Long-term Repopulation Ability Research Articles

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the invivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible forlifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration invivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.

In Vivo Selection Unmasks a Dormant Pool of Repopulating Hematopoietic Clones

Long-term clonal tracking studies utilizing hematopoietic stem and progenitor cells (HSPCs) in nonhuman primates receiving myeloablative transplantation demonstrate a successive pattern of repopulation: short-term repopulating cells are succeeded by long-term clones. However, the duration of short-term repopulation and the numbers of clones contributing to either short or long-term repopulation are unclear. Here, we tracked >11,000 unique clones in 8 pigtail macaques for up to 9 years following myeloablative transplantation with autologous, lentivirus gene-modified CD34+ HSPCs. Seven of these animals received cells expressing the P140K mutant methylguanine methyltransferase transgene, which is resistant to the combination of O6-benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy, thus conferring a selective advantage to gene-modified cells in vivo. After transplantation and before in vivo selection with O6BG/BCNU, we observed a successive pattern of hematopoietic reconstitution, with short-term clones declining within 100 days after transplantation. Within the first year after transplant, the percent of persistent clones varied from animal-to-animal, ranging from 8% to 54% of clones detected at a >1% frequency, and remained stable in the absence of selective pressure. Importantly, when animals engrafted with P140K-expressing cells were administered O6BG/BCNU we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving ≤12x106 CD34+ cells/kg at the time of transplant (n = 4), chemotherapy also induced a re-emergence of previously declined short-term repopulating clones or a stabilization (i.e. decreased fluctuation) of repopulating clones identified between 100 days and 1 year after transplant. However, in animals receiving robust cell doses, ≥35x106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. In one animal receiving 22x106 CD34+ cells/kg at transplant, chemotherapy administration beginning <1 year (253 days) after transplant induced clonal stability, which was maintained through two additional chemotherapy treatments. These data suggest that some short-term repopulating clones may have long-term repopulation ability, but revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess repopulating cells results in early dormancy of a large proportion of repopulating clones. Together, these findings suggest that previous estimates of HSPC frequency based on clone tracking are an underestimate of true graft repopulation potential. DisclosuresNo relevant conflicts of interest to declare.

EPCR/PAR1 Signaling Navigates Long-Term Repopulating Hematopoietic Stem Cell Bone Marrow Homing to Thrombomodulin-Enriched Blood Vessels

Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) is an active and essential first step in clinical stem cell transplantation. EPCR is expressed by murine BM LT-HSCs endowed with the highest repopulation potential and its ligand, activated protein C (aPC), has anticoagulant and anti-sepsis effects in EPCR+/PAR1+ endothelial cells. We recently found that signaling cascades, traditionally viewed as coagulation and inflammation related, also independently control EPCR+ LT-HSC BM retention and recruitment to the blood via distinct PAR1 mediated pathways. EPCR/PAR1 signaling retains LT-HSCs in the BM by restricting nitric oxide (NO) production and Cdc42 activity, promoting VLA4 affinity and adhesion. Conversely, thrombin/PAR1 signaling overcome EPCR+ LT-HSC BM retention by initiating NO production, leading to TACE-‎mediated EPCR shedding, CXCR4 and PAR1 upregulation and parallel CXCL12 secretion by PAR1+ BM stromal cells, enhancing stem cell migration and mobilization.Since EPCR shedding is essential for BM LT-HSC recruitment, we tested EPCR role in LT-HSC BM homing. EPCR+ LT-HSC exhibited reduced in vitro migration towards CXCL12 and enhanced CXCL12-dependent adhesion to fibronectin. Unexpectedly, transplanted EPCR+ LT-HSCs preferentially homed ‎to the host BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing antibody treatment, which blocks binding to aPC, leading to attenuated EPCR+ LT-HSC homing to the BM but not to the spleen. Importantly, short term aPC pretreatment inhibited NO production and dramatically increased EPCR+ LT-HSC BM homing. Since EPCR navigates LT-HSC to the BM, we studied the role of EPCR signaling in LT-HSC BM repopulation. Mimicking EPCR signaling by in vivo NO inhibition induced preferential expansion of blood and bone-forming stem cells and gave rise to higher donor type EPCR+ LT-HSCs in competitive repopulation assays. Similarly, repeated treatment with aPC expanded BM EPCR+ stem cells and increased competitive LT-repopulation. Importantly, loss of EPCR function reduced HSC long-term repopulation ability while maintaining their short-term repopulation activity. BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with normal stem cells in competitive long-term repopulation assays. Consistent with inferior HSC BM repopulation, Procrlow mice exhibited reduced numbers of BM LT-HSC with reduced adhesion capacity. Additionally, these mice displayed increased HSC frequencies in the blood circulation and the spleen, which were pharmacologically corrected by inhibiting NO generation with L-NAME treatment. BM retention is essential for quiescent HSC protection from chemotherapy. Mice treated with NO donor SNAP, or with blocking EPCR antibody as well as Fr2-/-mice lacking PAR1 expression, were more susceptible to hematological failure and mortality induced by 5-FU treatment compared to control mice. Together, these results indicate a functional aPC/EPCR/PAR1 signaling pathway, regulating EPCR+ LT-HSC BM homing, adhesion and long-term repopulation potential. The thrombin-thrombomodulin (TM) complex converts protein C to its activated form aPC, facilitating high affinity binding to its receptor EPCR. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by a unique BM endothelial cell (BMEC) subpopulation, but not in the spleen. Moreover, EPCR+ LT-HSCs were found adjacent to TM+/aPC+ BMECs, imposing their adhesion and retention. Interestingly, similar to BMECs, BM EPCR+ LT-HSC also express surface TM, implying the possibility of autocrine aPC generation.Herein we define EPCR as a guidance molecule, navigating slow migrating LT-HSC in the blood flow specifically to TM+ BMEC supporting niches, maintaining NOlow stem cell retention, long-term blood production and protection from myelotoxic insult. Conversely, thrombin/PAR1 signaling oppositely increase NO generation and EPCR shedding allowing increased CXCR4-dependent LT-HSC migration and mobilization. Harnessing EPCR signaling may improve clinical stem cell transplantation, increasing LT-HSC specific BM homing and repopulation by aPC pretreatment, as well as potentially to overcome malignant stem cell chemotherapy resistance. DisclosuresNo relevant conflicts of interest to declare.

Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures.

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.

In vivo time-lapse imaging shows diverse niche engagement by quiescent and naturally activated hematopoietic stem cells

AbstractHematopoietic stem cells (HSCs) maintain the turnover of mature blood cells during steady state and in response to systemic perturbations such as infections. Their function critically depends on complex signal exchanges with the bone marrow (BM) microenvironment in which they reside, but the cellular mechanisms involved in HSC-niche interactions and regulating HSC function in vivo remain elusive. We used a natural mouse parasite, Trichinella spiralis, and multipoint intravital time-lapse confocal microscopy of mouse calvarium BM to test whether HSC-niche interactions may change when hematopoiesis is perturbed. We find that steady-state HSCs stably engage confined niches in the BM whereas HSCs harvested during acute infection are motile and therefore interact with larger niches. These changes are accompanied by increased long-term repopulation ability and expression of CD44 and CXCR4. Administration of a CXCR4 antagonist affects the duration of HSC-niche interactions. These findings suggest that HSC-niche interactions may be modulated during infection.

Biological effects of T315I-mutated BCR-ABL in an embryonic stem cell–derived hematopoiesis model

The occurrence of T315I mutation during the course of targeted therapies of chronic myeloid leukemia is a major concern because it confers resistance to all currently approved tyrosine kinase inhibitors. The exact phenotype of the hematopoietic stem cell and the hierarchical level of the occurrence of this mutation in leukemic hematopoiesis has not been determined. To study the effects of T315I-mutated breakpoint cluster region-abelson (BCR-ABL) in a primitive hematopoietic stem cell, we have used the murine embryonic stem cell (mESC)-derived hematopoiesis model. Native and T315I-mutated BCR-ABL were introduced retrovirally in mESC-derived embryonic bodies followed by induction of hematopoiesis. In several experiments, T315I-mutated and nonmutated BCR-ABL-transduced embryonic bodies rapidly generated hematopoietic cells on OP-9 feeders, with evidence of hematopoietic stem cell markers. After injection into NOD/SCID mice, these cells induced myeloid and lymphoid leukemias, whereas transplantation of control (nontransduced) hematopoietic cells failed to produce any hematopoietic reconstitution invivo. Moreover, the expression of native and T315I-mutated BCR-ABL conferred to mESC-derived hematopoietic cells a self-renewal capacity demonstrated by the generation of leukemias after secondary transplantations. Secondary leukemias were more aggressive with evidence of extramedullary tumors. The expression of stem cell regulator Musashi-2 was found to be increased in bone marrow of leukemic mice. These data show that T315I-mutated BCR-ABL is functional at the stem cell level, conferring to mESC-derived leukemic cells a long-term hematopoietic repopulation ability. This model could be of interest to test the efficiency of drugs at the stem cell level in leukemias with T315I mutation.

RUNX1a Enhances Hematopoietic Lineage Commitment From Human Embryonic Stem Cells and Inducible Pluripotent Stem Cells

Abstract 347Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are pluripotent stem cells (hPSCs) with potential to differentiate into all types of somatic cells. Patients suffering from blood disorders can be cured with hematopoietic cell transplantations (HCT). Technical advancements in hPSC production and handling have revolutionized their potential applications in regenerative medicine and provided enormous hope for patients who may need HCT. hiPSCs derived from autologous cells could provide unlimited leukocyte antigen matched blood cells on a patient-specific basis. A remaining hurdle in this process remains the need for efficient and effective generation of specific blood cells from hPSCs for therapeutic use. Transcription factors play key roles in regulating maintenance, expansion, and differentiation of blood cells from hPSCs. Studies have shown that transcription factor RUNX1 is required for the formation of definitive blood cells. There are several alternatively spliced isoforms of the RUNX1 protein, including the shortest form RUNX1a and two longer forms RUNX1b and RUNX1c. Based on known properties of RUNX1 proteins, we hypothesized that RUNX1a promotes the production of therapeutic hematopoietic stem cells from hPSCs. By employing ectopic expression of RUNX1a on different human ESC and iPSC lines (H9, BC1, iCB5) under a defined hematopoietic differentiation system, we aimed to identify function of RUNX1a on lineage commitment and molecular mechanisms of RUNX1 activity in differentiation of PSCs to hematopoietic cells. We demonstrated that expression of endogenous RUNX1a parallels lineage commitment and hematopoietic emergence from hPSCs. During differentiation process RUNX1a enhanced the expression of several mesoderm and hematopoietic differentiation related factors, including KDR, SCL, GATA2, and PU.1. In addition, over-expression of RUNX1a in embryoid bodies (EBs) showed more efficient and earlier emergence of typical sac structures, which predicts cell lineage commitment and germ layer development at the early stage of EB differentiation. At day 7, EBs derived from hPSCs was dissociated into single cells for flow cytometry analysis. The mean frequency of CD31+CD34+CD45− and total CD34+ cells with hemato-endothelial cell features are 35.1% and 67.1% from RUNX1a-overexpressing EBs, and 8.7% and 24.1% from vector control EBs. Immunohistochemistry analysis of EBs at day 9 of differentiation confirmed that expression of RUNX1a accelerated mesoderm commitment and emergence of hemato-endothelial precursors. Flow cytometry analysis on EBs collected at days 9, 11, 13 showed that ectopic RUNX1a induced a robust increase in the frequency of hematopoietic progenitor cells in all hPSC lines examined. At Day 9, RUNX1a-overexpressing EBs generated 48.5% CD43+CD45+ cells, 45.1% CD34+CD45+ cells, and 8.5 folds higher CD43+ cells than vector EBs. Later at Day 13, 80% CD45+ and 75% CD43+/CD34+CD45+ hematopoietic stem/progenitor cells (HSPCs) achieved from dissociated EBs. In liquid culture, RUNX1a HSPC showed strong expansion and high percentage of CD235a+CD45− (20%) and CD71+CD235a+ (16%), markers for erythroid populations. Flow cytometry and western blots on RUNX1a-EB formed colonies showed significantly higher β-globin production than that of the vector, suggesting expression of RUNX1a in HSPC enhanced definitive hematopoiesis. RUNX1a-hPSCs derived HSPCs possess self-renewal capability and are capable of differentiating into multi-lineages ex vivo. Furthermore HSPCs generated from RUNX1a-EBs possessed the capacity of interacting with surrogate niche and showed long-term repopulation ability under LTC-IC (Long-Term Culture-Initiating Cell Assay) condition. Colonies generated from HSPC of RUNX1a-EBs after 3 week bulk LTC-IC culture showed 300 folds higher than vector control. RUNX1a-hPSCs derived CD34+CD45+ cells could maintain a non-adherent population in ouldCD45+ sEBsND THIS SENTENCE5 week culture on stromal cell M210. In summary we identified that RUNX1a enhances derivation of definitive hematopoietic cells from human PSCs. Our study provides an important and useful system to enhance specificity and efficiency of generating functional blood cells and further differentiated cells from human PSCs, which may provide valuable source for future clinical applications in patients with hematologic disorders. Disclosures:No relevant conflicts of interest to declare.

Metabolic Cross Talk Between Foxo3 and mTOR Is Essential for Hematopoietic Stem Cell Function

Abstract 856Unbalanced accumulation of reactive oxygen species (ROS) compromises self-renewal of hematopoietic stem cells (HSC). Nonetheless, survival of HSC in the hypoxic niche requires mitochondrial ROS-mediated activation of hypoxic response and significant metabolic adaptation. Hypoxia favors glycolysis to mitochondrial oxidative phosphorylation for energy production. In agreement with this, increased mitochondrial function results in defective maintenance of HSC as observed in Tsc1−/− mice through overactivation of mammalian target of rapamycin (mTOR) signaling pathway. Mechanisms that regulate the strict coordination of mitochondrial function with upstream metabolic pathways that is required for HSC maintenance and balancing ROS remain largely unknown. Foxo3 Forkhead transcription factor is a strong candidate for coordinating metabolic pathways in HSC. Evidence from our laboratory and others has identified Foxo3 as a critical regulator of HSC quiescence and a key modulator of oxidative stress in HSC. To address whether Foxo3 has a more global metabolic control of HSC activity, we investigated the mitochondrial function in Foxo3 mutant HSC. To achieve this, we measured ATP content and oxygen consumption, two major defining mitochondrial parameters, using ATP Bioluminescence Assay and Oxygen Biosensor analysis respectively in freshly isolated Lin−Sca-1+cKit+(LSK) CD34−Flk2− bone marrow cells isolated from wild type and Foxo3−/− mice. We showed that loss of Foxo3 leads to significant mitochondrial defects in HSC as indicated by a strong decrease in both ATP content and oxygen consumption. In agreement with this, the glycolytic flux in Foxo3 mutant HSC, as analyzed by 13C lactate production using gas chromatography-mass spectrometry, was increased indicating a shift in the ATP production from mitochondria to the cytosolic glycolysis in Foxo3 mutant HSC. In addition, mitochondrial mass and membrane potential, that is generated during mitochondrial oxidative phosphorylation for energy production, were assessed by flow cytometry in WT and Foxo3−/− LSK using mitotracker green and JC-1 probes respectively. Loss of Foxo3 increased both mitochondrial mass and membrane potential, likely reflecting a compensatory mechanism to the defective mitochondria in Foxo3−/− HSC. These anomalies may partly contribute, in addition to the known defective ROS detoxification, to the increased ROS levels previously observed in Foxo3−/− HSC. Importantly, the mitochondrial dysfunction was not due to the abnormal increase of ROS observed in Foxo3−/− HSC since in vivo treatment of Foxo3−/− mice with ROS scavenger N-Acetyl-Cysteine (NAC) for two weeks, did not revert the increased mitochondrial membrane potential. Altogether these results strongly suggest that Foxo3 is critical for the regulation of mitochondrial function in HSC. mTOR signaling controls major cellular metabolic processes and is critical for the regulation of mitochondrial function in HSC. We have previously found that mTOR signaling is amplified by a redox-mediated mechanism in Foxo3−/− hematopoietic progenitors (Yalcin et al., 2010). Thus, we asked whether mTOR signaling is involved in the Foxo3 regulation of mitochondria function in HSC. Interestingly, in vivo treatment of Foxo3−/− mice for two weeks with rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1) activity, as measured by the decrease in phosphorylation of ribosomal protein S6 in HSC, mitigates the increased mitochondrial membrane potential and normalizes ROS levels in Foxo3−/− HSC, suggesting that Foxo3 regulation of mitochondria is mediated by mTOR signaling in HSC. Notably, rapamycin treatment rescued partially Foxo3−/− HSC pool and function as measured by the number and frequency of LSK in Foxo3−/− mice, as well as their long-term repopulation ability measured by the capacity of CD48−CD150+LSK cells to repopulate the hematopoietic compartment in lethally irradiated recipient mice within 8 weeks. Taken together, our findings reveal a new function for Foxo3 in the control of mitochondria in HSC and support a model in which mitochondria is key to the maintenance of HSC. We propose that a Foxo3-mTOR signaling node partly controls mitochondrial function in HSC. These findings are likely to have an important impact on our understanding of the metabolic regulation of hematopoietic and leukemic stem cells and may be of therapeutic value. Disclosures:No relevant conflicts of interest to declare.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Abstract 2315SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures:No relevant conflicts of interest to declare.

Enhanced Angpt1/Tie2 signaling affects the differentiation and long-term repopulation ability of hematopoietic stem cells

Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4 week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage(-)Sca-1(+)c-Kit(+) (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Hematopoietic Stem Cells Survive Circulation Arrest and Reconstitute Hematopoiesis in Myeloablated Mice

Hematopoietic stem and progenitor cells (HSPC) for bone marrow transplantation are currently obtained directly from living voluntary donors or from cord blood units. However, a suitable donor is not always found. Because HSPC are known for their relative resistance to hypoxia, using an experimental murine model, we explored cadaveric bone marrow (BM) as their alternative source. After donor mice were sacrificed, BM was left in intact femurs at 37°C, 20°C, or 4°C under ischemic conditions, resulting in combined oxygen and metabolic substrate shortage and the accumulation of metabolic waste products. BM cells were harvested after a set time period ranging from 0 to 48 hours. To determine the impact of delayed harvesting on the transplantability of HSPC, a competitive repopulation assay using a murine Ly5.1/Ly5.2 congenic model in 2 different settings was used: after submyeloablative (6 Gy) or myeloablative (9 Gy) total-body irradiation, Ly5.2 hosts received cadaveric Ly5.1 cells or a mixture of cadaveric Ly5.1 cells and fresh Ly5.2 cells in a 1:1 ratio. Chimerism resulting from cadaveric donor cells, followed up to 6 months after transplantation, proved that the long-term repopulation ability of HSPC was fully preserved for 2 hours, 6 hours, and 12 hours at 37°C, 20°C, and 4°C of ischemia, respectively. A colony-forming unit-spleen (CFU-S) clonogenic assay revealed a higher sensitivity of proliferating hematopoietic progenitors to ischemia compared to repopulating cells (STRC and LTRC). Flow cytometry analysis of apoptosis in cadaveric BM demonstrated that the LSK (Lin(low)Sca-1(+)c-Kit(+)) subpopulation, enriched in HSPC, contained less apoptotic and dead cells than the BM as a whole. Furthermore, the number of LSK SLAM (CD150(+)CD48(-)) and LSK SP (side population) cells (fractions highly enriched in hematopoietic stem cells) decreased in parallel with BM transplantability. As well as cadaveric BM cells, we also tested the transplantability and survival of BM cells after storage in a suspension in vitro without specific hematopoietic growth factors. HSPC did not display any decrease in transplantability after 2 days of storage at 37°C or 4 days at 4°C. A higher sensitivity of progenitors to unfavorable conditions was observed again using CFU-S and granulocyte macrophage-colony forming cell (GM-CFC) assays, especially at 37°C. This paper shows that HSPC survive the cessation of circulation for a considerable time and maintain their engraftment potential. This time is significantly extended with in vitro storage compared to the cadaveric BM.

Severe Combined Immunodeficiency-Repopulating Cell Assay May Overestimate Long-Term Repopulation Ability

Severe Combined Immunodeficiency-Repopulating Cell Assay May Overestimate Long-Term Repopulation Ability

Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

Genetic regulation of hematopoietic stem cell exhaustion during development and growth

During aging, hematopoietic stem cell (HSC) exhaustion is more severe in BALB/cByJ (BALB) mice than in C57BL/6J (B6) mice. Our objective is to determine whether HSC exhaustion during development from fetus to adult also is more severe for BALB than for B6 mice. Hematopoietic stem cells from fetal liver cells (FLCs) and from young adult bone marrow cells (BMCs) were compared using the competitive repopulation assay to measure long-term repopulating ability (LTRA) and HSC expansion after serial transplantation. LTRAs were measured in repopulating units (RU), as the ability to produce donor-type erythrocytes and lymphocytes in lethally irradiated recipients relative to the congenic fresh marrow competitor. To test expansion, FLCs or BMCs were serially transplanted into lethally irradiated carriers whose marrow cells were compared using fluorescence-activated cell staining (FACS), and subsequently tested for LTRA. BALB and B6 FLCs, respectively, repopulated 2.6 and 13.5 times as well as BMCs. LTRAs correlated with HSC expansion for BALB, but not B6. Per million donor cells, CD34(-) HSC-enriched fractions (HEFs) and total RU values were 6.8 and 4.6 times higher for FLCs than for BMCs in BALB carriers, while these ratios were only 1.2 and 0.97 higher in B6 carriers. In B6 HSC development, LTRA is dissociated from expansion. Although 1 x 10(6) BMCs have much lower LTRA, they expand HSCs as well as 1 x 10(6) FLCs. HSC expansion is partly exhausted in BALB, but not B6, during development.

Bone Marrow Connexin-43 Expression Is Critical for Hematopoietic Regeneration After Chemotherapy

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.

A murine model of antimetabolite-based, submyeloablative conditioning for bone marrow transplantation: biologic insights and potential applications

Nonmyeloablative conditioning regimens for marrow transplantation are desirable in many settings. Because repeated doses of the antimetabolite 5-fluorouracil (5-FU) decreases marrow long-term repopulating ability (LTRA) upon transplantation into lethally irradiated hosts, we hypothesized that mice given sequential doses of 5-FU (termed paired dose 5-FU) may permit substantial syngeneic marrow engraftment. C57Bl/6 or X-linked chronic granulomatous disease (X-CGD) mice were administered 5-FU (150 mg/kg) on days -5 and -1. Assessment of host marrow phenotype and repopulating ability occurred on day 0. Transplantation of syngeneic donor marrow occurred on day 0 or day +15. We confirmed that the number of Sca-1+lin- cells and the LTRA of marrow from paired dose 5-FU-treated animals were diminished. C57Bl/6 hosts conditioned with paired doses of 5-FU followed by transplantation of 20 x 10(6) fresh B6.SJL marrow cells on day 0 displayed 44.9% +/- 7.1% donor chimerism 2 months posttransplant, and 34.4% +/- 8.6% donor chimerism 6 months posttransplant. In contrast, paired dose 5-FU-conditioned hosts transplanted with similar numbers of donor cells on day +15 exhibited only 3.4% +/- 1.2% donor chimerism at 2 months. Paired dose 5-FU-conditioned X-CGD hosts transplanted with MSCV-m91Neo-transduced X-CGD marrow averaged 6.6% +/- 2.3% (range, 4%-10%) NADPH oxidase-reconstituted neutrophils 12-16 months after transplant. These findings support the concept that impairment of host stem cell competitiveness may be an important mechanism for permitting engraftment of donor cells, and suggest that only a brief period of modest host stem cell impairment may be necessary to achieve substantial donor cell engraftment.

Granulocyte Colony-Stimulating Factor Prior to Low-Dose Total Body Irradiation as Conditioning Regimen in Hematopoietic Stem Cell Transplantation for Gene Therapy.

Genetic blood diseases can potentially be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. Nonmyeloablative conditioning regimens such as low-dose total body irradiation (LDTBI) can enhance competitive engraftment in the absence of a selective advantage for HSC. Granulocyte colony-stimulating factor (G-CSF) treatment of recipients prior to LDTBI has been shown to enhance engraftment of fresh bone marrow (BM) cells, but the underlying mechanisms and whether this combined approach can enhance long-term engraftment of genetically transduced donor cells have not been defined. We examined whether pre-treatment of recipient mice with G-CSF prior to LDTBI improves long-term donor engraftment of retroviral vector-transduced HSC in murine X-linked chronic granulomatous disease (X-CGD). In initial studies, we verified that pre-treatment of wild type mice with 4 ug G-CSF SC BID for 5 days prior to irradiation with either 160 cGy or 300 cGy significantly increased long-term engraftment of congenic fresh BM cells (see Table).Donor → HostCell doseLDTBI (cGy)Donor chimerism + vs - G-CSF (Mean ±SD)C57BL/6J → PtrcaPep3b/Boy J20 x 106 fresh BM16065.2±0.9% vs 43.4±3.5%, n=4 and 5 mice each; p=0.0002 (4 months after transplant)C57BL/6J-EGFP → C57BL/6J2.5 x 106 fresh BM30055.25±4.5% vs 28.7±5.3%, n=4mice each; p=0.0002 (6 months after transplant)C57BL/6J male X-CGD → C57BL/6J female X-CGD5 x 106 transduced BM30051.0±13.1% vs 30.4±7.8%, n=5mice each; p=0.02 (3 months after transplant)We next investigated 2 potential mechanisms by which G-CSF prior to LDTBI might enhance long-term engraftment of HSC. First, analysis of host BM 20 hours after transplantation showed a significantly lower frequency of homed donor HSC in G-CSF+160cGy treated mice compared to 160 cGy-conditioned mice (1 ± 0.13% vs 3.5 ± 0.7% in one experiment, and 3.4 ± 0.8% vs 9.5 ± 2.3% in another experiment, n= 20 mice). Second, in a competitive repopulation assay, we observed a substantial decrease in marrow long-term repopulating ability (LTRA) in mice treated with G-CSF + 160 cGy compared to 160 cGy alone (10.5% vs 48.6%, n= 5 mice in each group; p= 0.0002). These results suggest that administration of G-CSF prior to LDTBI does not increase initial homing of donor cells but further impairs LTRA of recipient BM compared to LDTBI alone. In the context of gene therapy, we transduced male X-CGD BM with MSCV-gp91Neo vector and infused these transduced cells into female X-CGD mice treated ± G-CSF + 300 cGy. Donor cell engraftment, measured by FISH for Y chromosome, at 3 months post-transplant was significantly increased in recipients pretreated with G-CSF (see Table). NADPH oxidase activity (by 123-dihydrorhodamine assay) was reconstituted in 9.75 ± 5.5% of neutrophils compared to 6.6 ± 2.5% without G-CSF pretreatment. The calculated fraction of donor cells with oxidase activity was similar in both groups (18.8 ± 6.6% vs 21.4 ± 11%, respectively) (p= 0.67). These results suggest that the use of G-CSF prior LDTBI provides a competitive advantage for long-term engraftment by decreasing LTRA in the host and may be a useful strategy to increase engraftment of fresh and retroviral transduced donor BMC.

Homing Efficiency in Two Models of Submyeloablative Conditioning and Correlation with Long-Term Engraftment.

Submyeloblative conditioning regimens for bone marrow transplantation (BMT) are desirable to reduce host toxicity in many settings. We previously demonstrated that hosts conditioned with 160 cGy or with two sequential doses of the antimetabolite 5-fluorouracil (5-FU) displayed ~50% and ~35% donor chimerism 6 months after transplantation of 20 x 106 fresh congenic bone marrow (BM) donor cells, respectively. Donor chimerism in 160 cGy- or 5-FU-conditioned mice was proportional to the reduction in host long-term repopulating ability (LTRA) caused by the conditioning regimen (~80–90% vs. ~55%, respectively). In this study, we sought to determine if donor marrow homing efficiency also correlated with long-term engraftment in these models of submyeloablative conditioning. We hypothesized that sequential doses of the cell cycle-specific agent 5-FU (5-FU x 2) may damage the marrow microenvironment less than low-dose radiation, resulting in more efficient homing in 5-FU x 2-conditioned mice. C57Bl6/J (Bl/6) hosts were conditioned with either 5-FU x 2 administered on days -5 and -1 (150 mg/kg IP) or with 160 cGy given the day of transplantation (day 0). Bl/6 (CD 45.2+) hosts were transplanted with 20 x 106 PKH-26 stained Bl/6 or with Boy J (CD 45.1+) whole BM cells via tail vein injection. The percentage of homed cells 20 hours after BMT in 5-FU x 2-conditioned mice was 3.53 % ± 3.1% (n = 13 from 3 independent experiments), while the percentage of homed cells in the 160 cGy group was 8.67 % ± 2.1% (n = 12; p = 0.0375). The fraction of donor cells which homed to the spleen or remained in the peripheral blood after 20 hours was similar between conditioning regimens. This difference in homing efficiency led us to consider other possible explanations as to why BM cells home less efficiently in 5-FU x 2-conditioned hosts than in low-dose irradiated mice. We hypothesized that 5-FU x 2 may have a more injurious effect on the marrow microenvironment than previously thought. Examination of marrow stromal cell potential using a colony-forming unit-fibroblast (CFU-F) assay revealed that marrow from 5-FU x 2-treated mice (n = 3) contained 1.1 ± 0.4 colonies/marrow femur equivalent (FE), whereas marrow from 160 cGy-treated mice (n = 3) contained 33.8 ± 13.4 colonies/FE. In comparison, marrow from untreated mice (n = 3) averaged 95.6 ± 27.5 colonies/FE. These data suggest that homing efficiency correlates with donor chimerism in these two models of submyeloablative conditioning, and establish at least two potential mechanisms (a lesser reduction of host LTRA, and decreased donor cell homing efficiency) for the lower level of long-term donor chimerism observed in 5-FU x 2- compared to 160 cGy-conditioned hosts. Furthermore, 5-FU x 2 conditioning appears to disrupt the marrow microenvironment to a greater degree than low-dose radiation. Current efforts are aimed at exploring the molecular interactions of homed donor cells with conditioned marrow.