Phenotypic Hematopoietic Stem Cells Research Articles

FoxO Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress.

Hematopoietic development and long-term homeostasis are tightly regulated by a dynamic balance of stem cell self-renewal and differentiation. To understand the role of the FoxO family of transcription factors in these processes, we studied the impact of somatic deletion of all FoxO genes in the adult hematopoietic system of mice engineered with the interferon-inducible Mx-Cre transgene and various conditional FoxO alleles: FoxO1L/L; FoxO3 L/L; and/or FoxO4 L/L. Cre-mediated excision of FoxO1, FoxO3 and FoxO4 (FoxO1/O3/O4L/L), resulted in myeloid lineage expansion, lymphoid developmental abnormalities and a marked decrease of the lineage-negative, Sca-1+, c-Kit+ (LSK) compartment, containing the hematopoietic stem cell (HSC) population, but normal numbers of myeloid progenitors. Furthermore, FoxO1/O3/O4L/L null bone marrow cells were defective in competitive and noncompetitive repopulation assays. This FoxO deficient defect correlated with enhanced cell cycling and increased apoptosis of HSC cells. Notably, there was an HSC-restricted elevation in the level of reactive oxygen species (ROS) that was not observed in committed progenitors, and was associated with decreased catalase and MnSOD expression. Furthermore, in vivo treatment with the anti-oxidative agent N-acetyl-cysteine (NAC) resulted in complete reversion of the FoxO deficient HSC phenotype. Taken together, these results demonstrate that, in the HSC compartment, FoxO proteins are mediators of quiescence and enhanced survival and play essential regulatory roles in the response to physiologic oxidative stress, a function that may contribute to the long-term regenerative potential of the hematopoietic stem cell compartment.

Rb is dispensable for self-renewal and multilineage differentiation of adult hematopoietic stem cells

Stem cells have been identified as essential for maintaining multiple organ systems, including the hematopoietic system. The distinct cell fates of self-renewal and differentiation of hematopoietic stem cells (HSCs) depend on cell division. Recently, several negative regulators of the cell cycle, such as the cyclin-dependent kinase inhibitors p21(Cip1), p27(Kip1), and p16(INK4a)/p19(ARF), have been demonstrated to have a role in regulating HSC fate decisions, suggesting that regulation of the G(1)-S phase transition can contribute to HSC self-renewal. Because the retinoblastoma protein, Rb, plays a central role in the regulation of the G(1)-S phase cell cycle, we sought to determine whether it has an intrinsic role in the regulation of HSC fate. Surprisingly, we found that HSC function was essentially normal in the absence of Rb. Rb(Delta/Delta) HSCs contributed normally to both myeloid and lymphoid lineages in both primary and secondary recipients, and no evidence of transformation was observed. Additionally, we observed a mild myeloid expansion and decrease in mature B cells within the Rb(Delta/Delta) bone marrow but a similar contribution to phenotypic HSC populations compared with nondeleted bone marrow. The Rb family members p107 and p130 were not deregulated in cells in which Rb had been deleted, as determined by quantitative RT-PCR on the highly enriched stem and primitive progenitor cell lin(-)c-Kit(+)Sca-1(+) population. These studies demonstrate that Rb is not intrinsically required for self-renewal and multilineage differentiation of adult HSCs.

QTL influencing baseline hematocrit in the C57BL/6J and DBA/2J lineage: age-related effects

Baseline serum hematocrit varies substantially in the population. While additive genetic factors account for a large part of this variability, little is known about the genetic architecture underlying the trait. Because hematocrit levels vary with age, it is plausible that quantitative trait loci (QTL) that influence the phenotype also show an age-specific profile. To investigate this possibility, hematocrit was measured in three different age cohorts of mice (150, 450, and 750 days) of the C57BL/6J (B6) and the DBA2/J (D2) lineage. QTL were searched in the B6D2F(2) intercross and the BXD recombinant inbred (RI) strains. The effects of these QTL were explored across the different age groups. On the phenotypic level, baseline serum hematocrit declines with age in a sex-specific manner. In the B6D2F(2) intercross, suggestive QTL that influence the phenotype were located on Chromosomes (Chr) 1, 2, 7, 11, 13, and 16. With the exception of the QTL on Chr 2, all of these QTL exerted their largest effect at 750 days. The QTL on Chr 1, 2, 7, 11 and 16 were confirmed in the BXD RIs in a sex- and age-specific manner. Linkage analysis in the BXD RIs revealed an additional significant QTL on Chr 19. Baseline serum hematocrit is influenced by several QTL that appear to vary with the age and sex of the animal. These QTL primarily overlap with QTL that have been shown to regulate hematopoietic stem cell phenotypes.

The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

Induction of Adipocyte Differentiation and Reduction of Hematopoesis-Supporting Activity of Mouse Stromal Cells by Suppression of Transcription Factor GATA-2.

(Introduction) Aplastic anemia (AA) is characterized by peripheral pancytopenia and fatty bone marrow. An immunological attack to hematopoietic stem cells has been thought to be responsible for the development of the disease. Previously, we reported the expression of transcription factor GATA-2 is significantly decreased in CD34 positive cells in AA. Together with the phenotypes of hematopoietic stem cells in GATA-2 hetero-knockout mice, GATA-2 down-regulation may play a role in the reduction of a stem cell pool observed in AA. On the other hand, GATA-2 has been shown to be essential for the maintenance of immaturity of preadipocytes. If a pathological immune response in AA decreases the level of GATA-2 expression in not only hematopoietic stem cells but also stromal preadipocytes, it may accelerate the maturation of preadipocytes, leading to the formation of fatty bone marrow. To explore this possibility, the phenotypic change of stromal preadipocytes by suppression of GATA-2 was examined in this study.(Method) The GATA-2 expression level was suppressed by using siRNA for GATA-2 in mouse stromal preadipocyte cell lines, TBR9 and TBR343. After the treatment with siRNA, the adipocyte differentiation was induced by the incubation with insulin and dexamethasone for 7days. Then, the maturation level was examined by oil drops formation judged by oil red staining, and by the expression level of adipcin and PPAR-γ mRNA. Supporting activity of hematopoietic colony formation was also evaluated by using mouse fetal liver cells after siRNA treatment.(Results) By using designed siRNA, the GATA-2 expression was suppressed to 30% of control, whereas the expression level of GATA-3, which is co-expressed in preadipocytes, was unchanged. When GATA-2 was suppressed by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated in both of stromal cells. Furthermore, the number of fetal liver hematopoetic colonies was significantly decreased by suppression of GATA-2, suggesting that GATA-2 down-regulation in stromal preadipocytes results in not only the acceleration of the maturation but also the reduced supporting activity of hematopoietic colony formation(Conclusion) These results suggest that suppression of GATA-2 in hematopoietic tissues induces the characteristic features of AA, i.e., decreased the number of hematopoietic stem cells and increased number of mature adipocytes.

Highly Purified Hematopoietic Stem Cells Convert to Cells with Myoblast Characteristics before Forming Myotubes.

We have previously shown robust conversion of marrow cells to skeletal muscle (up to 12 percent) in a regenerative environment. We have also recently shown the presence of marrow derived muscle colonies. Evaluation of GFP fluorescent intensity of fibers in individual colonies and comparing them with the single fibers outside the colonies strongly suggests a clonal origin for these colonies. Cotransplantation of marrow cells from Yellow and Cyan Fluorescent Protein transgenic mice also supported this theory. We studied the type of cells responsible for marrow to muscle conversion in an immunocompetent and an immunocomprosied model of marrow transplant. The choice of two models was to clarify any bias related to immunologic reactivity of GFP positive cells that has been well documented in the literature. In the first model, different population of GFP positive marrow cells were transplanted intravenously, after sublethal doses of radiation into C57BL/6 mice. One week after transplant their tibialis anterior muscle were injured with cardiotoxin and their muscles were evaluated for GFP positive, dystrophin positive and CD45 negative muscle fibers. In the second model, different population of GFP positive marrow cells were injected directly into tibilias anterior of Bege/Scid mice one day after cardiotoxin injury. Similar to the first model, GFP positive muscle fibers were evaluated 4 weeks post injury. For each population of marrow cells 3 to 6 animals were transplant with cells either positive or negative for the specific surface markers. Our data demonstrates that conversion potential of marrow cells to skeletal muscle fibers is highly enriched in a CD45 positive, c-Kit positive, Flk-2 positive and Sca-1 positive population. Furthermore, CD34 negative, CXCR4 negative and lineage negative cells have higher potential to participate in marrow to muscle conversion. This indicates that under the conditions of our experiments, mesenchymal stem cells are not the origin of marrow derived GFP muscle fiber. To follow the fate of marrow cells homed into muscle we transplanted GFP marrow cells into radiated C57BL/6 mice and at different timepoints before and after injury, harvested tibialis anterior muscle of these animals. Isolation of mononuclear cells from these samples revealed a new GFP+ CD45 negative population which was very rare at 1 week but more abundant at 4 weeks after transplant. Real time PCR analysis of these cells showed multiple stem cells markers including Sca-1, endoglin, c-Kit and CD34. They did not express any RNA markers for satellite cells or myoblasts (pax 7, MyoD, Myf 5) or hematopoietic cells (CD45, CD11b). We next injured GFP transplanted animals with cardiotoxin and isolated the same CD45-GFP+ population 3 days after injury. Real time PCR analysis at this point showed RNA activity for myoblast including Myf5, MyoD and desmin. Immunofluorescent staining of Lin-, Sca+ cKit+ marrow cells from GFP transgenic mice injected into cardiotoxin injured tibialis anterior muscle of Bege Scid mice confirmed our RT-PCR data and showed the presence of MyoD, Myf5, Myogenin and desmin in donor derived cells in recipient muscle. These data suggests that marrow derived mononuclear cells with hematopoietic stem cell phenotype in the recipient muscle lose their hematopoietic characteristics and obtain myoblast phenomenon after being exposed to injury.

The Stem Cell Continuum

Hematopoietic stem cells have been felt to exist in a hierarchical structure with a relatively fixed phenotype at each stage of differentiation. Recent studies on the phenotype of the marrow hematopoietic stem cell indicate that it is not a fixed entity, but rather that it fluctuates and shows marked heterogeneity. Past studies have shown that stem cell engraftment characteristics, adhesion protein, and gene expression varies with the phase of the cell cycle. More recently, we demonstrated that progenitor numbers and differentiation potential also vary reversibly during one cytokine-induced cell cycle transit. We have also shown high levels of conversion of marrow cells to skeletal muscle and lung cells, indicating a different level of plasticity. Recently, we demonstrated that homing to lung and conversion to lung cells in a mouse transplant model also fluctuates reversibly with cell cycle transit. This could be considered plasticity squared. These data indicate that marrow stem cells are regulated on a continuum related to the cell cycle both as to hematopoietic and to nonhematopoietic differentiation.

Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38– cells and SCID-repopulating cells

AbstractThe CD34+CD38– phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34+ cells, HSC content is difficult to measure since committed CD34+CD38+ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/severe combined immunodeficiency (SCID)–repopulating cells (SRCs). Contact coculture of fluorescence-activated cell sorter (FACS)–sorted bone marrow (BM) CD34+CD38– cells with human brain endothelial cells (HUBECs) supported a 4.4-fold increase in CD34+CD38– cells with a concordant 3.6-fold increase in SRCs over 7 days. Noncontact HUBEC cultures and the addition of thrombopoietin, stem cell factor (SCF), and macrophage colony stimulating factor I receptor (Fms)–like tyrosine kinase 3 (Flt-3) ligand supported further increases in CD34+CD38– cells (6.4-fold and 13.1-fold), which correlated with significant increases in SRC activity. Moreover, cell-sorting studies performed on HUBEC-cultured populations demonstrated that SRCs were significantly enriched within the CD34+CD38– subset compared with the CD34–CD38– population after culture. These results indicate that human HSCs can be identified and characterized by phenotype following expansion culture. These studies also demonstrate that HUBEC-elaborated soluble factors mediate a unique and potent expansion of human HSCs.

ABCG2 Expression as Prospective Marker for Hematopoeitic Stem Cell Purification.

ABCG2 (BCRP/MXR/ABCP) is an ABC transporter that effluxes Hoechst 33342 and is required for the side population (SP) phenotype of hematopoietic stem cells (HSCs). Although SP cells are enriched for HSC activity, they are not a pure stem cell population and contain a significant number of differentiated and committed cells. We hypothesized that expression of ABCG2 would provide a more specific marker for HSCs, given our prior results showing that other ABC transporters were expressed in committed SP cells. To test this hypothesis, we have generated a mouse strain in which an IRES-GFP element was inserted immediately after the stop codon of Abcg2, so that GFP and the endogenous Abcg2 gene were co-expressed from the same transcriptional regulatory elements. Immunohistochemical analysis using an anti-GFP antibody showed that Abcg2/GFP allele was strongly expressed in proximal tubule cells of kidney, in the epithelial cells of small intestine, and in vascular endothelium, recapitulating the normal expression pattern of Abcg2. Using flow cytometry to analyze GFP expression in bone marrow cells, we found that 48% of SP cells expressed GFP. In the distal tip of the SP region, previously shown to have the highest concentration of HSCs, 93% of cells expressed GFP. In gated Lin- GFP+ cells within the whole bone marrow population, 55 % of the cells expressed c-Kit and Sca1, a previously identified phenotype for HSCs. We sorted Lin- cells into GFP+ and GFP− subpopulations and determined repopulation frequencies in a limiting dilution assay. Fifteen percent of recipient mice were reconstituted with 10 Lin- GFP+ cells, and 50% mice with 60 Lin- GFP+ cells. In groups transplanted with 125 to 1000 Lin- GFP+ cells, 100% of the mice were reconstituted. In contrast, no reconstitution was seen in any mice transplanted with 60,000 Lin− GFP− cells, indicating that most if not all HSCs expressed the Abcg2/GFP allele. Secondary transplant experiments and individual lineage analysis of peripheral blood cells confirmed that the Lin- GFP+ cells were long-term HSCs. These results demonstrate that expression of Abcg2 in Lin- cells defines a highly enriched population of HSCs, and is a more specific HSC marker than the SP phenotype. We are now increasing the stringency of the GFP sorting gate and transplanting mice with single cells to determine if a pure HSC population can be isolated with this simple 2 marker system.

Maintenance of Earlier Hematopoietic Stem Cell (HSC) Phenotype and Improved NOD/SCID Engraftment for UCB Expanded Short-Term in Cytokines over a Human Mesenchymal Stem Cell (huMSC) Platform.

Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer:UnexpandedMSC+ cytokinesCytokinesCD340.68 (.068M)0.74 (3.63M)1.94 (5.39M)CD1335.69 (.569M)2.56 (12.54M)0.74 (2.06M)CD349.6 (4.96M)2.2 (10.78M)0.42 (1.17M)CD5617.4 (1.74M)2.71 (13.28M)1.06 (2.95M)CD690.80 (7.28M)7.28 (35.67M)24.4 (67.8M)UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

Expression Profile of Aging Hematopoietic Stem Cells.

A decline in stem cell function has been suggested to contribute to vertebrate aging. Several labs have documented a reduction in transplant efficiency and skewing in lineage contribution when murine bone marrow or hematopoietic stem cells (HSC) from old donors were transplanted into young recipients. Paradoxically, evidence from several labs including ours has shown that the percentage of phenotypically defined HSC in C57Bl/6 mice increases with age (Fig. 1). Within the serum of aged animals, systemic inflammatory markers such as C reactive protein in humans and IL-1 in mice have been shown to steadily increase with age, however it is unclear if systemic inflammation plays a role in these age-related HSC phenotypes. In order to investigate what might account for these functional changes in HSC aging, we have characterized gene expression in aged HSC using Affymetrix microarrays, examining expression profiles of HSC purified from C57Bl/6 mice that are 2-, 6-, 12-, 21-months old. Using polynomial regression over the time course, we have found more than 700 genes that are 2-fold up-regulated and more than 400 genes that are down-regulated with time. We used the Gene Ontology to categorize age-regulated genes, and have identified the category of ‘inflammatory response' to be significantly enriched in genes that are up-regulated with age. Strikingly, several NF-Kb regulated genes, previously associated in other tissues with aging and inflammation (e.g. P-selectin, Cox-2, and ICAM-1) are up-regulated in aged HSC. The expression patterns of more than 15 genes, including clusterin, serum deprivation response, and growth hormone receptor, have been validated by quantitative real-time PCR. Furthermore, expression of several surface markers, including P-selectin, a protein that plays a role in inflammation, has been validated at the protein level by flow cytometry. In order to determine if the gene expression changes observed in aged HSC is caused by intrinsic or extrinsic factors, we have transplanted HSC from 21-month-old mice into young recipients and are assessing changes in their expression profile compared to young transplanted HSC. These data demonstrate a role for inflammation in HSC-aging, and may identify genes involved in stem cell transplant efficiency, lineage specification, and the onset of organismal aging. [Display omitted]

Phenotypic and Molecular Characterization of Bone Marrow L-NGFR + Mesenchymal Stem Cells.

In a previous report we demonstrated that the immunomagnetic sorting of bone marrow (BM) cells labeled with low-affinity nerve growth factor receptor (L-NGFR) antibodies allows the selection of phenotypically and functionally homogeneous cells that are capable of expansion, self-renewal and differentiation into multiple mesenchymal cells lineages. Furthermore, we reported the presence of a subpopulation of L-NGFR+ cells coexpressing CD133 and CD34, markers associated with a primitive hematopoietic stem cell phenotype. In the present study we expanded on the phenotypic characterization of these cells and investigated their potential for multilineage differentiation.BM L-NGFR+ cells were analyzed by flow cytometry immediately after immunoseparation and the expression of a variety of stem cell markers was studied. In 12 subsequent experiments L-NGFR+ cells expressed CD45low (97.5% ±3), CD34 (19.9%±13), CD133 (10.4%±6), CD105 (46.8%±36%), P1H12 (50.5%±18), KDR (34%±18) and SSEA-3 (0.47%±0.41). In addition L-NGFR+ expressed high levels of the SCF ligand CD117 (40%±16%). As we previously demonstrated, L-NGFR antibodies identify a subpopulation of cells with a high proliferative capacity and potential for multilineage differentiation along the mesenchymal lineage. We now show, in accordance to these phenotypic data, that the L-NGFR+ cells in the presence of SCF (100 ng/ml) doubled the number of CFU-F and expanded both adipocytic and osteoblastic differentiation in comparison to mesenchymal cultures without growth factors or supplemented with Flt-3L+IL-6 (both 100 ng/ml). SCF seems therefore to act at least as a survival/proliferation factor for mesenchymal stem cells.Transdifferentiation potentialities towards endothelium were determined incubating L-NGFR+ cells in M199 supplemented with 10% FBS, 50 ng/ml VEGF, 1 ng/ml bFGF and 2 ng/ml IGF-1. At confluence, the cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads and expanded with VEGF. Immunophenotypic analysis of 8 samples showed a variable expression of endothelial markers: P1H12 ranging from 17 to 58%, CD105 from 98–100% and CD202b from 18 to 100%.L-NGFR+ cells, immediately after immunoseparation, were expressing Desmin but not MyoD, Miogenin, Mrf4, Myf5 by means of RT-PCR, while these cells were expressing NSE, TRKA and GalC, but not Nestin and GFAP. Experiments are ongoing to demonstrate muscle and neuron-glial differentiation in vitro using specific media (DMEM 10% FBS + 3 mM %-azacytidine, astrocyte conditioned media, neural stem cell conditioned media),In conclusion, the expression on NGFR+ cells of a variety of markers, not exclusively related to the mesenchymal lineage, and the reproducible ability to differentiate endothelial cells suggest that these cells may represent a subset of adult MSC with some multipotentiality.

Reversible Expansion of Hematopoietic Stem Cells (HSC) and CML-Like Disease in Transgenic Mice Expressing BCR-ABL under the Control of the SCL 3′ Enhancer.

Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs.Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by real-time PCR, suggesting that the SCL 3′ enhancer indeed directed BCR-ABL expression to the most primitive hematopoietic cells within the bone marrow. The percentage of HSC in the bone marrow was expanded 7- and 26-fold in double-transgenic as compared to single-transgenic or wild-type control mice within 12 and 21 days, respectively, after BCR-ABL induction. GMP were increased 2- and 3-fold while the number of CMP was decreased 2-fold after 12 days but was increased 1.5-fold after 21 days. MEP were decreased 3-fold at both time points. In keeping with these results, the percentage of Ter-119 positive erythroid cells was decreased while the percentage of Gr-1 positive granulocytic cells was increased in the bone marrow. To assess reversibility of the phenotype, we readministered tetracycline to abrogate BCR-ABL expression. Double-transgenic mice showed rapid clinical improvement, reversion of neutrophilia and leukocytosis, normalization of Gr-1/Mac-1 positive cells in the peripheral blood and spleen, and reversion of splenomegaly. In addition, in mice that had developed lymphoblastic disease, readministration of tetracycline led to disappearance of lymphomas and of B220/BP-1 positive lymphoblastic cells in the peripheral blood.Furthermore, expansion of the HSC compartment in the bone marrow was also reversible, and the percentage of HSC decreased to levels observed in control mice. Repeated induction of BCR-ABL expression by removal of tetracycline led to reappearance of the myeloid and lymphoid phenotype. Again, the disease was reversible, and none of the animals relapsed while on tetracycline, suggesting that the phenotype remained completely dependent on the expression of the oncogene.In conclusion, we present a model of BCR-ABL mediated CML-like disease with expansion of phenotypic hematopoietic stem cells and myeloid progenitor cells in the bone marrow. The target cell population in this model closely resembles the origin of transformation in patients with CML, allowing for in vivo monitoring of early molecular mechanisms of BCR-ABL transformation. We are currently studying the function of the expanded HSC and progenitor cells in transplantation experiments.

Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity

AbstractHuman hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture, purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin-) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDHhiLin-cells contained 22.6% ± 3.0% of the Lin-population and highly coexpressed primitive HSC phenotypes (CD34+CD38-and CD34+CD133+). In vitro hematopoietic progenitor function was enriched in the ALDHhiLin-population, compared with ALDHloLin-cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDHhiLin-cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID β2 microglobulin (β2M) null models demonstrated that 10-fold greater numbers of ALDHhi-Lin-cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID β2M null mouse, suggesting that the ALDHhiLin-population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. (Blood. 2004;104:1648-1655)

γ-Catenin contributes to leukemogenesis induced by AML-associated translocation products by increasing the self-renewal of very primitive progenitor cells

AbstractAcute myeloid leukemia (AML) is characterized by the block of differentiation, deregulated apoptosis, and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor α (PML/RARα), promyelocytic leukemia zinc finger (PLZF)/RARα (X-RARα), and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating γ-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore, we investigated how AATPs influence self-renewal of HSCs and evaluated the role of γ-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the γ-catenin promoter. The crucial role of γ-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of γ-catenin expression through RNA interference, and (ii) the augmentation of replating efficiency of HSCs upon overexpression of γ-catenin itself. In addition, the inoculation of γ-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML. (Blood. 2004;103:3535-3543)

922. Deriving Tissues from Embryonic Stem Cells

Top of pageAbstract Differentiation of embryonic stem (ES) cells in vitro yields abundant hematopoietic progenitors, but achieving stable hematopoietic engraftment of irradiated mice has proven difficult, begging the question of whether ES cells give rise to transplantable hematopoietic stem cells (HSCs) in vitro, and limiting the application of ES cells in experimental and therapeutic models. In an effort to characterize factors that differentiate primitive progenitors derived from yolk sac or ES cells into transplantable HSCs, we have examined the effect of conditional expression of several transgenes linked to leukemogenesis, cytokine signaling, or blood formation in model organisms. These genes include the oncoprotein BCR/ABL, the signal transducer STAT5, the bHLH transcription factor SCL, the homeotic selector gene HoxB4 implicated in self-renewal of definitive HSCs, and Cdx4, an upstream regulator of Hox gene expression. BCR/ABL enables engraftment of animals with hematopoietic cells that exhibit multi-lineage differentiation potential, but leukemia invariably develops. STAT5 activation enhances hematopoietic proliferation in vitro but enables only limited, transient multi-lineage engraftment after transplantation of irradiated mice. Induction of SCL appears to enhance hematopoietic commitment during ES differentiation but does not promote hematopoietic engraftment. Expression of HoxB4 in primitive progenitors combined with culture on hematopoietic stroma enables generates transplantable cells with a definitive HSC phenotype. These progenitors engraft lethally irradiated adults and contribute to long-term, multi-lineage hematopoiesis in primary and secondary recipients. Cdx4 further enhances the capacity for HSC generation from ES cells. We have used HoxB4 modification of ES-derived HSCs to enable therapeutic transplantation with products of ES cells, including a proof-of-principle application of therapeutic cloning to treat an immunodeficient Rag2−/− mouse, and efforts are underway in murine models of thalassemia. Our studies in the blood forming system are laying a foundation for similar studies in germ cell development, and to applications with human ES cells.

SCL is required for normal function of short-term repopulating hematopoietic stem cells

AbstractThe stem cell leukemia (SCL) gene is essential for the development of hematopoietic stem cells in the embryo. Here, we used a conditional gene targeting approach to examine the function of SCL in adult hematopoietic stem cells (HSCs). Flow cytometry of bone marrow from SCL-deleted mice demonstrated a 4-fold increase in number of Linneg c-kit+ Sca-1+ cells. Despite this increase in the number of phenotypic HSCs, competitive repopulation assays demonstrated a severe multilineage defect in repopulation capacity by SCL-deleted bone marrow cells. SCL-heterozygous cells also showed a mild repopulation defect, thus suggesting haploinsufficiency of SCL. The transplantation defect of SCL-deleted cells was observed within 4 weeks of transplantation, indicating a defect in a multipotent progenitor or short-term repopulating HSCs. Although the defect persisted in secondary transplants, it remained relatively stable, suggesting that SCL was not required for self-renewal of the HSCs. Generation of SCL-deleted cells within SCL-wild-type mice rescued the early repopulating defect. Together, our results suggest that SCL is required for the normal function of short-term repopulating HSCs. (Blood. 2004;103:3342-3348)

Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice.

To study the effects of transformation-related protein 53 (Trp53) and other genes on hematopoiesis and hematopoietic stem cells (HSCs). Frequencies of murine bone marrow cells (BMCs) with the Lin(-)Sca-1(+)c-kit(+)CD34- phenotype were analyzed by flow cytometry, and were increased in mice with germ-line deletion of the Trp53 (Trp53(-/-)) gene but not in 25 other deletions of genes involved in cell cycling, development, cancer, or hematopoiesis. Therefore, Trp53(-/-) and wild-type Trp53(+/+) mice were compared using the following assays: complete blood counts, day-9 colony-forming unit spleen (CFU-S), and competitive repopulation. In the latter assay, donor repopulating ability was analyzed at one, three, and five months, while recipient survival and recipient blood and bone marrow cell composition were analyzed at five months, after transplantation. In comparison to wild-type controls, Trp53(-/-) mice had normal blood and bone marrow cell counts, increased CD11b(+), and decreased CD45R(+) cell proportions in blood and bone marrow, twice as many Lin(-)Sca-1(+)c-kit(+)CD34(-) BMCs, and 37% more day-9 CFU-S. In the competitive repopulation assay, Trp53(-/-) BMCs engrafted lethally irradiated recipients two to four times better than Trp53(+/+) BMCs. The Trp53(-/-) engraftment advantage increased with time in the recipients. Recipients of Trp53(-/-) donors had two to three times more Lin(-)Sca-1(+)c-kit(+)CD34(-) BMCs than recipients of Trp53(+/+) donors at five months after transplantation. However, only 44% of recipients of Trp53(-/-) donors survived five months after trans-plantation, compared with 92% of recipients of Trp53(+/+) donors. The Trp53-null allele expands bone marrow Lin(-)Sca-1(+)c-kit(+)CD34(-) cells and the overall activity of HSCs; however, it increases recipient mortality.

Major age-related changes of mouse hematopoietic stem/progenitor cells.

To study age-related changes of mouse bone marrow (BM) cells and hematopoietic stem cells (HSCs), we isolated rhodamine-123(low) (Rh(low)) Thy1.1(low) Lin(-)Sca-1(+) (TLS) HSCs from the BM of old mice and compared their functional characteristics to cells of the same phenotype isolated from young mice. We observed impaired recovery of B lymphocytes and decreased self-renewal in recipients of old Rh(low) cells compared to young Rh(low) cells. Blockade of Rh efflux using verapamil improved lymphoid reconstitution by enriched HSCs, and isolation of aged HSCs based on efflux of a fluorescent multi-drug resistance (MDR) substrate (Bodipy-verapamil) resulted in enrichment of HSC activity equivalent to that obtained with Rh. These observations suggest a complex relationship between MDR activity and HSC function during aging. To address whether the difference between young and aged donors was intrinsic to the HSC compartment or was due to a shift in HSC phenotype, we co-transplanted normal BM derived from young or old donors and followed repopulation simultaneously in the same recipient animals. In a parallel experiment, we co-transplanted HSCs purified from old donors with BM derived from young donors. In both experiments, transplants were given to both young and old recipients. The results show a clear defect in B-cell engraftment from either BM or HSCs of old donors, irrespective of the age of the recipient. In contrast, myeloid engraftment was predominantly derived from BM or HSCs derived from aged donors, again irrespective of recipient age. These data suggest a stem cell basis for B-cell immuno-senescence and the increased incidence of myelocytic leukemia in elderly people.

Marrow stem cell potential within a continuum.

On the basis of our studies of the fluctuation of the hematopoietic stem cell phenotype with cell cycle trnsit, we hypothesize that the ability of marrow stem cells to convert to nonhematopoietic cells will also vary at different points in the cell cycle. The new biology of stem cells has an impact on many fields including developmental biology and stem cell biology and the clinical potential is enormous.