanced HSPC proliferation and differentiation.

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells.

Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.

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.

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.

The Role of Cbx Proteins in Human Benign and Malignant Hematopoiesis

Introduction:Hematopoietic stem cells (HSCs) need to properly balance self-renewal and differentiation to prevent malignant transformation or HSC exhaustion. We recently showed that the presence of specific Cbx proteins in the Polycomb Repressive Complex 1 (PRC1) plays a crucial role in balancing self-renewal and differentiation of murine HSCs. Whereas Cbx7 induces self-renewal, Cbx4 and Cbx8 instead induce differentiation. In this project, we investigate the role of various CBX Polycomb proteins in human normal and malignant hematopoiesis. We aim to identify genes which are controlled by CBX7 in normal and malignant human hematopoiesis.Methods:We overexpressed CBX2, -4, -6, -7 and -8 in human CD34+HSPCs. We assessed functional consequences by measuring cobblestone area-forming cell (CAFC), colony-forming unit (CFU) frequencies, and performed stem cell xenotransplantation studies in NSG mice. To identify genes which are controlled by CBX7 or CBX8, we performed RNA- and Chip-Seq in CD34+ HSPCs. To explore the role of CBX7 in leukemic cells, we performed short hairpin RNA-mediated knockdown of protein expression in leukemic cell lines.Results:Overexpression of CBX7 and CBX8 in human CD34+ HSPCs led to a significant 5-10-fold increase of week 5 CAFC (fold increase: 9,62 for CBX7; 4,92 for CBX8) and 1,5-fold increase CFU-frequencies (fold increase: 1,32 for CBX7; 1,41 for CBX8) after 14 days. In contrast, overexpression of CBX4 and CBX2 led to equal or decreased CAFC- and CFU-frequencies. Transplantation of CBX7 overexpressing human CD34+ HSPCs in NSG mice led to higher long-term engraftment in comparison to the empty vector control (p<0,05 after 18 weeks). Even after one week of in vitro culture of CBX7-overexpressing CD34+cells significant engraftment levels were obtained. Conversely, downregulation of CBX7 in various leukemic cell lines resulted in markedly decreased proliferation and strongly induced differentiation. Expression analysis showed that genes that are upregulated upon CBX7 overexpression are preferentially expressed in primitive stem cells, and are repressed in more differentiated cell types.Conclusions:Our study indicates that CBX7 and CBX8 regulate self-renewal of human HSPCs. Furthermore, our data show that repression of CBX7 markedly inhibits proliferation of leukemic cells and can release the differentiation block of leukemic cells in vitro. Collectively our results indicate that targeting CBX7 may be a viable strategy to induce terminal differentiation of leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Suppression of Luteinizing Hormone Enhances HSC Recovery after Hematopoietic Injuries

▪A tightly regulated network of intrinsic and extrinsic signaling pathways exists to preserve HSC pool size and function. This is particularly relevant during hematopoietic injuries when dormant HSCs transiently start to proliferate to replenish blood cells; as unbalanced HSC proliferation can lead to stem cell exhaustion, long-term myelosuppression and death. Although there has been growing interest in how circulating sex hormones influence HSC function (Nakada et al., 2014; Sanchez-Aguilera et al., 2014), this pathway remains poorly understood. Here we describe a heretofore unknown role for the upstream hormone regulator, luteinizing hormone (LH), in regulating HSC biology.We found that both human and mouse HSCs highly expressed the LH receptor, and its expression was decreased or nearly absent in downstream progenitors (Figure 1a). LH significantly promoted HSC colony forming potential in cobblestone area-forming cell and colony-forming cell assays that, together with expression of the receptor, suggested that LH increased HSC expansion in vitro by acting directly on the most primitive HSCs.To investigate whether LH levels could impact on HSC pool size during hematopoietic stress in vivo, we challenged mice using models that force HSCs out of their quiescent status, Poly I:C and sub-lethal dose of total body irradiation (SL-TBI, 550cGy). We found that ablation of LH production using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) retained significantly more HSCs in G0 in both models (Figure 1b,c).Previous reports have shown that induction of HSC quiescence after high-dose irradiation correlates with increased hematopoietic recovery and enhanced mouse survival (Chen et al., 2008; Himburg et al., 2014; Johnson et al., 2010). Given its effectiveness in promoting HSC quiescence and the fact that LHRH-Ant are widely available and clinically approved, we hypothesized that LHRH-Ant could represent a rational non-cellular medical countermeasure for mitigating radiation injury and promoting hematopoietic regeneration when administered after hematopoietic insult. To test this hypothesis, we used a lethal TBI (L-TBI) dose of 840cGy that mediated lethality in more than 90% of B6 male mice. We found that pharmacological inhibition of LH using LHRH-Ant 24h after L-TBI spared the most primitive long term HSCs (Figure 1d) thus promoting hematopoietic recovery and mouse survival (Figure 1e). Consistent with our original hypothesis we also found a significantly higher proportion of Ki-67− quiescent HSCs in the LHRH-Ant-treated group with fewer proliferative HSCs compared to controls. Given the wide-ranging hormonal changes induced by LHRH suppression and the previously reported effects mediated by sex steroid ablation on hematopoietic stem/progenitor cell (HSPC) compartment (Khong et al., 2015), we next evaluated whether the LHRH-Ant effects on mouse survival after L-TBI were independent from the suppression of the downstream sex steroids. Administration of LHRH-Ant improved survival rates in surgically castrated mice following radiation injury, while surgical castration alone did not, indicating that the regenerative effects were independent from downstream sex steroids. To confirm whether the protective effects of LHRH-Ant treatment depended on suppression of LH, we administered the LH receptor agonist human chorionic gonadotropin (hCG) to LHRH-Ant treated mice that had been given L-TBI one-day prior. Consistent with our hypothesis, administration of hCG abrogated the beneficial effects of LHRH-Ant on survival after radiation injury.Taken together our studies showed that HSCs are a physiological target of LH, which promotes their proliferation. Furthermore, pharmacological inhibition of LH signaling using a single dose of an LHRH-Ant represents a rational and feasible approach to preserve the HSC pool after high dose radiation, thereby mitigating acute hematopoietic radiation syndrome. [Display omitted] DisclosuresVan Den Brink:Seres: Research Funding; Novartis: Consultancy; Regeneron: Consultancy; Flagship Ventures: Consultancy; Boehringer Ingelheim: Consultancy; Merck: Consultancy.

Low Doses of Oxygen Ion Irradiation Cause Acute Damage to Hematopoietic Cells in Mice.

One of the major health risks to astronauts is radiation on long-duration space missions. Space radiation from sun and galactic cosmic rays consists primarily of 85% protons, 14% helium nuclei and 1% high-energy high-charge (HZE) particles, such as oxygen (16O), carbon, silicon, and iron ions. HZE particles exhibit dense linear tracks of ionization associated with clustered DNA damage and often high relative biological effectiveness (RBE). Therefore, new knowledge of risks from HZE particle exposures must be obtained. In the present study, we investigated the acute effects of low doses of 16O irradiation on the hematopoietic system. Specifically, we exposed C57BL/6J mice to 0.1, 0.25 and 1.0 Gy whole body 16O (600 MeV/n) irradiation and examined the effects on peripheral blood (PB) cells, and bone marrow (BM) hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) at two weeks after the exposure. The results showed that the numbers of white blood cells, lymphocytes, monocytes, neutrophils and platelets were significantly decreased in PB after exposure to 1.0 Gy, but not to 0.1 or 0.25 Gy. However, both the frequency and number of HPCs and HSCs were reduced in a radiation dose-dependent manner in comparison to un-irradiated controls. Furthermore, HPCs and HSCs from irradiated mice exhibited a significant reduction in clonogenic function determined by the colony-forming and cobblestone area-forming cell assays. These acute adverse effects of 16O irradiation on HSCs coincided with an increased production of reactive oxygen species (ROS), enhanced cell cycle entry of quiescent HSCs, and increased DNA damage. However, none of the 16O exposures induced apoptosis in HSCs. These data suggest that exposure to low doses of 16O irradiation induces acute BM injury in a dose-dependent manner primarily via increasing ROS production, cell cycling, and DNA damage in HSCs. This finding may aid in developing novel strategies in the protection of the hematopoietic system from space radiation.

Ex vivo Culture System of Single Human Hematopoietic Stem Cell Used to Screan the Small Molecular Compounds

To explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC). By using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test. The effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05). The rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.

Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors

Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure.