Fraction Of Hematopoietic Stem Cells Research Articles

Short-term migration of transplanted Lin–Sca-1+c-kit+ hematopoietic stem cells after hippocampal ischemic injury of mice

The study of migration and differentiation potential of different types of stem cells remains a problem for cell biology and regenerative medicine. The purpose of the study was to evaluate the ability of transplanted hematopoietic stem cells (HSC) of murine fetal liver to migrate into a zone of hippocampal ischemic injury at suboccipital intraventricular injection; and to assess their neural differentiation possibility in the early period after transplantation.Materials and methods. We modeled an ischemic injury of the hippocampus of FVB-wt mice and after 24 hours transplanted suboccipitaly fetal liver HSC of FVB-Cg-Tg(GFPU)5Nagy/J fetuses (transgenic by GFP). Sorting of Lin–Sca-1+c-kit+ HSC fractions was performed by FACS. After 7 and 14 days we performed immunohistochemical staining of brain slices for GFP, NeuN and GFAP markers.Results. On the 7th day after transplantation injected cells penetrated up to 100 µm from the wall of the 3rd ventricle, and on the 14th day single transplanted cells localized in the ischemic hippocampal CA1 region. Donor’s cells were round shape and did not express NeuN and GFAP markers. Features of reactive astrogliosis and neuronal death were kept in the hippocampal CA1 region of experimental animals, similar to the control group.Conclusion. Transplanted Lin–Sca-1+c-kit+ mice fetal liver HSC are able to survive and migrate to the area of hippocampal ischemic injury, but the possibility of their neuronal or astrocyte differentiation in 14-day time was not confirmed.

Complementary regulation of early B-lymphoid differentiation by genetic and epigenetic mechanisms

Although B lymphopoiesis is one of the best-defined paradigms in cell differentiation, our knowledge of the regulatory mechanisms underlying its earliest processes, in which hematopoietic stem cells (HSCs) enter the B lineage, is limited. However, recent methodological advances in sorting progenitor cells and monitoring their epigenetic features have increased our understanding of HSC activities. It is now known that even the highly enriched HSC fraction is heterogeneous in terms of lymphopoietic potential. While surface markers and reporter proteins provide information on the sequential differentiation of B-lineage progenitors, complex interactions between transcription factors have also been shown to play a major role in this process. Epigenetic regulation of histones, nucleosomes, and chromatin appears to play a crucial background role in this elaborate transcription network. In this review, we summarize recent findings on the physiological processes of early B-lineage differentiation, which provides a new paradigm for understanding the harmonious action of genetic and epigenetic mechanisms.

Effect of SNS-032 on biological activity of hematopoietic stem cells in mice

This study was aimed to investigate the effect of SNS-032 (C17 H24 N4O2S2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice. The self-renewal capability of bone marrow cells was measured by cobblestone forming cell test. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were detected by flow cytometry. The results showed that there was no significant difference of the frequency of HSC between SNS-032 and control group. The expressions of CDK1, CDK2, CDK7 and p27 decreased in HSC (P < 0.05) while the expressions of CDK4, CDK6, p21, p18, p19, Bcl-2, Bax, Puma, p53, Bim1, Sall4 and Notch1 showed no difference between SNS-032 group and control group (P > 0.05). The fraction of viable HSC in each phase of cell cycle remained unchanged after the treatment of SNS-032 (P > 0.05). Furthermore, there was no statistical difference in the apoptotic fractions between control and drug-treated groups (P > 0.05). It is concluded that SNS-032 induce apoptosis of cancer cells. Interestingly, SNS-032 has no significant inhibitory effect on self-renewal and differentiation of normal HSC, as well as no obvious effect inducing apoptosis of normal HSC and HPC.

Methods of freezing cord blood hematopoietic stem cells

Cord blood (CB) is a valuable source of hematopoietic stem cells (HSCs). Extended storage of CB is possible provided that validated cryopreservation procedures are used. The study objective was to determine optimal methods of CB cryopreservation. In the study we 1) compared the effect of two-step cryopreservation and controlled-rate freezing method on the postthaw quality of CB (Study A) and 2) evaluated the postthaw quality of HSC fractions isolated from CB with various methods and frozen with controlled-rate freezing method (Study B). The same cryoprotectant mixture was used for 20 CB units (Study A) and 122 CB units (Study B). In Study A, 13.79 × 10(8) and 13.29 × 10(8) initial white blood cell (WBC) counts decreased to 6.38 × 10(8) and 6.02 × 10(8) after thaw for the two methods, respectively. The mononuclear cell (MNC) counts decreased from 5.90 × 10(8) to 3.71 × 10(8) and from 5.64 × 10(8) to 3.47 × 10(8) dependent on the method. MNC viability decreased from 99.0% to 97.4% for the former and from 98.5% to 97.2% for the latter method. The differences were insignificant. In Study B, postthaw WBC recovery in HSC fractions was 74.4% to 103.5%, MNC recovery 106.4% to 118.5%, CD34+ cell recovery 102.5% to 150.2%, and MNC viability 94.1% to 97.4%. Neither the cryopreservation procedure nor the freezing of isolated HSCs affected product quality, which may indicate that various freezing methods can be used for cell banking provided the they follow recommendations of good manufacturing practice and Directive 2004/33/EC.

Long-term repopulating hematopoietic stem cells and “side population” in human steady state peripheral blood

This report brings the first experimental evidence for the presence of long-term (LT) repopulating hematopoietic stem cells (HSCs) and Side Population (SP) cells within human steady state peripheral blood CD34+ cells. Ex vivo culture, which reveals the LT-HSC, also increases short-term (ST) HSC engraftment capacity and SP cell number (as well as the SP subpopulations defined on the basis of CD38, CD90 and CD133 expression) which are very low in freshly isolated cells. Thus, ex vivo incubation either allows the expansion of the small fraction of HSCs or reveals “Scid Repopulating Cells — SRC” that are present in the initial CD34+ cell population but unable to engraft. In addition, among these CD34+ cells, we confirm the presence of committed progenitors at frequencies similar to those found in cord blood CD34+ cells. These cells, obtained from leukoreduction filters (LRFs) and rejected in the course of the preparation of red blood cell concentrates, are an abundant and reliable material for obtaining committed progenitors, short- and long-term HSCs of therapeutic interest, especially after the ex vivo expansion phase. Our results open a perspective to set up new therapeutic protocols using expanded LRFs-recovered CD34+ cells as a source of HSCs for autologous or allogeneic transplantation.

Generation of an HIV Resistant T-cell Line by Targeted “Stacking” of Restriction Factors

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

System for Rapid and Reproducible Identification of MLL-AF9 Translocations Induced by Dietary Compounds and Nutritional Supplements

AbstractAbstract 1275▪▪This icon denotes a clinically relevant abstractInfant acute leukemias account for ∼30% of all malignancy seen in childhood across the Western world. They are aggressive and characterized by rapid onset shortly after birth. The majority of these (∼80% ALL and ∼60% AML) have rearrangements involving the MLL gene. Although MLL fusion to more than 75 genes have been identified, AF9 is one of its most common translocation partners. MLL breakpoint sequences associated with infant acute leukemia are similar to those in secondary AML following exposure to the topoisomerase II (topoII) poison etoposide. This similarity led to the hypothesis that exposure during preganancy to biochemically similar compounds may promote infant acute leukemia. Some studies have shown an epidemiological link between bioflavonoid intake and increased incidence of MLL-rearranged infant leukemias. These bioflavonoids have also been shown to inhibit topoII in in vitro DNA cleavage assays and produce MLL rearrangements by inverse PCR in mice. Hundreds of unregulated nutritional supplements are widely available and perceived to prevent cardiovascular disease, inflammation, and cancer; however, their potential to promote leukemic translocations should be determined. Our goal was to create a model system that allows for rapid quantifiable screening of a large number of compounds to determine their potential to promote MLL-AF9 bcr translocations, and does not rely on inverse PCR that requires elimination of artifacts or less physiologically relevant internal deletions or intronic alterations from analysis. Thus, we developed reporter stem cell lines that contain two transgene constructs—(1) the MLL bcr fragment containing a genetically-engineered GFP 5’ exon, and (2) the AF9 bcr containing a genetically-engineered GFP 3’ exon. A translocation between the two bcrs reconstitutes the full-length GFP transcript resulting in quantifiable green fluorescence. Cells were treated with etoposide, quercetin, genestein, luteolin, dipyrone, or benzoquinone for 1 hour at 25mM – 200mM concentrations then allowed to repair and proliferate in culture. GFP+ fluorescent colonies as a result of MLL-AF9 translocations were readily scorable by 96 hours in a dose-dependent manner. [Display omitted] [Display omitted] [Display omitted] As previously demonstrated, totipotent stem cell viability was extremely impaired by multiple concentrations of etoposide, but the surviving fraction of hematopoietic stem cells that differentiated into multiple lineage sub-types exhibited a significant number of MLL-AF9 translocations (frequency of 1.5×10−4). Bioflavonoids genistein and quercetin (75mM) were also potent promoters of MLL-AF9 translocations in totipotent stem cells and hematopoietic stem cells at roughly similar frequencies as etoposide. By contrast, more differentiated hematopoietic progenitor cells had a significantly decreased (3-log) number of colonies with MLL-AF9 translocations following exposure to multiple compounds. This decrease is likely due to differences in DNA damage and repair responses or the potential of cells carrying translocations to proliferate in culture. The accuracy and significance of this model system is apparent from treatment of stem cells with benzoquinone that was not sufficient to produce MLL-AF9 translocations following exposure to concentrations up to 125mM (frequency < 0.1×10−6). Benzoquinone is a non-bioflavonoid thought to have a distinct mechanism of action and clinically associated with multiple leukemias. This system is a direct measure of the sensitivity of the MLL and AF9 bcrs to topoII poisons and bioflavonoids independent of their normal chromatin context and independent of formation of a leukemic fusion protein. In addition, the system allows for rapid and reproducible screening of hundreds of compounds that may have the potential to promote leukemogenic translocations in early stem cell and more differentiated cell subpopulations analogous to the events observed in infant acute leukemias. Disclosures:No relevant conflicts of interest to declare.

JS5-5 - Hematopoietic Stem Cell is the Primary Oncogenic Target in Human Leukemias

ABSTRACT Malignant neoplasms might originate from a small abnormal cell population with deregulated self-renewal capacity, impaired differentiation activity and/or reinforced cell survival. This ‘cancer stem cell’ hypothesis is well supported in the field of hematologic disorders such as acute leukemias, based on identification of a small malignant stem cell fraction that can reconstitute human diseases in xenograft mouse models. In hematological malignancies, for perpetual propagation of leukemic mutations, these mutational events should occur in a cell population that can self-renew, otherwise the short life of a committed cell would not sustain these oncogenic events. We have successfully tracked the step-wise acquisition of mutations such as FLT3-ITD and AML1-ETO in hematopoietic stem cells (HSCs), which eventually developed de novo acute myelogeneous leukemia (AML). In contrast, in lymphoid malignancies, HSCs are not likely to play such a role in accumulation of mutations, because the clonality of lymphoid malignancies is usually found after the stage of rearrangement of T-cell receptor or immunoglobulin genes. However, we recently found that even in chronic lymphocytic leukemia (CLL), a mature B cell malignancy, the initial event for leukemic transformation could be traced up to HSCs. We found that mice transplanted with the CD34 + CD38- HSC fraction from CLL patients displayed expansion of monoclonal or oligoclonal CD5+ B cells. In contrast, normal HSCs always developed polyclonal CD5- B cells, suggesting that the primary deficit has occurred at the HSC stage in CLL patients. These data strongly support the hypothesis that HSCs are the cells that accumulate genetic abnormality to enable fully malignant transformation.

“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis

Abstract 2329Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications. Disclosures:No relevant conflicts of interest to declare.

Marking of Embryonic Precursors of Adult Hematopoietic Stem Cells At Gestational Day E7.5-E8.5 Using An Abcg2-CreERT2 Lineage Tracing Mouse Model

Abstract 1287Abcg2 is a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs), and is required for the side population phenotype of adult bone marrow HSCs as well as other adult tissue-specific stem cells. We have used these properties of Abcg2 expression for lineage tracing of stem cell development in mice, particularly for HSCs. An ires-CreERT2 cassette was inserted into the Abcg2 locus, down-stream of its stop codon but upstream of its endogenous polyadenylation site, so that both Abcg2 and CreER are co-expressed from a single bicistronic transcript. This design allows for minimum disruption of Abcg2 expression and tissue specific expression of CreERT2 under the control of endogenous Abcg2 transcription elements. The Abcg2CreER/CreER mouse was crossed with flox-STOP-flox-YFP (Rosa26RYFP/YFP) mouse to generate compound heterozygous Abcg2CreER/+ Rosa26RYFP/+ mice. Treatment of adult Abcg2CreER/+ Rosa26RYFP/+ mice with tamoxifen resulted in robust YFP expression in kidney proximal tubule cells and hepatocytes demonstrating the expected tissue-specific expression of the Abcg2CreER allele. We also observed tamoxifen-dependent appearance of YFP+ cells in all hematopoietic lineages in the peripheral blood and bone marrow, confirming our prior observations that Abcg2 is expressed in adult stem cells. Unexpectedly, we observed long term marking in intestinal epithelial cells and in seminiferous tubules 9 to 20 months after tamoxifen treatment, recapitulating classic progeny tracking patterns, proving that intestinal stem cells and spermatogonial stem cells express the Abcg2 marker. Pregnant females were treated with a single dose of 4-hydroxytamoxifen (4-HT) at gestational days E7.5 and E8.5 using overnight timed breeding pairs. We chose 4-HT rather than tamoxifen because 4-HT has been shown to decay relatively quickly in the fetus so that no recombination can occur 24 hours after the pulse. After maternal treatment, mice were born, grew to adulthood, and were analyzed 14–17 weeks after birth for expression of YFP in distinct peripheral blood lineages. In the majority of the 18 mice born from mothers treated with 4-HT at day E7.5 and from 17 mice born of mothers treated at day E8.5, a small but distinct YFP+ subpopulation could be clearly detected in all hematopoietic lineages (Figure A and B). The numbers of marked cells have been stable for approximately 4 months and are strictly dependent on 4-HT treatment of the mother. These results demonstrate that a precursor to adult hematopoietic stem cells exists at gestational day E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood. The low level of marking could reflect inefficient recombination due to either relatively low levels of expression of the recombinant allele in these embryonic HSC precursors or due to inefficient nuclear localization with the single 4-HT pulse. Alternatively, these marked embryonic HSC precursors may be generating only a minor population of adult HSCs that are competing against a larger fraction of HSCs that arise from precursors that originate later in gestation after the 24 hour 4-HT washout. We are in the process of determining the embryonic source of the E7.5 – E8.5 adult HSC precursor and have not yet determined whether it originates in the yolk sac, in another extra-embryonic source, or within the embryo proper. We are following these mice for longer periods of time to determine the stability of marking in primary and serial transplant experiments. Altogether, we expect that studies with this novel lineage tracing model will provide a better understanding of steady-state, uninterrupted embryonic hematopoietic development that does not require transplant assays to detect HSC activity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Disruption of Tet2 Leads to Enhanced Self-Renewal and Competitive Repopulating Capacity of Fetal Liver Hematopoietic Stem Cells

Abstract 2331TET2 (ten-eleven-translocation 2) gene has been reported to be frequently mutated in various human myeloid malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, acute myeloid leukemia, and chronic myelomonocytic leukemia. These observations suggest critical roles of TET2 dysfunction in molecular pathogenesis of myeloid malignancies. Recent studies using conditional knockout mouse model indicated that mouse Tet2 loss leads to clonal dominance of adult hematopoietic stem cells (HSCs) in competitive repopulation assay. However, self-renewal capacity of adult HSCs has never been addressed precisely by serial transplantation assay. In addition, the effect of Tet2 loss on hematopoietic stem/ progenitor cells was examined only in the BM, but not in the fetal livers (FLs). Since FL HSCs and adult HSCs differ in several aspects of their phenotypes and functions, we speculated that Tet2 might be involved differently in the regulation of FL and adult hematopoiesis.To address this issue, we analyzed E14.5 FL cells from Tet2 gene-trap (Tet2gt) mice. In these mice, gene trap-cassette was inserted into the second intron, just before the first coding exon. RT-PCR analysis showed that over 99% of Tet2 mRNAs from endogenous promoter were trapped by the gene-trap cassette in Tet2gt/gt mice, showing that Tet2gt allele can be considered as a null allele. Initial analysis showed that Tet2gt/gt embryos developed normally, but most Tet2gt/gt mice were perinatally lethal. Total numbers of FL cells and the numbers of committed progenitors in FLs as revealed by colony assays were not significantly different between each genotype. Interestingly, Tet2gt/gt embryos displayed significant increase in lineage (Lin)(-)Sca-1(+)c-Kit(+)(LSK) fraction compared to wild type (WT) (Tet2+/+) littermate (2.42±0.66% vs. 1.17±0.18%, p=0.02). In addition, common myeloid progenitor (CMP) fraction (IL7Rα(-), Lin(-), Sca-1(-), C-Kit(+), CD34(+), FcgRII/ III(low)) was significantly increased in Tet2gt/gt FLs compared to WT (9.04±1.09% vs. 6.26±0.53%, p=0.008). In serial transplantation assays, donor cells derived from Tet2+/gt and Tet2gt/gt FLs showed significantly higher peripheral blood chimerism in secondary and tertiary recipient mice as compared to that of WT cells, showing that disruption of Tet2 leads to the enhanced self-renewal capacity of FL HSCs. Moreover, donor-derived HSC fraction (CD34−LSK cells) was significantly expanded in the recipients of Tet2gt/gt FL cells, suggesting that increased self-renewal capacity is cell intrinsic to Tet2gt/gt HSCs. We have also examined differentiation of Tet2-mutant FL cells in the recipients’ peripheral blood, and found that Tet2gt/gt cells displayed impaired differentiation to Gr-1(+)CD11b(+) mature granulocytes (WT vs. Tet2gt/gt = 5.02±1.35% vs. 11.5±3.09% in the primary recipients) and slight, but significant increase of B cells. Liquid culture of FL cells with cocktails of cytokines in vitro demonstrated that Tet2gt/gt FL cells retained higher percentage and number of LSK, Lin- and c-Kit+ cells after the culture for 7-days compared to WT cells, showing enhanced resistance of Tet2gt/gt cells to differentiative stimuli in in vitro culture. It is of note that Tet2+/gt mice showed a significant increase in hematopoietic stem/progenitor fraction (LSK) in the BM compared to wild type littermate (0.48±0.11% vs. 0.32±0.04%, p=0.04). However, they presented no signs of extramedullary hematopoiesis such as splenomegaly and expansion of LSK cells in spleens during an observation up to 35-weeks.Taken together, we demonstrate that Tet2 critically regulates self-renewal and long-term repopulating capacity of FL HSCs and has pleiotropic functions in myeloid and lymphoid differentiation. These data strongly indicate that Tet2 is an essential regulator of BM and FL hematopoiesis. In addition, enhanced HSC self-renewal, expansion of HPC and myeloid progenitors and perturbed myeloid differentiation induced by TET2 ablation likely to set molecular basis for myeloid transformation, which explains high incidence of loss-of-function mutations of TET2 in myeloid malignancies. Disclosures:No relevant conflicts of interest to declare.

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

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

Enhanced Generation of Human Hematopoietic Stem/Progenitor Cells From ES and Patient Derived IPS Cells Using a Novel Compound Screen

Abstract 1294The ability to generate hematopoietic stem and progenitor cells from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to identify novel pathways involved in hematopoietic stem and progenitor cell generation and expansion from human ES and iPS cells. By using a small molecule compound library with our optimized iPS-2-blood lineage differentiation protocol, we have identified two novel chemical compounds that specifically enhance the generation of phenotypic adult hematopoietic stem cells. Compound A and compound B both enable increases of CD45/43+CD34+CD38-CD90+CD45RA- cells by 183+/−53%, n=5 and 275%, n=1, respectively. This is in comparison to DMSO carrier control wells, where the percentage of blood cells (CD45/43+) produced per total cells for these experiments was 73+/−2.9%, n=5. The increase in hematopoietic cell output using compound A was highly significant for the adult phenotypical hematopoietic stem cell fraction with a P-value of 0.03, n=5 (see Figure). Hematopoietic progenitor, CD45/43+ CD34+, counts were also slightly increased for compound A and B at 129+/−32%, n=5 and at 211%, n=1, respectively. Interestingly, no statistically significant increase in the number of total blood CD45/43+ cells was detected at the time of harvest, with either compound; at 108% +/− 8.4%, n=5, for compound A, and 164%, n=1, for compound B. In addition to the improvements, as measured by FACS, compounds A and B both increased the numbers of clonogenic progenitors as measured by CFU-assay, allowing for a 241+/−67%, n=4, and 443%, n=1, increase in total hematopoietic colony counts, respectively. These results identify 2 novel compounds with the ability to expand the more primitive fractions of hematopoietic cells with preferential expansion of the most primitive fraction of hematopoietic cells derived from human ES and iPS lines. We are currently performing transplantation experiments with cells generated using these compounds to assess their repopulating potential. Further studies are being performed to investigate the molecular mechanisms of these compounds for hematopoietic stem and progenitor cell generation from ES and iPS cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Hemp, an mbt domain-containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation

To clarify the molecular pathways governing hematopoietic stem cell (HSC) development, we screened a fetal liver (FL) HSC cDNA library and identified a unique gene, hematopoietic expressed mammalian polycomb (hemp), encoding a protein with a zinc-finger domain and four malignant brain tumor (mbt) repeats. To investigate its biological role, we generated mice lacking Hemp (hemp(-/-)). Hemp(-/-) mice exhibited a variety of skeletal malformations and died soon after birth. In the FL, hemp was preferentially expressed in the HSC and early progenitor cell fractions, and analyses of fetal hematopoiesis revealed that the number of FL mononuclear cells, including HSCs, was reduced markedly in hemp(-/-) embryos, especially during early development. In addition, colony-forming and competitive repopulation assays demonstrated that the proliferative and reconstitution abilities of hemp(-/-) FL HSCs were significantly impaired. Microarray analysis revealed alterations in the expression levels of several genes implicated in hematopoietic development and differentiation in hemp(-/-) FL HSCs. These results demonstrate that Hemp, an mbt-containing protein, plays essential roles in HSC function and skeletal formation. It is also hypothesized that Hemp might be involved in certain congenital diseases, such as Klippel-Feil anomaly.

MicroRNA-125b expands hematopoietic stem cells and enriches for the lymphoid-balanced and lymphoid-biased subsets

MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.

Hematopoietic Stem Cell Cycling Dynamics In Steady State and Upon Hematopoietic Challenge

AbstractAbstract 572Life-long blood production is maintained by a small fraction of hematopoietic stem cells (HSCs). Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assays with 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic mouse models (Wilson et. al., 2008; Foudi et. al., 2009). While the former studies showed that all HSCs equally divide and likely contribute to blood formation (clonal maintenance), the latter suggested that some HSCs divide frequently and contribute to blood formation until cell death or full differentiation, while some HSCs are quiescent and then get activated to follow the same fate as frequently dividing ones (clonal succession). However, due to low resolution, none of the labeling techniques used were able to track single cell divisions. Furthermore, methods used might have direct influence on cycling activity of HSCs. Thus it remains to be determined a) if HSC divide continuously, sequentially or repetitively and contribute to steady-state hematopoiesis, b) what is a relationship between divisional history and repopulating ability, and c) how self-renewal and differentiation capacity of HSC is impacted by naturally-occurring severe hematopoietic challenges as infections. To address this directly, we set up a high resolution non-invasive in vivo HSC divisional tracking assay with CFSE (carboxyfluorescein diacetate succinimidyl ester). We here show that i.v. transfer of CFSE-labeled HSCs into non-conditioned congenic recipient mice allows evaluation of steady-state HSC cycling-dynamics as CFSE is equally distributed to daughter cells upon cellular division. Transfer of Lin-c-kit+Sca-1+ cells (LKS) into non-irradiated mice revealed non- and 1–7x divided LKS in recipient bone marrow over 20 weeks. To test in vivo limiting dilution and single cell HSC potential, non- or ≥5x divided cells were sorted based on divisional history from primary recipients at different weeks after transplantation, and transplanted into lethally irradiated secondary recipients. Single non-divided LKS at 3 weeks post primary transfer was able to multi-lineage repopulate 24% of recipients long-term, while 50 of ≥5x divided LKS did not engraft. Interestingly, both non- and ≥5x divided LKS at 7 or 12–14 weeks after primary transfer engrafted and showed fluctuating contribution to multi-lineage hematopoiesis over serial transplantation. Mathematical modeling based on limiting dilution transplantation, revealed no evidence for a dichotomy of biologically defined HSCs in different groups. Instead, steady-state serial transplantation with temporary fast-cycling cells revealed that they can slow down over time, suggesting dynamically changing cycling activity of HSC. We next tested the effects of hemato-immunological challenge on HSC proliferation. Mice transplanted with CFSE-labeled LKS cells were repetitively treated with LPS. Analysis 8 days after final LPS injection, i.e. three weeks after steady-state transplantation revealed that all LKS entered cell cycle and the number of ≥5x divided LKS was increased. Secondary transplantation showed that 2–4 time and ≥5x divided LKS from LPS-treated mice reconstituted multi-lineage hematopoiesis whereas both fractions from control mice failed to engraft. This data clearly indicate that HSCs are activated from quiescence upon LPS challenge and provide evidence, that naturally-occurring hemato-immunological challenges, such as gram-negative bacterial infection induces proliferation and self-renewal of HSCs. Our data suggest in contrast to previously proposed concepts, a novel “dynamic repetition” model for HSC cycling activity and blood formation where some HSCs participate in hematopoiesis for a while, subsequently enter a resting phase and get reactivated again to contribute to blood formation in repetitive cycles, leading to homogenous total divisional history of all HSCs at end of life. These findings might represent a biological principle that could hold true for other somatic stem cell-sustained organ-systems and might have developed during evolution to ensure equal distribution of work-load, efficient recruitment of stem cells during demand, and reduction of risk to acquire genetic alterations or fatal damage to the whole HSC population at any given time. Disclosures:No relevant conflicts of interest to declare.

A Novel MBT-Containing Protein, Hemp, Plays Essential Roles In Skeletal Formation and Hematopoietic Stem Cell Function.

AbstractAbstract 1597Hemp (hematopoietic expressed mammalian polycomb, also denoted as mbt-containing 1) gene was originally identified in the hematopoietic stem cell (HSC)-enriched fraction of the mouse fetal liver (FL). It encodes a protein containing a putative Cys2-Cys2 zinc-finger region, followed by four tandem malignant brain tumor (MBT) repeats, which is frequently observed in polycomb gene (PcG) proteins. The structural characteristics strongly suggest that Hemp functions as an epigenetic regulator, but its biological role remains unknown. To address this issue, we generated hemp-deficient (hemp–/–) mice. Hemp–/– mice died soon after birth. Although no abnormalities were detected in internal organs, skeletal analysis exhibited a variety of malformations. Severe deformities were observed in the thoracic cavity, strongly suggesting that hemp–/– mice died of respiratory failure. Interestingly, they showed malformations of cervical and thoracic vertebrae, which were different from typical homeotic transformations observed in PcG-deficient mice. These results suggest that Hemp governs downstream target genes in distinct manners from conventional PcG proteins.The hematopoietic analysis of hemp in the FL showed that hemp is preferentially expressed in CD150+LSK and CD150–LSK HSC fractions in the hematopoietic hierarchy. Hemp–/– FL contained a significantly reduced number of hematopoietic cells and produced fewer number of hematopoietic colonies as compared to hemp+/+ FL. The decreases correlated with reduced number of CD150+LSK HSCs in hemp–/– FL, which generated much fewer hematopoietic colonies in the HPP-CFC assay. In addition, the competitive repopulation assay exhibited that the hematopoietic reconstitution ability of hemp–/– FL CD150+LSK HSCs was significantly impaired. Moreover, microarray analysis revealed that expression levels of several genes, such as Prdm16, Sox4, and Erdr1 were altered in hemp–/– FL HSCs.Since hemp–/– mice died at neonate, the role of Hemp in adult hematopoiesis remains to be elucidated. To address this issue, we generated hemp conditional knockout (cKO) mice. Acquired deletion of Hemp in the hematopoietic tissues was successfully achieved by crossing hempflox/flox mice with MxCre mice and stimulating the compound mice with pIpC. Analysis of the hematopoietic tissues revealed that the cell numbers of Mac+Gr1– and Mac+Gr1+ fractions in the hemp cKO bone marrow (BM) were significantly increased and decreased, respectively, as compared to those of the wild-type BM. However, no apparent differences have so far been observed between hemp cKO and wild-type littermates in functional analyses, such as colony forming activity and competitive repopulation ability of the BM cells.Here, we report that a novel MBT-containing protein, Hemp, plays essential roles in skeletal formation and HSC function during embryogenesis and also contributes to myeloid differentiation in adult hematopoiesis. Since Hemp likely functions as an epigenetic regulator, further studies will be required to clarify whether and what methylated lysine residues Hemp interacts with through the MBT repeats, what kind of genes are direct targets of Hemp, and how Hemp exerts its biological activity. Disclosures:No relevant conflicts of interest to declare.

Relationship between Radiosensitivity and Nrf2 Target Gene Expression in Human Hematopoietic Stem Cells

NFE2-related factor 2 (Nrf2), which belongs to the cap "n" collar family of basic region leucine zipper transcription factors, is a key protein in the coordinated transcriptional induction of expression of various antioxidant genes. The purpose of this study was to analyze the expression of Nrf2 target genes, such as heme oxygenase 1 (HO-1), ferritin heavy polypeptide 1 (FTH1), NAD(P)H dehydrogenase, quinone 1 (NQO1), glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione reductase (GSR) and thioredoxin reductase 1 (TXNRD1), after X irradiation of CD34(+) cells that were prepared from human placental/umbilical cord blood hematopoietic stem cells (HSCs). We evaluated the relationship between radiosensitivity and expression of Nrf2 target genes in HSCs. The number of colony-forming cells derived from 2-Gy-irradiated HSCs decreased to approximately 20% of the nonirradiated control. At the same time, the mRNA expression of HO-1, FTH1, NQO1, GSR and TXNRD1 was significantly increased after X irradiation. A statistically significant negative correlation was observed between the surviving fraction of HSCs and the intrinsic NQO1 mRNA expression, indicating that HSCs in which NQO1 mRNA levels are low may also be radioresistant. The present results suggest that the antioxidant system associated with Nrf2 is involved in the radiosensitivity of HSCs.

Abstract 2964: YB-1 expression in early hematopoiesis and leukemic cells

Abstract Hematopoietic transcription factors play a critical role in directing the commitment and differentiation of hematopoietic stem cells (HSC) along a particular lineage. Y box protein (YB-1), a cold shock family protein is a transcription factor which is widely expressed throughout development and has been implicated as a cell survival factor that regulates transcription and translation. YB-1 is known to be involved in erythroid cell development by interacting with Globin Transcription Factor (GATA); this study aims to investigate YB-1 expression in normal hematopoietic differentiation and leukemia. EML-clone 1 cells, a murine hematopoietic stem cell line, was used as a model for looking at the expression of YB-1 during myeloid differentiation by western blotting and quantitative RT-PCR. Fluorescence activated cell sorting was conducted to isolate lineage——/IL-7R—/c-kit+/Sca1+ (LKS) hematopoietic stem, lineage——/IL-7R—/c-kit+/Sca1— myeloid progenitor cells and granulocytes from mouse bone marrow to assess the YB-1 expression in vivo. YB-1 protein levels were analyzed in a panel of myeloid leukemic cell lines by immunoblotting. YB-1 mRNA and protein levels were high in the EML cells but the expression goes down in RA/IL-3 treated EML-clone 1 cells (myeloid progenitors) and was even more dramatically down-regulated in GM-CSF treated EML cells during the course of myeloid differentiation. Interestingly, LKS (enriched fraction of hematopoietic stem cells) and myeloid progenitor cells showed high level of YB-1 expression as compared to the differentiated cells like granulocytes. Further, we observed that YB-1 protein was expressed in several myeloid leukemic cell lines blocked at different stages of myeloid development. Thus, our data suggest that YB-1 is down-regulated during myeloid differentiation and it might be involved in hematopoietic differentiation. Aberrant YB-1 expression could be a contributing factor in the development of leukemia thus making it an excellent molecular target for therapy in myeloproliferative disorders and leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2964.

Preliminary findings on the use of pulsatile machine reperfusion of a placenta to improve the cord blood collection yield including primitive hematopoietic stem cell fractions

Umbilical cord blood (CB) use is limited in most adults since the mean amount of hematopoietic stem cells (HSCs) collected is generally insufficient to engraft successfully. This study demonstrates for the first time the feasibility of pulsatile machine placenta reperfusion (PMPR), which significantly improves CB collection quantity and quality compared to standard collection methods. PMPR was performed on eight delivered placentas up to 39 hours after venipuncture-based collection. PMPR was performed on average for 26 (20-30) minutes, 17 hours after delivery (6.25-39), using perfusate approved for donated organ reperfusion. Both PMPR and conventional collection-derived CB cells were analyzed using immunophenotyping and colony assays. The combination of PMPR and the conventional venipuncture method yielded a mean of 1.5-, 4.9-, 11-, 7.5-, 7.5-, and 7.7-fold increased number of total mononuclear, CD34+, CD34+/CD38-, CD133+, CD133+/CD34+, and CD133+/CD34- cells, respectively, compared to conventional venipuncture alone. CB cells obtained by PMPR alone generally demonstrated a significant increase in percentages of primitive HSC phenotype with equivalent cell viability between PMPR and venipuncture. This article demonstrates for the first time that CB can be collected up to 39 hours after delivery with PMPR while maximizing CB collection including HSCs with primitive phenotypes.