Residual Hematopoietic Stem Cells Research Articles

Guest editorial: leukemia stem cell

Leukemia is a clonal, malignant hematological disorder. Recent studies have shown that at least in acute myelogenous leukemia (AML), the clonogenic and leukemogenic activities reside in a minor, primitive fraction of the leukemia cell population. In 1996, Dick et al. showed that the CD34CD38, but not other fractions of bone marrow cells from AML patients can reconstitute human AML in immunodeficient mice, demonstrating direct evidence for the presence of leukemia stem cells (LSCs). After this finding, the concept of the LSC becomes critical to understand the pathogenesis of leukemia. In this issue, four excellent review articles [1–4] focus on developmental mechanisms of LSCs to discuss how to eradicate them to obtain absolute cure of leukemia. Cancer should result from the accumulation of multiple genetic abnormalities. In hematopoiesis, the cells accumulating such mutations need to survive for a long time in the human body, and therefore, the main target for mutations should be hematopoietic stem cells (HSCs) that are capable of self-renewal. HSCs harboring multiple mutations can produce a large number of proliferating progenitor populations with identical mutations, which further increases the chance to get another critical hit to transform into LSCs. This outline of leukemia development reasonably fits the concept of blastic crisis from chronic myelogenous leukemia (CML). In CML, a HSC acquires BCR-ABL, resulting in clonal expansion of BCR-ABLexpressing hematopoietic progenitors and mature cells. These HSCs accumulate additional mutations, and their expanding myeloid or lymphoid progenitors finally transform into LSCs of myeloid or lymphoid blastic clones, respectively. In this context, BCR-ABL is an early mutation, and HSCs in CML patients could be regarded as ‘‘preLSCs’’. Imatinib mesylate, an inhibitor for kinase activity of BCR-ABL, induces hematological and cytogenetic remissions in nearly every patient in chronic phase of CML, preventing the transformation into blastic crisis, but the cessation of this drug results in relapse of CML in the majority of patients. Therefore, it is important to understand the molecular mechanisms to maintain BCR-ABLexpressing residual HSCs (pre-LSCs) in CML. Ito [1] summarizes key players mediating maintenance of such CML stem cells. Recent technological advances in genomics have revealed recurrent driver mutations in AML, demonstrating existence of the similar evolutionary processes for LSC development, in which genetic diversity caused by multiple mutations and clonal selection of a population with growth advantage should be critical. In this selection process, it is now clear that pre-LSCs possessing multiple mutations but still retaining ability to differentiate into all mature blood cells progressively become dominant. Chan and Majeti [2] especially focus on mutations related to epigenetic regulation, one of important groups of driver mutations, and summarize the current understanding of their roles in leukemogenesis. Targeting the LSC should be the ultimate therapeutic strategy to cure leukemias. Xenogeneic transplantation of human cells into immunodeficient mice has enabled to identify cell fractions enriched for LSCs. Ishikawa [3] summarizes the history of xenotransplantation models, and describes his NSG mouse model that is a powerful tool to discover functional properties of LSCs including their cell cycle status and location in the bone marrow. Using the K. Akashi (&) Department of Medicine and Biosystemic Sciences, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan e-mail: akashi@med.kyushu-u.ac.jp

Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic STEM and Progenitor CELL Mutagenesis in Response to Irradiation

AbstractAbstract 3447Radiation-induced double-strand breaks (DSBs) represent a serious threat to the preservation of genetic information when it reaches hematopoietic stem cells (HSCs). Residual loss of HSC functions and increased risk of developing hematopoietic malignancies are two concerning complications of anti-cancer radiotherapy. Management of acute myelosuppression following radio- or chemotherapy has been significantly improved in recent years by the use of growth factors. However, how cytokine/environmental signals integrate the DNA damage responses in HSCs and regulate the long-term residual HSC defects following radio-or chemotherapy is unknown. Notably, the contribution of cytokines regulating HSC functions to HSC intrinsic DNA damage repair processes remains to be delineated. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal, survival and expansion posttransplantation. In this study, we uncover an unknown and unique function for TPO/Mpl in the regulation the DNA damage response. We show that DSB repair, measured by both γH2Ax foci resolution and neutral comet assays, following γ-irradiation (IR) or topoisomerase II inhibitor treatments, is defective in Mpl−/− and Mpl+/− HS and progenitor cells (HSPCs). Similar defects were found in wild-type cells treated in the absence of TPO. This indicates that the impaired DNA repair of Mpl−/− and Mpl+/− cells results from a specific loss of TPO-mediated DNA damage response signaling at the time of IR rather than from intrinsic constitutive differences. TPO stimulates DNA repair by increasing IR-induced DNA-PK phosphorylation at Ser2056 and Thr2609 and non-homologous end joining (NHEJ) efficiency in both HSPCs and the human UT7-Mpl cell line. This is to our knowledge the first demonstration that a cytokine involved in the homeostatic maintenance of HSCs may also regulate their response to external DNA damaging insults by controlling the DSB repair machinery. Short TPO treatment in vitro or single TPO injection to TPO/Mpl proficient mice prior to sublethal total body IR reduced IR-induced HSC genomic instability and loss of long-term reconstitution ability. This may open new avenues for administration of TPO agonists before radiotherapy to minimize radiation-induced HSC injury and mutagenesis. In addition, since Mpl is haploinsufficient in the regulation of DNA damage repair, these data suggest that Mpl might also act as a tumor suppressor in response to radiotherapy. Disclosures:No relevant conflicts of interest to declare.

Abstract 3303: Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Despite the power of next-generation DNA sequencing to describe AML genomes and to identify recurrent mutations, our fundamental understanding of the genomics of leukemogenesis is incomplete. Founder mutations in the majority of AML cases are unknown because pre-leukemic cells are clinically silent and are outcompeted by their malignant descendants. Our limited knowledge of founder mutations comes from infrequent cases of AML arising secondary to antecedent clonal bone marrow disorders or rare instances of inherited syndromes. Importantly, recent advances in AML stem cell biology now enable the prospective separation of residual non-leukemic hematopoietic stem cells (HSC) from AML cells. We previously showed that non-leukemic HSC contain clonal antecedents of AML in patients in long-term remission post-therapy, and have proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. Here, we investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual non-leukemic HSC. Using exome sequencing, we identified mutations present in eight individual AML genomes (mean 10 mutations per patient) and screened for them in the residual HSC. We identified several mutations present in residual HSC that retained normal multilineage differentiation in vivo. These putative “early” mutations detected in residual HSC include NPM1c, mutations in TET2, and novel AML mutations in genes involving the cell cycle and mRNA biogenesis. Putative “late” mutations absent from residual HSC and only found in leukemic cells include FLT3 ITD and IDH1 R132H. Finally, through single cell analysis, we determined that as we hypothesized, a clonal progression of multiple mutations occurs in non-leukemic HSC. Quantitative genetic analyses of the HSC compartment enabled us to reconstruct the subclonal architecture of normal and pre-leukemic stem cells. Consistent with mouse models, pre-leukemic HSC with TET2 mutations outcompeted their normal counterparts. However, in most cases, normal HSC were 6-50 times more numerous than pre-leukemic HSC. In two cases where sequential populations of pre-leukemic HSC were identified, the less mutated pre-leukemic HSC population was 10-25 times more numerous than its more mutated descendent. This result contrasts with the classical model of a linear succession of dominant pre-leukemic subclones. These pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations, suggest a potential mechanism contributing to relapse, and constitute a cellular reservoir that may need to be targeted for more durable remissions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3303. doi:1538-7445.AM2012-3303

Abstract SY35-01: Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Despite the power of next-generation genome sequencing to describe AML genomes and to identify recurrent mutations, our fundamental understanding of both the genomics of leukemogenesis and the genetic heterogeneity within the precursor cells is incomplete. Founding mutations in the majority of AML cases are largely unknown because pre-leukemic cells are clinically silent and are outcompeted by their malignant descendants. Our limited knowledge of founding mutations comes from infrequent cases of AML arising secondary to antecedent clonal bone marrow disorders or rare instances of inherited syndromes, but this does not include the large majority of de novo AML cases. Recently, advances in AML stem cell biology have enabled the prospective separation of residual hematopoietic stem cells (HSC) from AML cells. We previously showed that residual HSC contain clonal antecedents of AML in patients in long-term remission post-therapy, and have proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. Here, we investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual HSC. Using exome sequencing, we defined mutations present in individual AML genomes from cases harboring the FLT3-ITD mutation, and screened for these mutations in the residual HSC. We identified several mutations present in residual HSC retaining normal multilineage differentiation in vivo, including mutations in NPM1, TET2, and SMC1A, a newly identified recurrently mutated gene in AML. Finally, through single cell analysis, we determined that as we hypothesized, a clonal progression of multiple mutations occurs in HSC. These pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations, suggest a potential mechanism contributing to relapse, and constitute a cellular reservoir that may need to be targeted for more durable remissions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY35-01. doi:1538-7445.AM2012-SY35-01

Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia

Abstract 4▪▪This icon denotes a clinically relevant abstractAcute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Despite the power of next-generation genome sequencing to describe AML genomes and to identify recurrent mutations, our fundamental understanding of the genomics of leukemogenesis is incomplete. Founding mutations in the majority of AML cases are largely unknown because pre-leukemic cells are clinically silent and are outcompeted by their malignant descendants. Our limited knowledge of founding mutations comes from infrequent cases of AML arising secondary to antecedent clonal bone marrow disorders or rare instances of inherited syndromes, but this does not include the large majority of de novo AML cases. Previously, we showed that non-leukemic hematopoietic stem cells (HSC) contain clonal antecedents of AML in patients in long-term remission post-therapy, and have proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. More recently, we demonstrated the prospective separation of residual HSC from AML cells, based on differential expression of surface markers such as CD47 and TIM3.1,2 Here, we investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual non-leukemic HSC, speculating that these residual non-leukemic HSC might in fact constitute a reservoir of pre-leukemic HSC harboring founder mutations, but lacking the complete complement of abnormalities required to generate AML. Using exome sequencing, we identified mutations present in multiple individual AML genomes (mean 10 mutations per patient) and screened for them in the residual HSC. In most cases, we identified several mutations present in residual HSC that retained normal multilineage differentiation in vivo. These “early” mutations include NPM1c and novel AML mutations in genes involved in the cell cycle and mRNA biogenesis. Putative “late” mutations absent from residual HSC and only found in leukemic cells include FLT3 ITD and IDH1 R132H. Next, using custom-designed SNP Taqman genotyping assays, we analyzed single residual HSC for the presence of the identified “early” mutations. As hypothesized, we determined that a clonal progression of mutations occurs in non-leukemic HSC, based on the identification of individual cells containing subsets of these “early” mutations. Quantitative genetic analyses of the HSC compartment enabled us to reconstruct the subclonal architecture of normal and pre-leukemic stem cells. In all cases, normal HSC were 6–50 times more numerous than pre-leukemic HSC, and in one case where we identified two sequential populations of pre-leukemic HSC, the less mutated population was 25 times more numerous than its more mutated descendent. This result contrasts with the classical model of a linear succession of increasingly dominant pre-leukemic subclones, suggesting that the relationship between subclone size and clonal progression may be complex. In summary, our results show that pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations. Ultimately, these clonal antecedents of leukemia may prove to be clinically important. Indeed, some cases of relapsed pediatric ALL have been shown to arise from a clone ancestral to the presenting leukemia. The same may be true in AML, in which relapsed disease develops from a pre-leukemic HSC clone that acquires additional novel mutations resulting in a genetically divergent leukemic relapse. This possibility suggests that pre-leukemic HSC constitute a cellular reservoir that may need to be targeted for more durable remissions. Disclosures:No relevant conflicts of interest to declare.

TIM-3 Is a Promising Target to Eradicate Acute Myeloid Leukemia Stem Cells Sparing Normal Hematopoietic Stem Cells

Abstract 559Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for permanent cure. To selectively kill AML LSCs sparing normal hematopoietic stem cells (HSCs), one of the most practical approaches is to target the AML LSCs-specific surface or functionally indispensable molecules. Based on differential transcriptome analysis of prospectively-purified CD34+CD38− LSCs from AML patient samples and normal HSCs, we found that T-cell immunoglobulin mucin-3 (TIM-3) was highly expressed in AML LSCs but not in normal HSCs (Kikushige et al., Cell Stem Cell, 2010). In normal hematopoiesis, TIM-3 is mainly expressed in mature monocytes and a fraction of NK cells, but not in granulocytes, T cells or B cells. In the bone marrow, TIM-3 is expressed only in a fraction of granulocyte/macrophage progenitors (GMPs) at a low level, but not in HSCs, common myeloid progenitors, or megakaryocyte/erythrocyte progenitors. In contrast, in human AML, TIM-3 was expressed on cell surface of the vast majority of CD34+CD38− LSCs and CD34+CD38+ leukemic progenitors in AML of most FAB types, except for acute promyelocytic leukemia (M3). FACS-sorted TIM-3+ but not TIM-3− AML cells reconstituted human AML in the immunodeficient mice, indicating that the TIM-3+ population contains most of functional LSCs. To selectively eradicate TIM-3-expressing AML LSCs, we established an anti-human TIM-3 mouse IgG2a antibody, ATIK2a, possessing antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities in leukemia cell lines transfected with TIM-3. We first tested the effect of ATIK2a treatment on reconstitution of normal HSCs in a xenograft model. ATIK2a was intraperitoneally injected to the mice once a week after 12 hours of transplantation of human CD34+ cells. Injection of ATIK2a did not affect reconstitution of normal human hematopoiesis except removing TIM-3-expressing mature monocytes. In contrast, injection of TIM-3 to the mice transplanted with human AML samples markedly reduced leukemic repopulation. In some mice transplanted with AML bone marrow, only normal hematopoiesis was reconstituted after anti-TIM-3 antibody treatment, suggesting that the antibody selectively killed AML cells, sparing residual normal HSCs. To further test the inhibitory effect of ATIK2a on established human AML, eight weeks after transplantation of human AML cells, engraftment of human AML cells was confirmed by blood sampling and thereafter ATIK2a was injected to these mice. In all cases tested, ATIK2a treatment significantly reduced human TIM-3+ AML fraction as well as the CD34+CD38− LSCs fraction. In addition, to verify the anti-AML LSCs effect of ATIK2a treatment, human CD45+AML cells from the primary recipients were re-transplanted into secondary recipients. All mice transplanted from primary recipients treated with control IgG developed AML, whereas none of mice transplanted with cells from ATIK2a-treated primary recipients developed AML, suggesting that functional LSCs were effectively eliminated by ATIK2a treatment in primary recipients.Thus, TIM-3 is a promising surface molecule to target AML LSCs. Our experiments strongly suggest that targeting this molecule by monoclonal antibody treatment is a practical approach to eradicate human AML. Disclosures:No relevant conflicts of interest to declare.

Surprise! HSC Are Aberrant in Chronic Lymphocytic Leukemia

In this issue of Cancer Cell, Kikushige etal. report the surprising finding that, in human chronic lymphocytic leukemia (CLL), hematopoietic stem cells (HSC) aberrantly generate clonal B cells with CLL-like phenotypes, which implicate HSC in the pathogenesis of this mature lymphoid malignancy and has major implications for CLL therapies.

Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker

Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.

Use of MGMT Resistance to Enrich for Transduced Hematopoietic Cells in Gene Therapy for Fabry Disease.

Abstract 3584Poster Board III-521Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A (α-gal A) activity that results in the widespread accumulation of neutral glycosphingolipids. Renal failure, neuropathy, premature myocardial infarction, and stroke occur in patients with this condition due to deposition of globotriaosylceramide (Gb3) in vascular endothelial cells. Gb3 primarily originates from the breakdown of red blood cells. Currently, Fabry disease is mainly treated by enzyme replacement therapy (ERT). This treatment represents only a short-term therapeutic strategy and does not solve the basic genetic defect of the disorder. Fabry patients have also shown variable and muted response to ERT. Genetically-modified hematopoietic stem cells (HSCs) are attractive targets for durable transgene expression, however lower transduction efficiencies may be an obstacle to attainment of successful therapy. Therefore, our goal is to design and implement a recombinant lentiviral vector (LV) to confer drug tolerance to modified Fabry patient HSCs in order to enrich them in vivo. O6-methylguanine-DNA-methyltransferase (MGMT) overexpression provides cellular resistance to alkylating agents, which can be administered to kill residual untransduced HSCs while modified cells are protected. To this end, we have developed two different LV constructs. The first iteration was represented by a bicistronic LV encoding the human α-gal A cDNA followed by an EMCV-IRES element and a mutated, but functional, form of the selectable marker (MGMT-P140K). Next, a second iteration has been developed with the order of the transgenes reversed and carrying a codon-optimized copy of the human α-gal A cDNA. Expression and functionality of both cassettes were tested in the human erythroleukaemic cell line (K562) by intracellular MGMT staining and α-gal A activity assays, before and after selection with O6-benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that the second construct allowed sufficient MGMT expression for selection of transduced cells while maintaining α-gal A expression at appreciable levels. Flow cytometry analysis revealed that one drug treatment was sufficient to increase the MGMT-positive cell population from 15% to 90%, which resulted in a 2-fold increase in the enzyme activity. However, two treatments were required for achieving a similar result with the LV of the first iteration. The second LV also facilitated enrichment of transduced Fabry B cells in vitro. Future directions on this project include the modification of Fabry patient bone marrow cells, transplantation into a novel xenograft mouse model that we have recently created, and selection and amplification of the modified cells in vivo. Considering that clinical evidence suggests that even a slight increase in α-gal A activity may help to correct the disease, these results suggest that the MGMT-based drug selection system holds promise for providing a cure for Fabry Disease with a single gene therapy treatment. Disclosures:No relevant conflicts of interest to declare.

In Vivo Treatment with Chromatin Modifying Agents Dramatically Increases Hematopoietic Stem Cell Numbers.

Abstract 370▪▪This icon denotes an abstract that is clinically relevant.The effects of aging in hematopoietic stem cells (HSC) are still unclear. The experimental evidence suggests a progressive decline in immune function and a possible qualitative or quantitative defect of HSC with aging resulting in ineffective production of functional blood cells in some individuals. This is evidenced by the increased incidence of diseases (i.e. aplastic anemia, hypoplastic myelodysplastic syndrome) characterized by a failure to produce optimal numbers of functional blood cells with advancing age. In order to directly assess functional potency of HSC with aging, HSC from young (27 yo) and elderly (68 yo) healthy human subjects were injected simultaneously into humanized mice. Our results demonstrated that an equal dose of BM CD34+ cells (3.75 × 104) from young individuals always out competed grafts from elderly subjects in a true competitive repopulation assay. We have previously demonstrated that ex vivo treatment of cord blood (CB) CD34+ cells using a hypomethylating drug, 5aza-2-deoxycytidine (5azaD), and an HDAC inhibitor, trichostatin A (TSA), augments the number of repopulating HSC (Araki et al, Blood 2007). We hypothesized whether administration of chromatin modifying agents (5azaD/TSA) in vivo in non-toxic doses could similarly reactivate genes critical for expansion of residual HSCs of a host. To test this hypothesis we have examined whether administration of this therapy (5azaD/TSA) augments human hematopoietic engraftment established from a low number of transplanted HSC in a xenotransplantation assay (NOD/SCID mice). Our results indicate that sequential administration of 5azaD/TSA in vivo 4 to 5 weeks after transplantation can boost the degree of human HSC engraftment. Briefly, when we injected 2 × 104 CD34+CD90+ CB cells and examined the BM of NOD/SCID mice 8-12 weeks after transplantation, the human hematopoietic engraftment was 5.37% in control (n = 11) and 9.26% (n = 17) in mice receiving 5azaD/TSA as measured by flow cytometry (human CD45+, CD33+, CD71+ cells). Similarly when 4 × 104 CD34+CD90+ CB cells were injected the degree of hematopoietic engraftment in 5azaD/TSA treated mice was significantly greater than in the control group (n = 5 each group, 4.03% vs. 27.99%, p = 0.02). In additional experiments, in which the NK cell function of the host was blocked by TM-bf° antibody, the degree of multilinage hematoipoietic chimerism was more pronounced in mice treated with 5azaD/TSA (n = 5 each group, control = 0.88 % vs. 5azaD/TSA = 47.9%, p = 0.0009). In order to further confirm that in vivo administration of 5azaD/TSA is capable of augmenting the number of repopulating HSC, we performed serial transplantation studies. We observed that in vivo treatment of 5azaD/TSA of the primary host can cause successful human hematopoietic engraftment of the secondary (2 out of 4 mice) and tertiary hosts (2 out of 3 mice). These results are consistent with our earlier studies, in which ex-vivo treatment of CB HSC using 5azaD/TSA resulted in an absolute increase in the number of repopulating HSC which was associated with an increase in the transcript and protein levels of genes (HoxB4, Bmi-1 etc.) implicated in self-renewal of HSC (Araki et al. Blood 2007). In summary, our results indicate that in vivo intervention via epigenetic mechanisms may potentially provide new treatment modalities to boost the HSC pool in bone marrow failure or poor post transplant engraftment. Disclosures:No relevant conflicts of interest to declare.

N-Cadherin-Mediated Microenvironmental Interactions Protect CML Stem Cells from Imatinib Mediated Apoptosis.

The therapeutic success of Imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by incomplete elimination of malignant hematopoietic stem cells (HSC). The bone marrow microenvironment supports the self-renewal, proliferation and survival of HSC. We investigated whether microenvironmental interactions protected CML HSPC from elimination by IM. CML CD34+CD38− primitive and CD34+CD38+ committed progenitors were cultured for 96 hours with IM (5μM) and with or without irradiated hTERT-immortalized primary human marrow stromal cells. Stroma coculture effectively protected CML progenitors from IM-induced apoptosis (20±6% apoptosis of CD38− cells without stroma and 4±1% apoptosis with stroma, p=0.04, n=4; 13±4% apoptosis of CD38+ cells without stroma and 7±1% apoptosis with stroma, p=0.04, n=4). Stroma coculture did not affect IM-mediated inhibition of CML progenitor proliferation. CML CD34+ cells cultured with and without stroma were injected into irradiated NOD-SCID-IL2Rγ- chain deleted (NSG) mice. Prior coculture with stroma prior to injection resulted in significantly increased engraftment of CML cells in the BM and spleen of NSG mice both at 4 weeks (short-term repopulation) and 10 weeks (long-term repopulation) after transplantation, both with and without IM treatment (without IM, 53±9% human cells in BM after 4 weeks with stroma and 27±5% without stroma, p=0.02, n=4; with IM, 13±2% with stroma and 5±2% without stroma, p=0.01, n=4). Q-PCR analysis showed that stroma coculture was associated with increased engraftment of BCR-ABL+ cells both with and without IM treatment. Culture of CML CD34+ cells in Transwell inserts (0.45μm pores) over stromal layers resulted in partial reversal of protective effect of stroma, suggesting that direct stromal contact contributed to protection of CML progenitors from IM-induced apoptosis (13±2% apoptosis with stroma, 20±5% with Transwell, and 30±3% without stroma). N-cadherin, a cell-cell adhesion protein expressed on HSPC and stromal cells, may contribute to drug resistance in Ph+ lymphoblastic leukemia. Blocking antibodies (Ab) to N-cadherin partially reversed the protective effect of stroma on CML cells treated with IM (33±2% apoptosis with anti-N-cadherin Ab, 26±4% without Ab, p=0.01, n=3). Similarly an N-cadherin-blocking cyclical peptide enhanced apoptosis of IM-treated CML progenitors cultured on stroma (31±4% apoptosis with N-cadherin peptide, 26±3% with control peptide, p=0.03, n=3). CML progenitors cultured on N-cadherin coated plates showed modestly reduced apoptosis compared with controls indicating that N-Cadherin may also directly transduce anti-apoptotic signals (24±4% apoptosis with N-cadherin, 28±3% with BSA, p=0.001, n=4). A minority of CML progenitors expressed N-cadherin on the cell surface. Expression was higher in CD38− cells (17%) compared with CD38+ cells (6%). The percentage of cell surface N-cadherin +ve cells increased after IM treatment in the presence of stroma but not after culture with stroma or IM alone (no stroma no IM 6.5%, stroma alone 6.8%, IM alone 9.9%, stroma and IM both17.8%). We show that purified surface N-cadherin +ve CML CD34+ cells are capable of engrafting NSG mice (0.9±0.4% human cells for N-cadherin +ve and 0.6±0.2% for N-cadherin -ve cells, p=0.12, n=4). Interestingly, surface N-cadherin -ve cells also expressed N-cadherin RNA on Q-PCR, and antibody labeling after fixation and permeablization indicated that a large proportion of these cells expressed intracellular N-cadherin protein, suggesting that they could potentially express surface N-cadherin. In conclusion, our studies indicate that the marrow microenvironment enhances CML HSPC preservation and protects CML HSC from pro-apoptotic effects of IM treatment through direct cell-cell contact-mediated interactions mediated at least in part through N-cadherin. Measures aimed at interfering with N-cadherin mediated microenvironmental interactions could enhance eradication of residual malignant HSC in CML patients receiving IM treatment.

Enrichment of Hematopoietic Stem Cells from Normal and Trp53 Null Mice Using SLAM Receptor CD150 as a Positive Selection Marker.

CD150 is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors which can be used as a positive selection marker for hematopoietic stem cells (HSCs) from mouse bone marrow (BM) and fetal liver (Kiel et al., Cell 2005; Yilmaz et al., Blood 2006; Kim et al., Blood 2006). In the current study, we combined CD150 with a set of lineage-specific markers to enrich HSCs, comparing normal C57BL/6 (B6) mice with B6-Trp53-deficient (Trp53 null) mice which were previously shown to have elevated HSC activity. Using an anti-mouse CD150 antibody (Clone TC 15-12F 12.2, BioLegend), we defined a population of Lin−CD41−CD48−CD150+(SLAM) cells that is 0.0078 ± 0.0010% and 0.0135 ± 0.0010% of total BM cells from B6 and Trp53 null mice. The size of the SLAM cell fraction was strongly correlated (R2= 0.7116, P<0.0013) to the population of well defined Lin−Sca1+CD117+ (LSK) cells (at 0.0165 ± 0.0036% and 0.0276 ± 0.0036% respectively) for the same B6 and Trp53 null mice we have tested (results consistent with previous findings indicating that the HSC pool is larger in Trp53 null mice than in B6 mice). To test HSC function in vivo, we sorted SLAM cells and Lin−CD41−CD48−CD150+ (SLAM−) cells from B6 and Trp53 null donors and transplanted them into lethally irradiated (11 Gys) B6 recipients. In a day 12 colony forming unit-spleen (CFU-S) assay, both SLAM and SLAM−cells formed colonies but the SLAM cells contained 12–15 fold higher day 12 CFU-S than did SLAM−cells. In a competitive repopulation assay, when donor SLAM and SLAM−cells were mixed with fresh B6-CD45.1 BM cells and engrafted into lethally-irradiated B6 recipients for six months, the density of repopulating units (RUs) was 22.48 ± 12.05 and 11.06 ± 8.78 per thousand SLAM cells from B6 and Trp53 null donors, dramatically higher than the RU density of 0.43 ± 0.09 and 0.55 ± 0.43 per thousand SLAM−cells from the same B6 and Trp53 null donors. When BM cells from the competitive repopulation recipients were serially-transplanted into secondary recipients, the SLAM donor cell contribution increased while the SLAM−donor cell contribution decreased with time when measured at two, six and fourteen weeks in the secondary recipients, indicating that SLAM cells were the primitive HSCs that sustained hematopoiesis long-term. No recipient that received Try53 null SLAM or SLAM−cells died, significantly different from our previous observation that whole BM cells from Trp53 null donors yielded high donor reconstitution in a competitive repopulation assay but 56% recipient died within five months after reconstitution (TeKippe et al., Exp Hematol 2003). SLAM markers may have enriched HSCs by excluding any Try53 null BM cell responsible for early recipient death. Analysis of intracellular reactive oxygen species (ROS) revealed higher proportions of ROS−cells in the CD150+ than in the CD150− BM cell fraction in both B6 and Trp53 null mice, consistent with the hypothesis that HSCs are metabolically inactive and therefore contain lower level of ROS. While SLAM− cells may still contain some residual HSC activity, results from the present study are in strong support of utilization of SLAM receptor CD150 as a positive selection marker for HSCs. The Lin− CD41−CD48−CD150+SLAM combination provides a simple and effective method for HSC enrichment.

Therapeutic Effect of HOXB4-Expanded Stem Cells in Mice with Beta Thalassemia Given a Non-Myeloablative Conditioning Regimen.

The ultimate promise of gene therapy for patients with hemoglobinopathies depends on the development of safe strategies for achieving 2 goals. One is to obtain efficient and permanent correction of the gene defect in autologous hematopoietic stem cells (HSCs). The second is to develop methods for the pre-transplant amplification of transduced HSCs to high levels to ensure that they will outcompete the large residual endogenous HSC population remaining in non-myeloablated hosts (e.g. previous experiments have shown that a minimum of ~5 × 106 normal adult mouse bone marrow (BM) cells (~500 HSC) is required to achieve a level of chimerism of 20% in mice given 200 cGy). The ability of HOXB4 to promote HSC self-renewal divisions in short term culture prior to their use as transplants offers an attractive approach to achieve this latter goal. As a first test we transduced day-4 5FU BM cells from normal mice with a MSCV-HOXB4-IRES-GFP or control MSCV-IRES-GFP virus and then transplanted the cells either before or after 7 days maintenance in vitro into normal recipients given 250 cGy. Mice transplanted with an estimated 50 HSCs immediately after transduction with either virus reached equivalent low levels of chimerism (~10%) showing that HOXB4 does not impart an in vivo selective growth advantage under sublethal conditions. After ex vivo culture, the GFP transduced cells yielded an even lower level of chimerism (~5%), in contrast recipients of cultured HOXB4-transduced cells attained much higher stable levels of lympho-myeloid chimerism (~50%), indicative of a marked expansion of the HSCs pre-transplant and their retention of robust competitive repopulating potential. We then applied this approach to a gene therapy model of severe β-thalassemia in mice bearing a homozygous deletion of the β-major globin gene (β-MDD). To model a transplant of genetically corrected cells, BM cells were harvested from day-4 5FU pre-treated congenic wild-type donors and transduced with the HOXB4 virus. Cells were then cultured for 10 days and the progeny of 200K starting cells transplanted into 3 β-MDD and 4 normal recipients given 200 cGy. Transplantation of 500K freshly harvested day-4 5FU BM cells into 4 similarly conditioned control mice failed to produce significant chimerism (1–3% at 5 months). In contrast, all 4 control recipients of ex vivo expanded HOXB4-transduced cells exhibited significant stable chimerism (21±6% at 5 months). Similar levels of chimerism were also achieved in all 3 β-MDD recipients (18–76%), one of which was sustained at 34% at 5 months (52% in the RBCs). This was associated with substantial improvement in the Hct (36% vs 23% in untreated β-MDD), Hb (10.5 vs 5 g/dl) and RBC morphology. Southern blot analyses performed on 53 individual in vitro-expanded myeloid colonies generated from FACS-selected GFP+ marrow cells from this mouse 2 months post-transplant showed 19 distinct integration patterns indicating reconstitution from polyclonal expanded HSCs. This conclusion was further confirmed by proviral integration site analyses, which identified 13 separate integration sites from 9 colonies that had unique proviral patterns. These data demonstrate the curative potential of ex vivo expanded HSCs in a preclinical model of β-thalassemia treated with non-myeloablative conditioning. They also underscore the potential of HOXB4 as a potent tool to achieve the HSC expansions required.

Proliferative activity of hematopoietic stem cells in long-term cultures of mouse embryonic liver

The proliferative activity of hematopoietic stem cells was studied by the “thymidine suicide” method in organ cultures of mouse embryonic liver. Hematopoiesis was found to continue in these cultures for 2 months. The rate of proliferation starts to fall off only on the 50th day of culture; at the same time there is a sharp decrease in the number of residual hematopoietic stem cells in the cultures.