Hematopoietic Stem Cell Failure Research Articles

Hematopoietic Defects In ACD/TPP1-Deficient Mice Reveal An Essential Role for the Shelterin Complex In Blood-Forming Stem Cell Homeostasis

AbstractAbstract 882Bone marrow failure syndromes can be caused by loss-of-function mutations in telomerase components. Besides the enzymatic activity of telomerase, telomere maintenance requires the protective function of the shelterin protein complex. This complex consists of six proteins that bind to the telomere and prevent an inappropriate DNA damage response from being elicited by exposed telomeric DNA. Within this complex, TPP1, encoded by the gene Acd, plays a central role in complex organization as it links elements that bind the double-stranded portion of the telomere to those that bind the single-stranded overhang. We report an essential function for the shelterin complex and specifically for TPP1 in the maintenance of hematopoietic stem cells (HSCs). Utilizing adrenocortical dysplasia (acd), a spontaneous autosomal recessive mouse mutation causing profoundly hypomorphic Acd expression, we identified phenotypic and functional abnormalities in fetal hematopoietic progenitors. acd mutant Fetal liver HSCs were larger, more granular, and expressed higher levels of the cell surface protein Sca-1, as compared to wild-type counterparts. BrdU incorporation analysis showed an accumulation of Acd-deficient progenitors in the G2M phase of the cell cycle, consistent with G2M arrest. Absolute quantification of fetal liver HSCs revealed a significant reduction in stem cell numbers in acd mutants as compared to control littermates. p53 deficiency rescued the HSC depletion phenotype, but induced a paradoxical exacerbation of the increased progenitor cell size and Sca-1 expression. These findings suggest that p53-dependent and p53-independent elements contribute to the acd HSC phenotype. In competitive repopulation assays, acd HSCs failed to provide tri-lineage hematopoietic reconstitution in lethally irradiated recipients, indicating a profound functional defect. To further study the effects of Acd deficiency in adult HSCs, we generated an Acd allele in which exons 3–8 are flanked by loxP sites, allowing conditional inactivation following Mx1-Cre induction via poly(I:C) administration. Acd inactivation resulted in rapid HSC depletion within 2 weeks after initiation of poly(I:C) injections. To assess whether Acd exerted exclusively cell-autonomous effects in HSCs, we transplanted Acdf/–Mx1-Cre+ HSCs into wild-type recipients. Poly(I:C)-mediated Acd inactivation in these bone marrow chimeric mice resulted in rapid HSC loss and failure of tri-lineage hematopoiesis in animals reconstituted with Acd-deficient bone marrow. These data indicate an exquisite cell-autonomous requirement for Acd/TPP1 in adult mouse HSCs. In view of the extremely rapid HSC loss after Acd inactivation, these observations cannot be explained by telomere shortening. Together, our findings represent the first report of an essential role for Acd/TPP1 and the shelterin complex in HSC maintenance and hematopoietic homeostasis, independently of telomerase function. Disclosures:No relevant conflicts of interest to declare.

Adult hematopoietic stem cells require NKAP for maintenance and survival

AbstractSteady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.

Chemotherapy-induced mesenchymal stem cell damage in patients with hematological malignancy

Hematopoietic recovery after high-dose chemotherapy (HDC) in the treatment of hematological diseases may be slow and/or incomplete. This is generally attributed to progressive hematopoietic stem cell failure, although defective hematopoiesis may be in part due to poor stromal function. Chemotherapy is known to damage mature bone marrow stromal cells in vitro, but the extent to which marrow mesenchymal stem cells (MSCs) are damaged by HDC in vivo is largely unknown. To address this question, the phenotype and functional properties of marrow MSCs derived from untreated and chemotherapeutically treated patients with hematological malignancy were compared. This study demonstrates a significant reduction in MSC expansion and MSC CD44 expression by MSCs derived from patients receiving HDC regimens, thus implicating potential disadvantages in the use of autologous MSCs in chemotherapeutically pretreated patients for future therapeutic strategies. The clinical importance of these HDC-induced defects we have observed could be determined through prospective randomized trials of the effects of MSC cotransplantation on hematopoietic recovery in the setting of HDC with and without hematopoietic stem cell rescue.

Mechanisms of hematopoietic stem cell aging

New blood cells are continually produced from the hematopoietic stem cells (HSCs) that reside in the bone marrow. Throughout the life-span of the organism, this stem cell reservoir sustains life. Although HSCs can persist in vivo longer than one life-span ( Harrison et al., 1978), with aging, HSC regenerative potential diminishes and skewing from lymphopoiesis toward myelopoiesis occurs. The expansion in the HSC pool with aging provides sufficient, yet abnormal, blood production. Examination of gene expression changes in aged HSCs has provided a link between aging and genomic instability. Furthermore, studies on the effects of reactive oxygen species (ROS) on HSC aging has given more insight into the reasons for HSC failure. Understanding of the interactions between niche cells and HSCs and changes in them with aging, may give us insights into the lineage skewing phenotype observed in the aged, and also other immune dysfunctions.

Cytokinesis Failure in Fanconi Anemia Pathway Deficient Murine Hematopoietic Stem Cells.

Abstract 495Fanconi Anemia (FA) is a rare recessive chromosomal-instability disorder characterized by congenital malformations, a high predisposition to cancer, and progressive bone marrow failure. FA is genetically heterogeneous and, to date, thirteen FA genes have been identified (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N). The thirteen encoded FA proteins cooperate in a common DNA repair pathway active during the Synthesis (S) phase of the cell cycle. DNA damage detected during replication results in the monoubiquitination of two FA proteins, FANCD2 and FANCI, that translocate into chromatin-associated DNA repair foci where they colocalize with downstream components of the pathway. Partial colocalization with BLM, the RecQ helicase mutated in Bloom's syndrome, has also been described. How disruption of this pathway leads to bone marrow failure is a critical unanswered question. Interestingly, FA cells also have abnormalities that suggest a defect in mitosis, including micronuclei and multinucleation. The objectives of this study were to 1) investigate the role of the FA pathway in normal mitosis and 2) determine whether defects in this function underlie the bone marrow failure of FA patients. For this study, we used HeLa cells transiently or stably knocked down for FA genes, FA patient derived cell lines and hematopoietic stem cells from Fanconi mice models generated in our laboratory (Fancd2-/- and Fancg-/-). First, a polyclonal antibody was raised against FANCI and, together with an anti-FANCD2 antibody, used to investigate the localization of the FANCD2-I complex throughout the cell cycle by immunostaining. FANCI and FANCD2 colocalized to discrete foci on condensed chromosomes in a population of cells in Mitosis (M) phase, consistent with results of Chan et al. (Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol. 2009;11:753-760), Naim and Rosselli (The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol. 2009;11:761-768). These foci were dependent on an intact FA pathway, but did not localize at centromeres and did not increase when the spindle assembly checkpoint was challenged. By immunofluorescence, we showed an increase in the presence of Hoechst positive DNA bridges and PICH positive / BLM positive DNA bridges (Hoechst positive and negative) in anaphase and telophase of FA deficient cells compared to FA proficient cells. This increase of DNA bridges between separating sister chromatids in FA deficient cells correlated with an increase of multinucleated cells. Multinuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Furthermore, inhibition of apoptosis increased the number of binucleated cells, suggesting that cytokinesis failure led to apoptosis. Importantly, an increase in binucleated cells was also observed in the hematopoietic stem cells population from Fancd2-/- and Fancg-/- mice, compared to wild-type sibling mice, and this increase correlated with elevated apoptosis in those cells. Based on these new findings, we conclude that the Fanconi pathway is required for normal mitosis and hypothesize that apoptosis induced by cytokinesis failure of hematopoietic stem cells may cause the bone marrow failure commonly found in FA patients. Disclosures:No relevant conflicts of interest to declare.

Regulation of Reactive Oxygen Species and Genomic Stability in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are defined by their ability both to self-renew and to give rise to fresh blood cells throughout the lifetime of an animal. The failure of HSCs to self-renew during aging is believed to depend on several intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) factors. In this review, we focus on how dysregulation of reactive oxygen species (ROS) and disruptions of genomic stability can impair HSC functions. Recently, it was shown that long-term self-renewing HSCs normally possess low levels of intracellular ROS. However, when intracellular ROS levels become excessive, they cause senescence or apoptosis, resulting in a failure of HSC self-renewal. Repression of intracellular ROS levels in HSCs by treatment with an antioxidant that scavenges ROS can rescue HSC functions, indicating that excess ROS levels are at the root of HSC failure. Products of numerous genes that are involved in either DNA-damage responses or longevity-related signaling contribute to the maintenance of the HSC self-renewal capacity. Further investigations on the molecular mechanisms of ROS regulation and on the manipulation of excess ROS levels could lead to the development of novel therapeutics for hematopoietic diseases, regenerative medicine, and the prevention of leukemia.

S-phase fraction as a useful marker for prognosis and therapeutic response in patients with aplastic anemia

The functional definition of aplastic anemia (AA) is the failure of hematopoietic stem cells to proliferate. The aim of the present study was to analyze the S-phase fraction (SPF) (proliferative activity) in patients with AA at diagnosis to explore its relationship with disease characteristics and its value in discriminating among patients with different prognoses. We also investigated whether the SPF value influenced the response to immunosuppressive therapy in AA patients. The analysis of SPF at the time of diagnosis was carried out by flow cytometry on peripheral blood samples from 53 consecutive patients with AA and 30 age- and sex-matched controls. All patients were given cyclosporine and followed up periodically to determine response to therapy. Based on the median SPF, AA patients were divided into two groups: patients with SPF < 0.59% (n = 27) and patients with SPF > 0.59% (n = 26). An SPF > 0.59% was associated with advanced age (P = .02) and elevated serum LDH level (P = .01). Patients with an SPF > 0.59% also had a higher incidence of paroxysmal nocturnal hemoglobinuria and cytogenetic abnormalities. During a median follow-up of 18 months, 3.7% of patients with SPF < or = 0.59 and 11.5% of patients with SPF > 0.59% developed dysplasia and one patient with SPF > 0.59% converted into AML. A significantly higher (P = .018) overall response rate of 53.9% was found in patients with SPF > 0.59% versus 22.2% of patients with SPF < or = 0.59% at 6 months. Independently of the peripheral blood count, the SPF at diagnosis may provide information on the expected response to immunosuppressive therapy and the propensity for disease to evolve into MDS/AML. Hence, SPF may serve as an early indicator for the evolution of MDS/AML in patients with AA and thus contribute to therapeutic decisions.

Hematopoietic stem cell and multilineage defects generated by constitutive β-catenin activation

Gain of Wnt signaling through beta-catenin has been ascribed a critical function in the stimulation of hematopoietic stem cell self-renewal, whereas loss of beta-catenin is reportedly dispensable for hematopoiesis. Here we have used conditional mouse genetics and transplantation assays to demonstrate that constitutive activation of beta-catenin blocked multilineage differentiation, leading to the death of mice. Blood cell depletion was accompanied by failure of hematopoietic stem cells to repopulate irradiated hosts and to differentiate into mature cells. Activation of beta-catenin enforced cell cycle entry of hematopoietic stem cells, thus leading to exhaustion of the long-term stem cell pool. Our data suggest that fine-tuned Wnt stimulation is essential for hematopoiesis and is thus critical for therapeutic hematopoietic stem cell population expansion.

Idiopathic CD4+ Lymphocytopenia May Be due to Decreased Bone Marrow Clonogenic Capability

Background: Idiopathic CD4+ lymphocytopenia is defined by a stable decrease of CD4+ T cells in the absence of any known cause of immune deficiency. The mechanisms responsible for the immunological impairment are still unknown, but a regenerative failure of hematopoietic stem/progenitor cells has been hypothesized. Methods: We evaluated in the bone marrow (BM) of 5 patients with idiopathic CD4+ lymphocytopenia the phenotype of BM progenitor cells, their differentiation capacity with colony-forming cells and long-term culture-initiating cell assays, in parallel with the spontaneous IL-7 production in the patient sera. Results: Compared with controls, a regenerative failure of hematopoietic stem cells has been observed, both in ‘committed’ and in ‘uncommitted’ progenitor cells, despite high IL-7 serum levels. The percentage of phenotypically primitive CD34+CD38–DR+ cells (this includes the lymphoid precursor cells) was decreased, suggesting an involvement of the more primitive BM compartment in the de novo T cell generation. Conclusions: Despite the low number of patients, due to the low incidence of the disease, the decrease of primitive precursors sustains the possibility that diminished stem cell precursors might contribute to the development of CD4+ T cell depletion.

Hmgb3 deficiency deregulates proliferation and differentiation of common lymphoid and myeloid progenitors

AbstractHmgb3 is an X-linked member of a family of chromatin-binding proteins that is expressed in primitive hematopoietic cells capable of long-term hematopoietic repopulation. To examine the role of Hmgb3 in adult hematopoiesis, we generated Hmgb3-deficient (Hmgb3–/Y) mice, which are viable but erythrocythemic. Hmgb3–/Y mice contain normal numbers of hematopoietic stem cells (HSCs), which generate fewer than normal numbers of common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) and greater than normal numbers of more mature progenitors. Although fewer Hmgb3–/Y primitive progenitor cells are in the G2/M cell cycle phase, bromodeoxyuridine (BrdU) incorporation demonstrated enhanced proliferation compared with their wild-type counterparts. Hmgb3–/Y HSCs have increased levels of Gata-2 and c-myb mRNA. We propose that Hmgb3 deficiency leads to a failure of HSCs to expand into normal numbers of CLPs and CMPs. This defect is compensated for by the ability of Hmgb3–/Y progenitors to expand rapidly and differentiate into normal numbers of hematopoietic cells.

Lack of Functional DNA-PKcs and DNA Double-Strand Break Repair Leads to a Repopulating Stem Cell Defect in Scid Mice.

Intact function of DNA repair gene is required for maintenance of genomic stability and long term survival of stem cells. We hypothesize that DNA-PKcs, a key factor for DNA double-strand break (DSB) repair, is critical for hematopoietic stem cell (HSC) function. Expression level of DNA-PKcs mRNA monitored by RT-PCR was high in kit+lin− and sca+lin− cells, low in sca+kit+lin− cells and not seen in lin+ cells, implying its role in highly proliferative progenitors. To assess the function of HSCs deficient in DSB repair, serial transplantation capacity of scid (DNA-PKcs−/−) BM cells into lethally irradiated recipients was compared to wildtype BM. Primary transplants of scid BM died after treatment with 2Gy irradiation 4 wks post-transplantation (n=3). In contrast, parental scid mice survived 3Gy irradiation, implying radiation hypersensitivity of scid BM cells after transplantation. No changes were found in the telomere length, cell cycle distribution and apoptosis between the wildtype and scid BM cells after primary transplantation. Scid BM cells failed to repopulate recipients after the third round of transplantation (n=8). To assess competitive repopulating capacity, mixtures of wildtype and scid cells were transplanted into lethally irradiated recipients. BM CFU of primary recipients were predominantly wildtype (8 mice for C3H background, total CFU=262; 5 mice for C56B/6 background, total CFU=336; n>15 per mouse). Scid cells with two independent genetic backgrounds caused consistent repopulation defects, confirming repopulation defect is caused by DNA-PKcs deficiency. All five primary recipients with C56B/6 background was repopulated predominantly by wildtype CFU (wt CFU 93±5% vs. wt CFU of input; 60±31%, p<10−4). Six of eight primary recipients with C3H background had BM cells repopulated by wildtype CFUs (wt CFU 93±9 % vs. wt CFU of input; 65+13 %, p<10−4), and two of eight primary recipients (wt CFU 67±10 %, p>0.05) had BM cells repopulated similar to donor mixture of wildtype and scid. BM cells of all eight primary recipient mice with C3H background were transplanted into secondary recipients. In all cases, including recipients of the primary cells with the mixed chimera, most BM CFU of secondary recipients originated from wildtype (wt CFU 96±7.8 %, total 16 mice, total CFU=511, and CFU=192 from the mixed chimera). Sca+kit+lin− cells were isolated from the secondary recipients, cultured for 2wks and genotyped. All sca+kit+lin− cells were originated from wildtype (total n=73, 6 mice), implying DNA-PKcs function for HSC proliferation. This confirmed that primary recipients had reconstituted with 100% wildtype HSCs and that the mixed chimera reverted to 100% wildtype. Frequency of sca+kit+lin− cells in scid BM was significantly higher than wildtype (scid 1.94±0.5x10−4, n=4 vs. wt 0.92±0.4x10−4, n=4; p=0.017). Frequency of sca+kit+lin− cells in scid secondary recipients became similar to wildtype secondary recipients (scid 0.61±0.2x10−4, n=4 vs. wt 0.48±0.02x10−4, n=3; p=0.25), implying decreased self-renewal of scid HSCs during repetitive transplantation. This indicates that deficiency in DNA double-strand break repair (scid) leads to HSC failure during repetitive transplantation. Thus, intact DNA repair is essential for maintenance and genomic stability of HSCs.

Rescue of lethal c-KitW/Wmice by erythropoietin

AbstractHomozygous natural white-spotted (W) mutations in the gene encoding the receptor tyrosine kinase c-Kit are associated with hypoplastic bone marrow, severe macrocytic anemia, and lethality during early postnatal life. c-KitW/Wmice can be rescued by wild-type hematopoietic stem cells (HSCs), but it is not known whether the lethality of c-KitW/Wmice is the result of HSC failure or defects specific for erythropoiesis. Here we show that transgenic expression of erythropoietin (EPO) can overcome the lethality caused by the c-KitW/Wmutation. In W mutant mice rescued by EPO, termed WEPO, erythrocyte colony-forming units (CFU-Es) are rescued to normal frequencies. Hence, Epo receptor signals can partially bypass the strict requirement for c-Kit signaling in erythropoiesis in the absence of c-Kit in vivo. Using a series of W and rescue mouse strains, we define here the erythropoietic threshold permitting survival in vivo. The lethality of c-KitW/Wmice has precluded analysis of this crucial receptor-ligand pair in adult stem/progenitor cells. Our strategy to generate viable c-KitW/Wmice will be useful to analyze the role of this important receptor tyrosine kinase in adult life in vivo. (Blood. 2004; 104:1688-1695)

Cancer predisposition and hematopoietic failure in Rad50(S/S) mice.

Mre11, Rad50, and Nbs1 function in a protein complex that is central to the metabolism of chromosome breaks. Null mutants of each are inviable. We demonstrate here that hypomorphic Rad50 mutant mice (Rad50(S/S) mice) exhibited growth defects and cancer predisposition. Rad50(S/S) mice died with complete bone marrow depletion as a result of progressive hematopoietic stem cell failure. Similar attrition occurred in spermatogenic cells. In both contexts, attrition was substantially mitigated by p53 deficiency, whereas the tumor latency of p53(-/-) and p53(+/-) animals was reduced by Rad50(S/S). Indices of genotoxic stress and chromosomal rearrangements were evident in Rad50(S/S) cultured cells, as well as in Rad50(S/S) and p53(-/-) Rad50(S/S) lymphomas, suggesting that the Rad50(S/S) phenotype was attributable to chromosomal instability. These outcomes were not associated with overt defects in the Mre11 complex's previously established double strand break repair and cell cycle checkpoint regulation functions. The data indicate that even subtle perturbation of Mre11 complex functions results in severe genotoxic stress, and that the complex is critically important for homeostasis of proliferative tissues.