Enrichment Of Hematopoietic Stem Cells Research Articles

Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.

Establishment of a Labeling System for Hematopoietic Stem Cells by Nucleostemin Enhancer/Promoter-GFP Transgenic Mice.

A labeling system for tissue stem cells is a powerful tool for stem cell research and for development of stem cell-based regenerative medicine to human disease. Here we generated transgenic mice expressing green fluorescent protein (GFP) under control of enhancer/promoter activity to Nucleostemin (NS) gene. NS, a nucleolar GTP-binding protein, expresses at high levels in embryonic stem cells and neural stem cells, suggesting that NS expresses in various tissue stem cells. In fact, we found that NS highly expressed in mouse immature hematopoietic cells containing hematopoietic stem cells (HSCs) and progenitor cells compared with differentiated hematopoietic cells. To examine whether HSCs could be labeled by using enhancer/promoter activity to NS gene, we generated transgenic mice harboring putative genomic enhancer/promoter region of NS gene followed by GFP cDNA-polyA in the 3′ end (NS-GFP tg mice). Fluorescence activated cell sorting (FACS) analysis indicated that most bone marrow (BM) mononuclear cells (MNCs) showed green fluorescence in NS-GFP tg mice. To validate the relationship between fluorescent intensity of GFP and expression levels of endogenous NS mRNA, BM MNCs from NS-GFP tg mice were separated into four fractions depending on their fluorescent intensity (i.e., GFP−, GFP+, GFP++, and GFP+++) by FACS, and these cells were subjected to quantitative real-time PCR analysis. The fluorescent intensity of GFP certainly reflected the amount of endogenous NS mRNA, indicating that the enhancer/promoter region used in this study is sufficient for monitoring the expression levels of endogenous NS mRNA in vivo. We next characterized expression levels of cell-surface markers on the four fractions of BM MNCs. FACS analysis showed that the GFP+++ cells, but not the other cell populations, lost the expression of mature hematopoietic cell markers (myeloid, B cells, T cells and erythroid cells). The fluorescent intensity of GFP in c-Kit+ Sca-1+ Lineage− (KSL) cells, which represent a primitive hematopoietic cell fraction containing HSCs and progenitor cells, was much higher than that in the other cell populations, indicating that the immature hematopoietic cells highly express GFP. To investigate the biological properties of the GFP-high expressing cells, we further performed colony-forming assay in vitro and BM transplantation assay in vivo. When the sorted BM MNCs were cultured in methylcellulose medium supplemented with cytokines, GFP+++ cells showed the highest number of colony formation. To assess the repopulating capacity as stem cell function, the sorted BM MNCs were transplanted into irradiated recipient mice. Notably, only GFP+++ cells had long-term reconstitution capacity of hematopoiesis in the recipient mice on the competitive reconstitution assay. In addition, the GFP+++ cells that were sorted from the GFP+++ cells-transplanted mouse retained the reconstitution capacity after secondary BM transplantation. The reconstitutive capacity of the GFP+++ cells into multi-lineages was confirmed by detection of donor-derived B, T, and myeloid cells in the recipient mice. These results demonstrate that HSCs with self-renewal capacity and differentiation potential for multi-lineages are enriched in GFP+++ cell population. The NS-GFP tg mouse system may provide a useful tool for effective enrichment of HSCs in combination with other cell-surface markers for HSCs.

Junctional Adhesion Molecule-A (JAM-A/JAM-1/F11R) Marks Long-Term Repopulating Hematopoietic Stem Cells.

Junctional adhesion molecule-A (JAM-A/JAM-1/F11R) is a cell adhesion molecule expressed in epithelial cells, endothelial cells and also hematopoietic cells such as leukocytes, platelets and erythrocytes. However, the expression of JAM-A in hematopoietic stem cell (HSC) had not been known. Here we show that JAM-A is expressed at a high level in the enriched HSC fraction, i.e. CD34+ c-Kit+ cells in embryonic day 11.5 (E11.5) aorta-gonod-mesonephros (AGM) and E11.5 fetal liver (FL) as well as c-Kit+ Sca-1+ Lineage- (KSL) cells in E14.5 FL, E18.5FL and adult bone marrow (BM). While the percentage of JAM-A+ cells in those tissues decreases during the development, the expression in HSC fraction is maintained throughout life. In fact, c-Kit+Sca-1+ cells are enriched approximately 200-fold from whole BM cells by anti-JAM-A antibody alone, i.e. the c-Kit+Sca-1+ cells in whole BM was about 0.15% and that in JAM-A+ cells was about 30%. Colony forming assays reveal that multi-lineage colony forming activity (CFU-mix) in JAM-A+ cells is higher than that in JAM-A- cells in the enriched HSC fraction in all those tissues. HSC transplantation assay revealed that long-term repopulating HSC (LTR-HSC) activity is present in the mice received 100 JAM-A+ KSL (7/9) cells and 300 KSL cells (5/6). Since the ratio of JAM-A+ cells to JAM-A- cells in the KSL fraction is about 6.5:3.5, 100 JAM-A- KSL cells are equivalent to 285 total KSL cells. In contrast to JAM-A+ cells, the mice received 1000 JAM-A- KSL cells, which are equivalent to more than 2850 total KSL cells, failed to engraft for long-term (0/6). These data revealed that long-term repopulating HSC (LTR-HSC) activity is present exclusively in the JAM-A+ cells, but not in JAM-A- cells. Moreover only 100 JAM-A+ cells isolated from whole BM cells by anti-JAM-A antibody alone reconstituted the hematopoietic system for long-term (4/7). Together these results indicate that JAM-A is expressed on hematopoietic precursors in various hematopoietic tissues and is an excellent and convenient marker to enrich LTR-HSC from BM cells.

CD61 enriches long-term repopulating hematopoietic stem cells

Among the subsets that define hematopoietic stem cells (HSCs), CD34 − c-kit + Sca-1 + lineage marker − (CD34 −KSL) cells are regarded as one of the populations that have the highest enrichment of HSCs in adult mouse bone marrow. Here, we demonstrate that long-term repopulating hematopoietic stem cells (LTR-HSCs) have high expression of CD61 (integrin β 3) within the CD34 −KSL population. Approximately 60% of CD34 −KSL cells showed high expression of CD61. CD61 HighCD34 −KSL populations also exhibited significantly greater properties of HSC, such as expression of HSC markers, the side population (SP) phenotype, and ability for long-term repopulation. In both SP cells and non-SP (NSP) cells, CD61 HighCD34 −KSL cells also contained significantly more LTR-HSCs than CD61 Low/−CD34 −KSL cells. Our results indicate that CD61 is exploitable for HSC enrichment as a supportive positive cell surface marker.

HOXB4's road map to stem cell expansion.

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Time-Dependent Effects on Coronary Remodeling and Epicardial Conductance after Intracoronary Injection of Enriched Hematopoietic Bone Marrow Stem Cells in Patients with Previous Myocardial Infarction

Bone marrow (BM) cells may interact with coronary endothelium and modulate coronary atherosclerosis. We investigated the time course of coronary luminal loss and changes in conductance after intracoronary injection of enriched hematopoietic BM stem cells in patients with previous myocardial infarction (MI). Among 24 patients with acute MI, 13 were randomized to early (<7 days) and 11 to late (4 months) intracoronary injection of CD133+ cells after the infarction. Segmental quantitative coronary angiography and fractional flow reserve (FFR) measurements of the infarct-related (IR) artery (A) and contralateral artery (control) were performed. In the early group, at 4 months, cumulative luminal loss (LL) of the minimal luminal diameter (MLD) of the IRA distal to the stented segment was -0.39 (-0.51-0.10) mm (p < 0.05 vs. control). There was no further change in LL between 4 and 8 months [-0.09 (-0.26-0.15) mm]. In parallel, FFR decreased at 4 months [-0.16 (-0.26-0.001), p < 0.05 vs. control] but slightly increased from 4 to 8 months follow-up [+0.05 (-0.10-0.09)]. In the late group, LL of the MLD of the IRA distal to the stented segments was -0.12 (-0.47-0.07) mm (NS vs. control) at 4 months and further -0.07 (-0.25-0.05) mm (NS) between 4 and 8 months. At 8 months, the total LL of the MLD in the early and late group was only slightly higher compared to control [-0.34 (-0.48--0.16), -0.36 (-0.69--0.09), and -0.12 (-0.39-0.05) mm, respectively, NS]. Early intracoronary administration of hematopoietic BM stem cells in patients with previous MI is associated with accelerated luminal loss and reduced conductance of the infarct-related artery.

The combined use of Hoechst efflux ability and aldehyde dehydrogenase activity to identify murine and human hematopoietic stem cells

In murine hematopoietic tissue, direct repopulation experiments have demonstrated that the side population (SP) represents a remarkable enrichment of hematopoietic stem cells. Human SP has been phenotyped as negative for lineage antigens as well as CD34. However, in the 9 years since the original publication, no long-term hematopoietic reconstitution has been reported for the adult human SP/CD34(-) subset. Elevated levels of aldehyde dehydrogenase (ALDH) have been demonstrated in murine and human progenitor cells when compared to other hematopoietic cells. Here, we report the phenotype of human cord blood SP cells. We established the technique of simultaneous phenotyping, Hoechst exclusion, and ALDH labeling on murine tissues. We then performed the simultaneous analysis of phenotype, SP, and ALDH activity on human cord blood and bone marrow cells. Finally, we analyzed the phenotype and functional potential of human cord blood ALDH(+) cells to determine whether Lin(-)/CD34(-) cells are identified via this technique. We demonstrate that human Lin(-)/CD34(-)/ALDH(+) cells are capable of long-term repopulation. Although the SP technique identifies cells that overlap with the ALDH(+) cell population, this is restricted to the CD34(+) cell subset. Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells.

Isolation of Hematopoietic Stem Cells from Human Cord Blood

Enrichment of hematopoietic stem cells is based on the expression of certain surface antigens, such as CD34 and CD133, or on the lack of expression of lineage-specific antigens. Immunomagnetic positive selection of CD34(+) or CD133(+) cells is performed using paramagnetic microbeads conjugated to specific monoclonal antibodies (anti-human CD34 or anti-human CD133). In negative selection of lineage-negative (Lin(-)) cells, the unwanted cells are labeled with antibodies against known markers for mature hematopoietic cells (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A) and retained in the column. Unlabeled cells pass through the column and are collected as the Lin(-) cell fraction. Immunomagnetic cell sorting system MACS is a fast and gentle method to enrich hematopoietic stem cells. Viable and highly pure cells can be separated to be used in various downstream applications, such as flow cytometry and cell culture.

Zebrafish kidney marrow contains ABCG2-dependent side population cells exhibiting hematopoietic stem cell properties

Zebrafish ( Danio rerio) has emerged as a powerful genetic model for the study of vertebrate hematopoiesis. However, methods for detection and isolation of hematopoietic stem cells (HSCs) have not yet been reported. In mammals, the combination of Hoechst 33342 staining with flow cytometry can be used for separation of a bone marrow side population (SP), which is highly enriched for HSCs. We applied a similar procedure to hematopoietic kidney marrow cells from adult zebrafish, and identified a segregated cohort of SP cells, which demonstrate a set of features typical of stem cells. SP cells show extremely low scatter characteristics, and are small in size with a minimum of cytoplasm. Treatment of zebrafish kidney marrow cells with reserpine or fumitremorgin C, which inhibit the ABCG2 transporter responsible for Hoechst 33342 efflux, caused a clear reduction in the number of SP cells. Consistent with the quiescent state of HSCs, the SP cells are strongly resistant to the myelosuppressive agent 5-fluorouracil. In addition, SP cells specifically demonstrate higher expression of genes known to be markers of HSCs of mammals. Hence, our results show that the SP phenotype is conserved between mammals and teleosts, and the properties of the zebrafish SP cells indicate a significant enrichment for HSCs. These rapid flow cytometric methods for purification of HSCs from zebrafish may greatly facilitate genetic analysis of stem cells using the advantages of this vertebrate model.

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF-alpha for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5+/-10-fold (mean+/-sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation

AbstractIn utero hematopoietic-cell transplantation (IUHCT) can induce donor-specific tolerance to facilitate postnatal transplantation. Induction of tolerance requires a threshold level of mixed hematopoietic chimerism. CD26 is a peptidase whose inhibition increases homing and engraftment of hematopoietic cells in postnatal transplantation. We hypothesized that CD26 inhibition would increase donor-cell homing to the fetal liver (FL) and improve allogeneic engraftment following IUHCT. To evaluate this hypothesis, B6GFP bone marrow (BM) or enriched hematopoietic stem cells (HSCs) were transplanted into allogeneic fetal mice with or without CD26 inhibition. Recipients were analyzed for FL homing and peripheral-blood chimerism from 4 to 28 weeks of life. We found that CD26 inhibition of donor cells results in (1) increased homing of allogeneic BM and HSCs to the FL, (2) an increased number of injected animals with evidence of postnatal engraftment, (3) increased donor chimerism levels following IUHCT, and (4) a competitive engraftment advantage over noninhibited congenic donor cells. This study supports CD26 inhibition as a potential method to increase the level of FL homing and engraftment following IUHCT. The resulting increased donor chimerism suggests that CD26 inhibition may in the future be used as a method of increasing donor-specific tolerance following IUHCT.

Age-Dependent Increase in Side Population Distribution Within Hematopoiesis: Implications for Our Understanding of the Mechanism of Aging

It is thought that, as we age, damage to our stem cells may lead to diminished stem cell pool function and, consequently, a reduced organ regeneration potential that contributes to somatic senescence. Stem cells have evolved many antitoxicity mechanisms, and certain mechanisms may be utilized to isolate hematopoietic stem cells. One method exploits the activity of the ATP-binding cassette/G2 transporter to efflux Hoechst 33,342 and results in a stem cell population known as the side population (SP). The SP subset represents a remarkable enrichment for hematopoietic stem cells and provides an opportunity to re-evaluate age-based changes in hematopoietic stem cells. We report here that the frequency of SP cells steadily increases with age, as does the proportion of Lin(-)/Sca-1(+)/c-kit(+) cells that is capable of Hoechst efflux. Phenotyping, progenitor, and long-term repopulation assays have indicated that SP cells in older mice are still stem cells, albeit with a lower homing efficiency than SP cells from younger mice. Analysis of apoptosis within SP cells has revealed an apoptosis-resistant population in SP cells from old mice. Gene expression analysis has determined that SP cells from old mice have a reduced expression of apoptosis-promoting genes than SP cells from young mice. This increase in SP cells with age seems to be an intrinsic property that may be independent of the age of the microenvironment (niche), and our data might provide some clues as to how this alteration in the proportion of stem/progenitor cells occurs. A possible selection-based mechanism of stem cell pool aging is discussed.

Evidence for an Adaptive Immune Barrier after in Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative approach that takes advantage of normal immunologic development to achieve donor specific tolerance. Despite the many potential advantages of the fetal recipient, IUHCT across MHC barriers has been limited by low levels of engraftment and the inability to consistently achieve allochimerism.Although the immature immune system of the developing fetus has long been appreciated as a principal advantage of IUHCT, the competence of the fetal immune system to act as a barrier to IUHCT has been a source of debate. Until now, comparisons of allogeneic and congenic engraftment have been inconclusive due to methodologic limitations resulting in minimal and inefficient engraftment. In this study, a new intravascular technique that allows definitive administration of much higher doses of donor cells was employed to directly compare the incidence and levels of engraftment following in utero transplantation of either congenic or allogeneic bone marrow (BM) or enriched hematopoietic stem cells (HSCs). 20E+06 B6 GFP BM cells (H2Kb+, GFP+) or 1E+05 cKit+Sca-1+Lin- B6 GFP cells (H2Kb+, GFP+) were intravenously injected via the vitelline vein into gestational day 14 Balb/c (H2Kd+, allogeneic) or C57Bl/6 (H2Kb+, congenic) fetal mice. The peripheral blood (PB) of recipients was serially analyzed by flow cytometry for GFP+ donor cells at 1, 2, 4 and 6 months of age. A separate group of animals was harvested at 1 week of age (2 weeks after injection) to assess donor chimerism in PB and BM. Our results demonstrate that 100% of surviving recipients of whole BM demonstrate engraftment at 1 week of age, but that 70% of allogeneic recipients lose engraftment by 1 month of age, and 80% ultimately fail to sustain long-term chimerism. In contrast, all congenic recipients maintain engraftment at 6 months of age (Table 1). Chimerism levels in allogeneic recipients drop significantly after 1 month of life while those in congenic recipients remain stable. This results in a significant difference in engraftment levels in allogeneic and congenic recipients beyond 1 month of life (Fig 1). Similar results were seen when enriched HSCs were the donor cell source. 100% (2/2) of congenic recipients of enriched HSCs demonstrated stable low level PB engraftment up to 6 months of life (0.14–0.55% GFP+ donor cells). In contrast, no allogeneic recipients (0/9) of enriched HSCs were chimeric from 1 to 6 months of life. In combination, these results demonstrating a 100% efficiency of long-term engraftment in congenic recipients and loss of engraftment by 1 month of age in the majority of allogeneic recipients strongly implicate an adaptive immune barrier to allogeneic engraftment after IUHCT. Better understanding of the immune mechanisms limiting allogeneic engraftment after IUHCT is required to allow the development of successful strategies for IUHCT.Efficiency of Engraftment after IUHCT in Congenic and Allogeneic Recipients1 week of age (BM)1 week of age (PB)1 month of age (PB)6 months of age (PB)congenic100% (8/8)100% (8/8)100% (25/25)100% (25/25)allogeneic100% (8/8)100% (8/8)29% (9/31)19% (6/31) [Display omitted]

AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.

Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.

Abcg2 Expression Marks Neural Stem Cells in the Murine Cerebellum.

Neural stem cells have been identified in both the cerebellum and forebrain of fetal and adult mice. These cells can form neurospheres in culture and differentiate into both glia and neurons either in vitro or in vivo. In embryonic day 14 forebrain, neural stem cells are found to exist exclusively in a subpopulation with the Side Population (SP) phenotype, and express Abcg2, a member of the ABC transporter family that is responsible for the SP phenotype in hematopoietic stem cells (HSCs). The expression of Abcg2 in stem cells in the cerebellum has not been characterized. We have generated an Abcg2/GFP knock-in mouse model in which expression of GFP is under control of the endogenous Abcg2 locus and used this model to demonstrate that Abcg2 expression can be used for HSC enrichment. Here we report the use of this mouse model to explore the relationship between Abcg2 expression and neural stem cell function in neonatal cerebellum. Single cells were prepared from cerebellum of 4–9 day old mice by digesting with papain. We then stained the cells with anti-CD45 and anti-Ter119 antibody to exclude the resident hematopoietic cells in subsequent flow cytometry analysis and cell sorting. We found that a small but consistent subpopulation of cells, comprising 0.7±0.12% of total CD45−Ter119- single cell preparations, expressed the Abcg2/GFP allele. To determine whether these GFP+ cells were enriched for neural stem cells, we sorted the CD45−Ter119- cells into GFP+ and GFP− subpopulations and analyzed for their neurosphere forming activity in the presence of epidermal growth factor and basic fibroblast growth factor. We found that the GFP+ subpopulation formed 21 fold more neurospheres compared with the GFP− subpopulation. These neurosphere forming cells can self-renew as evidenced by their capacity to form secondary neurospheres when replated. These results demonstrate that similar to what is seen with HSCs and with embryonic forebrain cells, Abcg2 is expressed in the neural stem cells in neonatal cerebellum, and Abcg2/GFP expression in this mouse model could also be used as a marker to prospectively purify neural stem cells from cerebellum. Ongoing studies are focused on defining the in vivo multilineage differentiation potential of the Abcg2/GFP+ cells and determining whether Abcg2 expression could be used as a marker for purification of medulloblastoma stem cells.

Toll-like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus

The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.

Prospective Isolation of Murine Hematopoietic Stem Cells by Expression of an Abcg2/GFP Allele

Stem cells from a variety of tissues can be identified by a side population (SP) phenotype based on Hoechst 33342 dye efflux. The Abcg2 transporter is expressed in hematopoietic stem cells (HSCs) and confers this dye efflux activity. To further explore the relationship among Abcg2 expression, the SP phenotype, and HSC activity, we have generated mice in which a green fluorescent protein (GFP) reporter gene was inserted into the Abcg2 locus. In these mice, the majority of bone marrow (BM) cells that expressed the Abcg2/ GFP allele were Ter119(+) erythroid cells. The Abcg2/GFP allele was also expressed in approximately 10% of lineage-negative (Lin(-)) and in 91% of SP cells using stringent conditions for the SP assay. Flow cytometric sorting was used to isolate various Abcg2/GFP(+) BM cell populations that were then tested for HSC activity in transplant assays. There was significant enrichment for HSCs in sorted Lin(-)/ GFP(+) cells, with a calculated HSC frequency of approximately one in 75. There was no HSC activity detected in Lin(-)/GFP(+) cells. Altogether, these results show that Abcg2 is expressed on essentially all murine BM HSCs and can be used as a prospective marker for HSC enrichment.

Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

Expression of Stem Cell Markers on Mononuclear Cells Derived From Heparinized Cadaveric Organ Donors Before and After Disconnection From the Respirator

We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45−). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.