SCID-repopulating Cells Research Articles

The AC133+CD38−, but not the rhodamine-low, phenotype tracks LTC-IC and SRC function in human cord blood ex vivo expansion cultures

AbstractPhenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture, better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs), we demonstrated that the rhodamine-low phenotype was lost, whereas AC133 expression was retained throughout culture. Furthermore, the AC133+CD38− subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number, and limiting dilution analysis in NOD/SCID mice, showed a 43-fold expansion of the AC133+CD38− subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation, respectively. Thus, AC133+CD38− is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures.

Intra-bone marrow transplantation of human CD34+ cells into NOD/LtSz-scid IL-2rγnull mice permits multilineage engraftment without previous irradiation

Non-irradiated immunodeficient recipients provide the best physiologic setting for revealing hematopoietic stem cell (HSC) functions after xenotransplantion. An approach that efficiently permits the detection of human hematopoietic repopulating cells in non-irradiated recipients is therefore highly desired. We compared side-by-side the ability to reconstitute hematopoiesis via intra-bone marrow transplantation (IBMT) in three commonly used mouse strains avoiding previous irradiation. Non-irradiated NOD/SCID and NOD/SCID (beta2m-/- mouse strains prevent engraftment even after IBMT. In contrast, combining the robustness of the NOD/SCID IL-2Rgamma-/- recipient with the sensitivity of IBMT facilitates the detection, without previous host irradiation, of human SCID-repopulating cells 10 weeks after transplantation. The level of chimerism averaged 14% and multilineage engraftment (lymphoid dominant) was observed consistently in all mice. Analysis of injected and non-injected bones, spleen and peripheral blood demonstrated that engrafting cells were capable of in vivo migration and expansion. Combining the robustness of the NOD/SCID IL-2Rgamma-/- mouse strain with the sensitivity of IBMT strongly facilitates long-term multilineage engraftment and migration for human CD34(+) cells without the need for previous irradiation.

CD318/CUB-domain-containing protein 1 expression on cord blood hematopoietic progenitors

CUB-domain-containing protein 1 (CDCP1)/CD318 is a single transmembrane molecule highly expressed in colorectal cancer and leukemia. It has also been shown to be expressed in hematopoietic progenitor cells. In this study, we analyzed the expression of CD318 on cord blood hematopoietic stem and progenitor cells. Cord blood mononuclear cells were depleted of mature blood cell linage (Lin)-positive cells and then Lin-negative cells were sorted by flow cytometry based on the expression of CD34 and CD318. Analysis of sorted cells by colony-forming assay showed that CD34(+)CD318(+) cells produced more mixed colony forming units and erythroid burst forming unit-derived colonies than CD34(+)CD318(-) cells. These colonies were also produced by CD34(-)CD318(+) and CD34(-)CD318(-) cells, but were generally fewer in number. When sorted cells were cultured on a monolayer of human mesenchymal stem cells, CD34(+)CD318(+) cells proliferated more abundantly than CD34(+)CD318(-) cells, while CD34(-)CD318(+) and CD34(-)CD318(-) cells failed to proliferate. Transplantation of CD34(+)CD318(+) cells into non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice resulted in efficient reconstitution of human cells, indicating that CD34(+)CD318(+) cells possess strong SCID-repopulating cell activity. These findings suggest that the co-expression of CD34 and CD318 identifies the immature character of hematopoietic stem cells.

High Resolution Purification and Characterization of Human Cord Blood-Derived CD34–negative SCID-Repopulating Cells with a Very Immature Phenotype.

Abstract 815We have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with an extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In that study, 13 lineage specific antibodies (Abs), including anti-CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41, CD56, and CD235a, were used to purify CD34− SRCs. A limiting dilution analysis demonstrated the frequency of CD34− SRCs in the 13 lineage-negative (Lin−) CD34− cells to be approximately 1/25,000. In this study, we added 5 more lineage specific Abs, including anti-CD11b, CD33, CD45RA, CD66c, and CD127, in order to highly purify CD34− SRCs. The 18 Lin−CD34− cells showed a homogeneously blast-like morphology, and their incidence in the CB-derived nucleated cells ranged from 0.0002 to 0.001%. The colony-forming capacity of these highly purified 18 Lin−CD34− cells was quite unique, since 50% of the total colony-forming cells (CFCs) were mixed colony-forming cells (CFU-Mix). In contrast, the 18 Lin−CD34+ cells formed myeloid, erythroid, and mixed colonies, however, only <10% of the total CFCs were CFU-Mix. The phenotypic and functional characterizations of these 18 Lin−CD34− cells were then further investigated by cocultures with the HESS-5 murine stromal cell line in the presence of a cocktail of cytokines, such as SCF, flt3 ligand, TPO, IL-3, IL-6, and G-CSF. After 7 days of coculture, the total number of cells was observed to expand by 20 to 30 folds, 40 to 60% of which were consisted of CD34+ cells. Next, we investigated the SRC activity of these 18 Lin−CD34− cells using NOD/Shi-scid mice. When 4×104 18 Lin−CD34− cells were transplanted using IBMI, all 4 mice were highly repopulated with human CD45+ cells, including CD19+ B-lymphoid and CD33+ myeloid cells. In addition, the level of human cell engraftment in the bone marrow (BM) ranged from 93.3 to 97.5% (median, 96.8%), at 12 weeks after the transplantation. Interestingly, the BM cells obtained from the primary engrafted NOD/ Shi-scid mice that received transplants of 1,000 to 2,000 18 Lin−CD34− cells showed a secondary repopulating capacity. Furthermore, a limiting dilution analysis demonstrated the frequency of CD34− SRCs in these 18 Lin−CD34− cells to be approximately 1/1,000. The next approach to characterize the CD34− SRCs with respect to the self-renewal potential as well as the long-term repopulating potential, was to serially analyze the kinetics of engraftment for 24 weeks in the NOD/Shi-scid/IL-2Rgcnull (NOG) mice that received transplants of 2,000 18 Lin−CD34− cells containing only 2 CD34− SRCs (estimated number). All 6 mice showed signs of human cell repopulation (0.4 to 28.9%, median 2.5%) at 5 weeks after the transplantation at the contra-lateral sites of IBMI. The repopulation rates gradually increased, and reached a high level of repopulation (47.0 to 87.9%, median 72.0%) at 18 weeks after the transplantation. These results indicated that CD34− SRCs could thus sustain a long-term human cell repopulation in NOG mice, thereby suggesting that CD34− SRCs are a distinct class of primitive HSCs in comparison to the previously reported CD34+ SRCs. Disclosures:No relevant conflicts of interest to declare.

Sequential Treatment of CD34+ Cells From Patients with Primary Myelofibrosis with Chromatin Modifying Agents Alters the Behavior of JAK2V617F Positive NOD/SCID Marrow Repopulating Cells.

Abstract 1910Poster Board I-933Primary myelofibrosis (PMF) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes which silence critical genes influencing cell proliferation, differentiation, survival and trafficking. The abnormal trafficking of CD34+ cells in PMF has been attributed to reduced expression of CXCR4 (Shi et al, Cancer Res, 2007; Wang et al, Cancer Res, 2009). We examined the effects of treatment with chromatin modifying agents (CMA), including sequential treatment with a DNA methyltransferase inhibitor, decitabine “5-aza-2'-deoxycytidine (5azaD)”, followed by a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA) (5azaD/SAHA), or trichostatin A (5azaD/TSA) on the in vitro and in vivo behavior of PMF CD34+ cells (PCC). Sequential treatment with 5azaD/SAHA resulted in a reduction in the number of total cells, CD34+ cells and assayable hematopoietic progenitor cells (HPC) as compared with PCC treated with cytokines alone. By contrast, treatment of PCC with 5azaD/SAHA led to the generation of 2.4-fold greater numbers of CD34+CXCR4+ cells as compared to PCC exposed to cytokines alone and a 15.8-fold increase of the number of CD34+CXCR4+ cells when compared to input PCC (P<0.05). The 5azaD/SAHA treated PCC had a greater migration capacity (20.2±6.9%) toward SDF-1 as compared with PCC exposed to cytokines alone (6.2±2.9%) or primary PCC (2.0±0.5%). These findings suggest that the abnormal trafficking of PCC might be corrected by treatment with 5azaD/SAHA, which is similar to what we had previously reported with 5azaD/TSA. Exposure of PCC to 5azaD/SAHA resulted in a reduction in the proportion of JAK2V617F+ HPCs (p=0.08, 5azaD/SAHA vs cytokines alone; p<0.05, 5azaD/SAHA vs primary PCC) and a reduction in the proportion of JAK2V617F homozygous HPCs in 3 of the 4 patients studied. We next evaluated the effect of sequential treatment with CMA on PMF SCID repopulating cells (SRC) by transplanting primary PCC, PCC treated with cytokines alone, 5azaD/SAHA or 5azaD/TSA and determined the JAK2V617F allelic burden of hCD45+ cells in the marrow of NOD/SCID/IL2Rγnull mice. Two months following transplantation, 2.9% of the marrow cells were hCD45+ cells in mice receiving primary PCC as compared to 0.2% in mice receiving cells treated with cytokines alone or 5azaD/TSA. The engrafted cells in the BM of recipient mice receiving primary PCC were composed of myeloid cells belonging to multiple hematopoietic lineages (CD33+ cells, 2.8%; Glycophorin A+ cells, 0.5%; CD41a+ cells, 0.1% and CD34+ cells, 0.1%) but not CD19+ or CD3+ cells. The JAK2V617F allelic burden of hCD45+ cells isolated from the BM of these mice was analyzed using real-time quantitative PCR. The percentage of JAK2V617F/JAK2total in the gDNA of hCD45+ cells was 71.5% in mice transplanted with primary PCC, 34.7% in mice transplanted with cells treated with cytokines alone and 6.8% in mice transplanted with PCC treated with 5azaD/TSA. Six months following transplantation, a similar degree of human cell chimerism was detected in the BM of recipient mice receiving primary PCC, cells treated with cytokines alone or 5azaD/SAHA. However, the percentage of hCD45+ cells in peripheral blood (PB) of mice transplanted with either primary PCC (0.25%) or PCC treated with cytokines alone (0.1%) was greater than that observed in mice transplanted with PCC treated with 5azaD/SAHA (0.05%). HCD34+ cells were detected in the PB of mice when primary PCC or PCC treated with cytokines alone were transplanted, 2.8% and 1.7%, respectively, but were not detected in mice transplanted with PCC treated with 5azaD/SAHA. These findings suggest that the abnormal trafficking of hCD34+ cells which characterizes PMF, can be recapitulated in NOD/SCID mice. Furthermore, the percentage of JAK2V617F/JAK2total in the gDNA of BM CD45+ cells was 82.5% and 97.6% in mice transplanted with primary PCC as compared to 27.1% and 41.5% in mice transplanted with PCC treated with cytokines alone but was dramatically reduced to 3.6% and 7.4% in mice transplanted with PCC treated with 5azaD/SAHA. These findings suggest that SRC in PMF are JAK2V617F+ and that JAK2V617F+ SRC can be preferentially suppressed by sequential treatment with CMA, leading to engraftment with JAK2V617F- SRC. Sequential treatment with CMA therefore represents a novel means of treating PMF at the level of a JAK2V617F+ SRC, a primitive HPC which shares many properties with the putative PMF stem cell. Disclosures:No relevant conflicts of interest to declare.

Irradiated Third Party Peripheral Blood Mononuclear Cells Enhance Umbilical Cord Blood Engraftment.

Abstract 3533Poster Board III-470Umbilical Cord Blood (UCB) is a readily available, alternate donor source for allogeneic Hematopoietic Stem Cell (HSC) transplantation and can be used with lower stringency HLA-matching between donor and host. However, only limited numbers of cells are available and non-engraftment or delayed engraftment occurs often when recipients receive single UCB units that contain < 2.5 × 107 TNC/kg recipient body weight. This excludes single UCB as a donor source for many adult patients who might otherwise benefit from an UCB transplant. Both immunologic and non-immunologic mechanisms may contribute to UCB HSC engraftment failure. UCB T-cells are phenotypically and functionally immature with a high percentage of CD45RA+ T cells and T cells are known to facilitate engraftment in human HSC transplantation. Co-transplantation of irradiated T-cells supported engraftment in an immunocompetent mouse transplant model without increasing GvHD (Waller et al. Blood94: 3222,1999).The working hypothesis for his study is that the irradiated third party peripheral blood mononuclear cells (PBMNCs) induce a cytokine or cellular response that facilitates hematopoietic engraftment. Long-term culture initiating cell (LTC-IC) frequencies determined with limiting dilution assays were statistically higher when either purified UCB derived CD34+ cells or CB MNCs cells were plated with irradiated PBMNCs (Figure 1). [Display omitted] To assess the impact of irradiated third party PBMCs on UCB engraftment we utilized sub-lethally irradiated NOD/SCID-IL2Rgnull mice in the presence and absence of irradiated third-party human PBMNCs. After sublethal irradiation (240 cGy) female 8 week-old animals were transplanted with either UCB MNCs corresponding to 8×103 (n=5) or 2.66×103 (n=9), or 8.88×102 CD34+ cells (n=9) with or without co-transplantation of 1×106 irradiated (2500 cGy) PBMNCs, and 4 animals received only irradiated PBMNCs as the control group. Engraftment levels are compared by calculating SCID repopulating cell (SRC) frequency. Co-transplantation of PB MNCs with UCB MNCs provided a steady, statistically significant increase in both short-term and long term hematopoietic engraftment (Figure 2). [Display omitted] In these pre-clinical studies, we have confirmed in vitro effects on LTIC formation and in vivo UCB graft-enhancing effects of irradiated PBMNCs. If applied in clinic, this cellular therapy option might overcome a major barrier to successful allogeneic UCB HSC transplantation: engraftment failure, making the procedure more applicable and safer. Disclosures:No relevant conflicts of interest to declare.

Ex Vivo Expansion of Human Hematopoietic Stem Cells by a Small-Molecule Agonist of c-MPL.

Abstract 500The thrombopoietin (TPO) signal via its receptor, myeloproliferative leukemia virus protooncogene (c-MPL), not only regulates platelet production but also plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). TPO has been characterized as a key factor for human HSCs and applied to ex vivo HSC expansion and gene transduction. However, to our knowledge, the effects of small molecule c-MPL agonists on ex vivo hematopoietic stem cell expansion and long-term hematopoietic reconstitution in vivo have not been explored in detail. In this study, we assumed that some small molecule c-MPL agonists may preferentially activate signals that facilitate self-renewal of HSCs. Thus, we have screened such small-molecule compounds and identified NR-101 as a novel c-MPL agonist compound that selectively stimulates c-MPL signaling. NR-101 exhibited in vitro potency to conduct megakaryocyte differentiation at a comparable level to that of recombinant human TPO (rhTPO). We also evaluated the effects of NR-101 on the ex vivo expansion of CD34+ hematopoietic stem and progenitor cells from human cord blood (hCB). We cultured hCB CD34+ cells in serum-free medium supplemented with rhTPO or NR-101 for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assay. Although the total number of cells cultured with NR-101 was similar to that cultured with rhTPO, the cultures with NR-101 showed >2-fold increase in the number of CD34+CD38- cells and contained 2-fold more high-proliferative-potential colony-forming cells (HPP-CFCs ; >1mm in diameter) compared to those with rhTPO. Correspondingly, SCID-repopulating cells (SRCs) were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34+ cells, and 2.3-fold compared to that with TPO. These data suggest that NR-101 promotes the net expansion of hematopoietic stem and progenitor cells more efficiently than TPO, the natural ligand of c-MPL. To investigate the molecular mechanism of hematopoietic stem and progenitor cell expansion mediated by NR-101, we compared the signaling cascades stimulated by NR-101 with those by rhTPO in UT7/TPO cells (a human megakaryoblastic leukemia cell line that expresses c-MPL). When UT7/TPO cells were stimulated with rhTPO, c-MPL, JAK2, STAT3, STAT5, AKT and p44/p42 MAP kinase were immediately activated by 5 min after stimulation. By contrast, in cells stimulated with NR-101, these signaling molecules except for STAT3 became active at later time point (30 min) and showed sustained activation for significantly longer periods. Of interest, NR-101 scarcely induced STAT3 activation. Gene expression profiling by quantitative real-time PCR revealed that the major targets of the TPO/c-MPL signal are similarly upregulated in UT7/TPO cells stimulated with NR-101. However, HIF-1α protein was accumulated at a higher level and expression of its downstream target genes, including VEGF and Glucose transporter genes, was upregulated more in both UT-7/TPO and hCB CD34+ cells stimulated with NR-101 than those with TPO. Furthermore, we examine the cell cycle status of NR-101-treated cells by detecting BrdU incorporation. The population of CD34+CD38- cells in the G0/G1 phases was significantly greater in cultures with NR-101 than in those with rhTPO. These results indicated that NR-101 activates c-MPL in a manner different from TPO does. In conclusion, we have identified NR-101, a novel nonpeptidyl small-molecule compound, which exhibits a selective and sustained activation of c-MPL. The results reported here demonstrate that NR-101 is sufficient to induce the expansion of human HSCs. The approach using NR-101 will provide a wider range of options and be useful for the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures:No relevant conflicts of interest to declare.

In vivo dynamics of human cord blood-derived CD34− SCID-repopulating cells using intra-bone marrow injection

The identification of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. This study investigated the long-term repopulating capacity and redistribution kinetics of human cord blood-derived CD34(-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) and compared them with those of CD34(+)CD38(+) and CD34(+)CD38(-) SRCs using the intra-bone marrow injection (IBMI) to clarify the characteristics of CD34(-) SRCs. On the basis of the limiting dilution analyses data, estimated numbers of CD34(+)CD38(+), CD34(+)CD38(-), and CD34(-) SRCs were transplanted to NOD/SCID mice by IBMI. The human cell repopulation at the site of injection and the other bones were serially investigated. Interestingly, CD34(+)CD38(+), CD34(+)CD38(-), and CD34(-) SRCs began to migrate to other bones 2 and 5 weeks after the transplantation, respectively. Accordingly, the initiation of migration seemed to differ between the CD34(+) and CD34(-) SRCs. In addition, CD34(+)CD38(+) SRCs only sustained a short-term repopulation. However, both CD34(+)CD38(-) and CD34(-) SRCs had longer-term repopulation capacity. Taken together, these findings showed that CD34(-) SRCs show different in vivo kinetics, thus suggesting that the identified CD34(-) SRCs are a distinct class of primitive HSCs in comparison to the CD34(+)CD38(+) and CD34(+)CD38(-) SRCs.

Low‐oxygen and high‐carbon‐dioxide atmosphere improves the conservation of hematopoietic progenitors in hypothermia

During short-term storage of hematopoietic cells (HCs) at 4°C a substantial decline in number and in functional capacity of progenitors occurs after 3 days. We hypothesized that physiologic O2 and CO2 concentrations of hematopoietic tissue microenvironment (approx. 3% O2 and approx. 6% CO2) could improve cell viability and functionality during storage at 4°C. Mobilized peripheral blood (PB) CD34+ cells from multiple myeloma or non-Hodgkin's lymphoma patients were stored in flasks containing air (approx. 20% O2 and approx. 0.05% CO2) or 3% O2/6% CO2 atmosphere, for 3, 5, and 7 days at 4°C. The total number of cells, the number of cells in G0 or G1 phase of cell cycle, and the apoptosis rate were determined. The functional capacity of stored cells was assessed by the capacity of progenitors to form colonies in methylcellulose (colony-forming cells [CFCs]) and of stem cells to repopulate the bone marrow (BM) of immunodeficient mice (SCI D-repopulating cell [SRC] assay). The total number of viable cells and cells in G1 phase as well as the number of total CFCs were significantly higher at 3% O2/6% CO2 than in air at all time points. Cells in G0 phase and SRC were equally preserved in both conditions. Atmosphere with low O2 and high CO2 concentration (3% O2/6% CO2) in hypothermia (+4°C) during 7 days of storage prevents cell damage and preserves a high number of functional HSCs and progenitors mobilized in PB by granulocyte-colony-stimulating factor.

Wnt/β-Catenin Pathway Affects the Cell-Fate Decisions of Hematopoietic Stem/Progenitor Cells Modulating the Epigenetic States of Various Essential Transcription Factors

Several reports have implicated canonical Wnt/β-catenin pathway in murine and human hematopoietic stem/progenitor cells (HSC/HPCs) ex vivo expansion. In addition, it was demonstrated augmentation of hematopoietic repopulating ability in vivo by post transplantation treatment with an ATP-competitive GSK-3 inhibitor, which leads to activation of intrinsic β-catenin. Conversely, it is also reported that constitutive activation of β-catenin enforced cell cycle entry of murine HSCs, thereby, exhausting the long-term repopulating cell pool and leading to hematopoietic failure associated with loss of multilineage differentiation. In this way, the precise roles of individual molecules concerning in canonical Wnt/β-catenin pathway for normal hematopoiesis have not been elucidated. In this study, we examined the effects of GSK-3 inhibition on stem-cell maintenance, progenitor cell expansion, and lineage decisions of murine and human HSC/HPCs. At first, the expression and localization of β-catenin in human CD34+ HSC/HPCs treated with GSK-3 inhibitor 9 (6-bromoindirubin-3-oxime) (GI9) was observed with confocal microscopy. After the treatment for 24 hrs, expression of β-catenin in vehicle-treated (negative control; NC) cells was scarcely detected except for the membrane-bounded form. On the other hand, in GI9-treated cells, β-catenin accumulated in their nucleus in a dose dependent manner. These results suggested that GI9-treatment activates intrinsic β-catenin in human HSC/HPCs. Next, CD34+ HSC/HPCs were cultured for 7 days in a serum-free medium containing with cytokines (SCF, FL, TPO, IL-6 and sIL-6R) and also with 2μM, 10μM of GI9 or vehicle. After 7 days culture, total viable cells and CD34+ cells were expanded 31.6±4.6 and 17.9±3.8 fold in NC cells, respectively (n=3). However, GI9-treatment could not maintain a proportion of CD34+ cells compared with NC significantly caused the growth inhibition in a dose dependent manner. From the analysis of cumulative distribution of first cell division among the cells treated with GI9 or vehicle, GI9-treatment caused delayed cell cycling especially in fractionated immature CD34+CD38− cells. In addition, GSK-3 inhibition lost SCID repopulating cells (SRCs) as tested in the NOD/SCID mouse model (SRCs was calculated to be 1 in 8,452 NC cells vs. in 45,503 GI9-treated cells using limiting dilution methods). These results suggested that activation of intrinsic β-catenin followed GSK-3 inhibition suppressed self-renewal of immature hematopoietic cells via modulating its cell cycle kinetics. Next, as for the multipotency of HSC/HPCs after the culture, the distribution pattern of immunophenotype and the colony forming ability were evaluated. About 80% of expanded cells expressed myeloid marker, CD33 in our culture system, however, GI9-treatment perturbed myeloid differentiation of CD34+ HSC/HPCs but induced the differentiation toward to megakaryocyte and erythroid lineages. Furthermore, in methylcellulose assay, although expanded cells with GI9-treatment generated all types of progenitors, GI9-treatment was inferior significantly in terms of expansion rate of myeloid progenitor, CFU-GM and superior in formation of erythroid progenitor, BFU/CFU-E compared with NC (No. of CFU-GM/1000 cells 151±65.8 vs. 284±17.0, No. of BFU/CFU-E/1000 cells 132±18.5 vs. 32.7.±7.0, respectively) (p<0.05, n=3). Similarly, in murine model, GI9-treatment tended to convert differentiation potential of common myeloid progenitor (CMP) from granulocyte and macrophage progenitor (GMP) to megakaryocyte and erythroid progenitor (MEP). As for this mechanism, we found that activated β-catenin suppresses the transcriptional activity of C/EBPα, which is essential transcription factor for granulocyte development, while it promotes the function of GATA1, essential transcription factor for megakaryocyte and erythrocyte development during the differentiation of HSC/HPCs. In addition, β-catenin competitively impeded the interaction between C/EBPα and its transcriptional coactivator, CBP/p300 in coimmunoprecipitaion analysis. Together, these results indicated that intrinsic β-catenin was supposed to play an important role in self-renewal and multipotency of HSC/HPCs and control the balance of lineage commitment of HSC/HPCs for normal hematopoiesis, presumably by regulating the interaction with essential transcription factors.

Expansion of Cord Blood Stem Cells Mediated by Epigenetic Mechanisms Remain Permissive to External Environmental Cues

Widespread use of ex vivo expanded hematopoietic stem cells (HSC) has been largely limited by the lack of proper understanding of factors regulating symmetric self-renewing HSC divisions. We have previously reported that the addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase inhibitor, trichostatin A (TSA) in the culture is capable of expanding cord blood (CB) HSC as detected by in vivo SCID repopulating cells (SRC) assay in immunodeficient mice. The increase in SRC during ex vivo expansion culture was associated with greater transcript and protein products of genes implicated in HSC self-renewal (Araki et al. Blood 2007). In order to determine whether variation of exogenous cytokine cocktails added in the culture influences the degree of expansion of HSC treated with 5azaD/TSA, we have cultured CD34+ CB cells in the presence of various cytokine combinations. Interestingly, despite treatment of CB cells with 5azaD/TSA the expansion of stem/progenitor cells varied greatly, depending on the combinations of cytokines used in the culture, ranging between 5 to 12 fold differance. The cytokine combination containing stem cell factor (SCF), Flt3-ligand (FL) and thrombopoietin (TPO) was found to promote maximal expansion of primitive CD34+CD90+ cells following treatment with 5azaD/TSA in comparison to other cytokine combinations used (GM-CSF+SCF+IL-3+IL-6+EPO, SCF+FL+TPO+IL-3, SCF+FL+TPO+IL-6, SCF+FL+TPO+IL-3+IL-6, SCF+IL-3+IL-6). Our results also indicate the importance of sequential addition of 5azaD followed by TSA for the net expansion of HSC. Reversal of the sequence of addition of 5azaD and TSA (TSA followed by 5azaD) resulted in almost complete abrogation of the expansion of primitive CD34+CD90+ cells, and this loss of expansion corresponded with decreased acetylation of histone H4. We have further demonstrated that despite pre-treatment with sequential 5azaD/TSA, various cytokine cocktails in the culture can affect the rate of CD34+CD90+ cell divisions which influences both in vitro clonogenic potential and in vivo SRC potential. The higher in vivo hematopoietic engraftment potential of 5azaD/TSA treated cells in the presence of the optimal cytokine combination (SCF+FL+TPO) is likely due to expansion of a relatively primitive HSC population in the culture which divides slower than the cells expanded in the presence of other cytokine combinations (i.e. SCF+FL+TPO+IL-3+IL-6). Further studies will be needed to understand the molecular mechanism of the loss of functional potential depending on culture conditions. Thus far, in a transwell culture system, CD34+CD90+ cells that have been expanded with 5azaD/TSA show greater migration potential towards stroma derived factor (SDF-1) than CD34+CD90+ cells that have been expanded in cytokines alone without 5azaD/TSA treatment. Most importantly the fraction of migrating cells present in the 5azaD/TSA treated expanded culture was comparable to unmanipulated primary CB CD34+ cells, a likely factor contributing to better engraftment in an immunodeficient mouse model. Our current studies indicate that HSC remain responsive to external humoral influences even after treatment with chromatin modifying agents. The relatively slower cell division rate of CB cells in the presence of 5azaD/TSA might be a critical determinant for the retention of HSC functional capability following ex vivo expansion.

Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells

Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI 112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.

Mesenchymal Stromal Cell Co-Culture Enhances Insulin-Like Growth Factor Binding Protein 2 - Augmented Ex Vivo Cultures of Human Cord Blood in a Viability Supporting, Contact-Dependent Process Involving the Rescue of Cells from Early Apoptosis

The use of cord blood (CB) for adult transplantation is limited by the number of hematopoietic stem (HSC) and progenitor cells (HPC) residing in the CB. Successful ex vivo expansion of HSC and HPC may overcome this limitation. An ex vivo expansion system (Zhang et. al., 2008, Blood) based on the additional of Angiopoietin-like 5 and/or Insulin-like Growth Factor Binding Protein 2 (IGFBP2) to a standard cytokine cocktail of stem cell factor, thrombopoietin and FLT3-ligand has recently been reported to expand CB HSC by ~20-fold in a SCID-repopulating cell (SRC) assay. In this study, we further investigated if co-culture of the CB cells with mesenchymal stromal cells (MSC) would enhance the ex vivo expansion of CB in IGFBP2-augmented cultures. Using thawed unselected CB cells, IGFBP2-augmented cultures were able to expand CD45(+)/CD34(+) cells 49-fold and retain in vitro colony-forming units granulocyte-macrophage (CFU-GM) colony size with a concomitant 15-fold increase in number. Under the same conditions, co-culture with MSC resulted in a further 2.9-fold enhancement of CD45(+)/CD34(+) cell expansion (144-fold expansion) and a 2.2-fold enhancement of CFU-GM expansion (33- fold expansion). The numbers reflect true expansion of cell numbers as no prior CD34/ CD133 selection was employed. We further studied the mechanism of MSC enhancement of HSC and HPC expansion. Rather than an increase in cellular proliferation rate, the MSC enhancement effect was associated with 2- to 5-fold increase in viability of a population of lymphocyte-like cells [FS(low) SS(low) CD45(+)] in CB, as determined by annexin-V/7AAD staining. Moreover, direct contact with MSC was necessary for optimal expansion and viability support as shown by use of conditioned media and transwell assays. Thawed mononucleated CB cells exhibited a high level of positivity for Annexin-V, which translated to subsequent 7AAD positivity during ex vivo expansion. MSC co-culture resulted in reversion of Annexin-V positivity within 3–6 hours, and reached a maximum on day 3 (80–90% viability with MSC versus 40–60% without, as defined by double negative staining for Annexin-V and 7AAD). Concomitantly, the loss of mitochondrial membrane potential as determined by JC-1 was also reduced by MSC co-culture. There was a reduction of Caspase 9 activity (12.7% with MSC versus 23.8% without) at day 3 and a reduction in Caspase 3 and 7 activity (5.5% with MSC versus 15.5% without) at day 7 with MSC co-culture. In further experiments, an increase of lysosomal activity in CB, as determined by acridine orange staining, was found to be reduced in MSC co-culture. The use of a non-viable MSC layer did not support CB viability and subsequent expansion. We hypothesized that cytosolic transfer of nutrient or other survival factors from MSC to CB could be a potential mechanism responsible for the rescue of thawed CB from loss of viability and subsequent enhancement in CB ex vivo expansion. Using genetically modified stromal cells expressing with the fluorescent protein containing mitochondria targeting sequence (DsRed-mito) and time-lapse (>10 hours) confocal imaging, we observed potential cytosolic transfer activities between the stromal cells. In conclusion, IGFBP2-augmented cultures result in successful CB expansion, which is further enhanced by MSC co-culture in a viability supporting, contact-dependent process that could involve reversal of cells from early apoptosis possibly mediated through cytosolic transfer of nutrient or cellular material. Further in vivo experimentation is in progress to verify the effect of our combined MSC-IGFBP2 culture system on HSC expansion.

Experimental Double Cord Blood Transplantation (DCBT) of An Expanded Unit Together with a Non-Manipulated Unit in NOD/SCID Mice.

We found that nicotinamide (NAM), a form of VitB3 and a recognized inhibitor of SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase, inhibits in vitro differentiation and promotes expansion of hematopoietic progenitor cells. Cord blood (CB)-derived CD34+ cells cultured with cytokines (FLT3, TPO, IL-6, SCF; 50ng/ml) and NAM (2.5mM) (Sigma Aldrich, catalog number N5535) display enhanced in vitro migratory activity toward SDF-1 and home to the BM (24hr following infusion in vivo) with higher efficacy than cells cultured with cytokines only. The number of SCID-repopulating cells increased by 9- and 7.6-fold in cultures treated with NAM relative to non-cultured cells and cytokine only cultured cells, respectively. This net increase in repopulation potential was sustained in competitive transplant experiments where cultured cells were infused along with non-cultured competitor cells derived from the same CB unit. Several experimental clinical protocols of CB-derived expanded cells involve co-transplantation of cultured and non-cultured cells in a double CB transplantation (DCBT). We therefore sought to investigate the engraftment potential of NAM-treated cultured cells in a DCBT setting in NOD/SCID mice, using two CBUs marked as “unit-1” and “unit-2”. Two similar experiments were conducted, each experiment had 4 groups of mice (n=10/ experimental group):non-cultured cellscells cultured with NAM transplanted along with the CD34 negative cell fraction from the same unit that was kept frozen till the day of transplantation.groups a + bnon-cultured cells from unit-1 transplanted along with non-cultured-cells from unit-2.Mice were transplanted with similar number of nuclear cells from the cultured or non-cultured units in the single or the DCBT groups. In Experiment-1, non-cultured cells were derived from one unit-1 and NAM-treated cultured cells were derived from another unit-2. In Experiment-2, we switched between the units (such an experiment was possible since our CBUs are frozen in several portions). Level of engraftment was evaluated two weeks post transplantation by FACS analysis of human CD45+ cells while the contribution of each unit to engraftment was measured by quantitative PCR for informative short tandem repeat (qSTR) regions that distinguished the units. The results show that in both experiments, the level of engraftment in the DCBT cohort of a cultured unit transplanted along with a non-cultured unit (12.8±1.8 %) was similar to the level of engraftment of the cultured unit when individually transplanted (15.4 % ±3, p>0.05). This level of engraftment was 9.8-fold higher (p<0.05) than the level of engraftment obtained in the DCBT cohort of two non-cultured units (1.3 % ±0.2). Chimerism analysis indicated that in recipients transplanted with two non-cultured units, engraftment was predominantly derived from unit-1, while in the two experiments of DCBT of a cultured unit transplanted along with a non-cultured unit the engraftment was predominantly derived from the cultured unit. In conclusion, the outstanding engraftment advantage of NAM cultured cells may explain their predominance in a DCBT setting.

Improved Granulocyte Colony-Stimulating Factor Mobilization of Hemopoietic Progenitors Using Cytokine Combinations in Primates

Peripheral blood stem cells (PBSCs), usually mobilized with granulocyte colony-stimulating factor (G-CSF) alone or in combination with chemotherapy, are the preferred source of cells for hemopoietic stem cell transplantation. Up to 25% of otherwise eligible transplant recipients fail to harvest adequate PBSCs. Therefore it is important to investigate existing and novel reagents to improve PBSC mobilization. Because of marked interindividual variation in humans, we developed a robust nonhuman primate model that allows the direct comparison of the efficacy of two PBSC mobilization regimens within the same animal. Using this model, we compared pegylated G-CSF (pegG-CSF) with standard G-CSF and compared the combination of G-CSF and pegylated megakaryocyte growth and development factor (pegMGDF) with G-CSF plus stem cell factor (SCF) by measuring the levels of CD34(+) cells, colony-forming cells (CFCs), and SCID repopulating cells (SRCs) before and after cytokine administration. Mobilization of CD34(+) cells, CFCs and SRCs using pegG-CSF achieved similar levels to those resulting from 5 days of standard G-CSF. The combination of G-CSF+pegMGDF mobilized progenitors to levels similar to G-CSF+SCF but greater than standard G-CSF for CD34(+) cells and CFC. This first direct comparison of PBSC mobilization in individual primates demonstrates that peg-G-CSF is equivalent to daily G-CSF and that the addition of pegMGDF to G-CSF improves mobilization. In light of the development of new thrombopoietin agonists, these data offer the potential for improved stem cell mobilization strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues

AbstractPrevious studies revealed that mAb BB9 reacts with a subset of CD34+ human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map BB9 expression throughout hematopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and surrounding endothelial cells are BB9+. Thereafter, BB9 is expressed by primitive hematopoietic cells in fetal liver and in umbilical cord blood (UCB). BB9+CD34+ UCB cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+BB9− counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population. Protein microsequencing of the 160-kDa band corresponding to the BB9 protein established its identity as that of somatic angiotensin-converting enzyme (ACE). Although the role of ACE on human HSCs remains to be determined, these studies designate ACE as a hitherto unrecognized marker of human HSCs throughout hematopoietic ontogeny and adulthood.

Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation

AbstractHematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)–repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.

Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab

AbstractBlockade of CD49d-mediated lymphocyte trafficking has been used therapeutically for certain autoimmune diseases, such as multiple sclerosis (MS). In addition to negative effects on the trafficking of mature lymphocytes to sites of inflammation, CD49d blockade in mice and monkeys rapidly mobilizes hematopoietic stem/progenitor cells (HSPCs) capable of short- and long-term engraftment. Here we aimed to ascertain the effects of treatment with antifunctional anti-CD49d antibody in humans (MS patients receiving infusions of the CD49d-blocking antibody natalizumab) on levels of circulating HSPCs after a single dose of antibody or after long-term treatment. On average, 6-fold elevated levels of circulating CD34+ cells and colony-forming unit-culture (CFU-C) were achieved within 1 day of the first dose of natalizumab, and similar levels were continuously maintained under monthly natalizumab infusions. The blood of natalizumab-treated subjects also contained SCID-repopulating cells. The fate of these circulating HSPCs and their clinical relevance for MS patients remains to be determined.

Hematopoietic Stem/Progenitor Cells (HSPC) Mobilization Parameters in Patients Chronically Treated with the CD49d Blocking Antibody Natalizumab.

Blockade of CD49d-mediated lymphocyte trafficking has been used therapeutically for certain autoimmune diseases. In addition to effects on mature lymphocytes, in mice and monkeys CD49d blockade also mobilizes immature hematopoietic cells from bone marrow (BM), capable of complete long-term engraftment despite a partial BM seeding defect. The aim of these studies was to ascertain effects on the biology of HSPC mobilization of single or chronic CD49d blockade in Multiple Sclerosis (MS) patients receiving disease-modifying treatment with the anti-functional anti-CD49d antibody, Natalizumab. These studies represent the first observations on HSPC mobilization by anti-CD49d antibodies in humans and on chronic exposure to a mobilizing agent. Circulating CD34+ cells (1.8±0.4/μL) and CFU-C (638±128/mL) were normal in MS patients (n=9) prior to the first Natalizumab infusion (normal controls: 1.3±0.1/μL CD34+ cells, 608±129/mL CFU-C). In chronically treated patients (i.e patients who had received ≥5 prior doses of Natalizumab) HSPC numbers were significantly elevated 30 days after the last infusion (CD34+ cells 9.0±1.2/μL, CFU-C 3243±332/mL, n=10, p<0.005). The blood of chronically Natalizumab-treated subjects also contained SCID-repopulating cells (n=3). After the first Natalizumab infusion, peripheral blood CD34+ cells and CFU-C were significantly increased over pre-treatment values (CD34+ cells 1.6±0.2/μL vs. 8.0±2.1/μL, CFU-C 414±161/mL vs. 2560±726/mL, n=7, p<0.005), indicating that a single dose of Natalizumab was sufficient to elicit similar mobilization levels as in chronic recipients. Renewed Natalizumab infusion in “chronic” Natalizumab-recipients did not result in additional mobilization, compared to values from just before that infusion (CD34+ cells 7.9±1.7/μL vs. 7.9±0.9/μL, CFU-C 3133±335/mL vs. 3525±305/mL, n=4). These data indicate that in the absence of CD49d-mediated BM retention a new equilibrium is established between HSPC in BM and in circulation. In long-term Natalizumab recipients a total of ca. 50x10E6 CD34+ cells (8 CD34+ cells/μL * 6L of blood) are in circulation at any given time. Considering the documented short half-life of circulating HSPC, (100 min in the mouse) this implies that approximately 700x10E6 CD34+ cells/day are trafficking in the average Natalizumab recipient. Whether the elevated numbers of circulating HSPC play a clinically relevant role for Natalizumab-treated MS patients is currently unclear. Given reports of MS flares under G-CSF, we propose that “steady-state” apheresis without additional mobilizing agents may be a safe and effective alternative to G-CSF if MS patients on Natalizumab are considered for autologous HSPC transplantation, an emerging treatment for refractory MS. The usefulness of Natalizumab as a mobilizing agent is likely restricted to the autologous setting, given the risk of immunization against Natalizumab and its prolonged effects. Instead, short-acting small-molecule inhibitors of CD49d could be developed as a mobilizing strategy for patient groups intolerant or unresponsive to G-CSF, alone or in combination with other novel mobilizing agents.

Normal Short-Term but Reduced Long-Term Engraftment Capacity of CML Hematopoietic Cells with Skewed Myeloid Lineage Differentiation Is Seen in an Improved Mouse Model of Human Hematopoiesis.

The NOD/SCID mouse model has been widely used to assay human hematopoietic cells capable of long-term multilineage engraftment (SCID-repopulating cells or SRC). Leukemia cells from CML patients can also engraft in NOD-SCID mice. However the utility of this model for studying CML stem cells is limited by inconsistent and low levels of engraftment, and lack of a leukemia-related phenotype of engrafted cells. Since NOD/SCID IL2Rγchain KO (NOG) mice support superior engraftment of human hematopoietic cells compared with NOD/SCID mice, they may allow for improved evaluation of CML stem cells. In the current study we investigated engraftment of CML CD34+ cells in the NOG mouse model. CD34+ cells were transplanted into irradiated (300 cGy) 8 wks old NOG mice via tail vein injection. Blood samples were obtained at 4 wks intervals to monitor human cell engraftment. At the end of the maintenance period of 16–18 wks, animals were euthanized and marrow content of femora, spleen cells and peripheral blood were obtained. Human cell engraftment was analyzed by flow cytometry after labeling cells with anti-human CD45 mAb and specific human cell subsets detected by staining with human CD34, CD33, CD14, CD11b, CD19 and CD3 mAbs. Establishment of human hematopoiesis was consistently seen 4 weeks after injection. Larger numbers of CML (1–2 x106) compared to normal bone marrow (1–8x105) CD34+ cells were required to establish engraftment, which is consistent with previous reports. Interestingly CML CD34+ cells established their highest levels of engraftment in blood 4 wks after transplantation (14.9±5.9% CD45+ cells, n=13) and engraftment levels were significantly lower at 8 wks (1.8±0.3%) and beyond. However engraftment was consistently detected in blood up to 16–20 wks. The engraftment kinetics of CML cells was strikingly different from that of normal CD34+ cells which while showing similar levels of engraftment at 4 wks (12.6±3.8%, n=16), showed considerably higher engraftment at 8 wks (19.2±3.8%) with a gradual decline subsequently (11.5±2.7% at 12 wks). Analysis after autopsy at 16–18 wks revealed higher levels of human cell engraftment in mouse BM and spleen compared with blood. CML CD34+ cells showed reduced BM engraftment compared with normal CD34+ cells (7.3±4.8% vs. 22.1±9.0% CD45+ cells, n=7, p<0.01). CML CD34+ cells engrafted with myeloid lineage cells and minimal lymphoid engraftment was seen (CD33: 5.2±3.7%, CD11b: 2.6±1.6% and CD19: 0.1±0.1%), whereas normal CD34+ cells engrafted with both myeloid and lymphoid cells (CD33: 7±3%, CD11b: 1.6±0.6% and CD19: 11±4.3%). We also observed reduced frequency of CD34+ cells in mice receiving CML compared with normal cells (2.6±2.5% vs. 6.2±3.0%, p<0.05). In conclusion we show, using an improved mouse model of human hematopoiesis, that CML SRC have similar short-term but reduced long-term engraftment capacity compared with normal SRC, possibly indicating a preponderance of short-term repopulating cells or increased HSC turnover. CML SRC demonstrated predominantly myeloid differentiation compared with balanced myeloid and lymphoid engraftment of normal SRC. These results support the utility of the NOG mouse model to investigate functional characteristics of CML stem cells and study the activity of specific therapeutic interventions against primitive CML cells.