Primitive Human Hematopoietic Cells Research Articles

Single-Cell Analysis of Human B Lymphoid and Neutrophil/Monocyte Lineage Restriction

Lifelong production of most types of mature blood cells is sustained by a small population of cells with extensive regenerative potential. However, the detailed steps that restrict multipotent or even bipotent human hematopoietic cells to any single lineage remain poorly understood. Those that segregate the human B-lymphoid and neutrophil/monocyte (NM) lineages are of particular interest as these appear to identify a stage that might control the different properties of human leukemias that display perturbed NM and/or B-cell programs. To undertake a refined analysis of this normal lineage restriction process in human cells, we first devised a culture system that permits it to be tracked clonally and efficiently (50%). Initial experiments showed this could be achieved using a combination of multiple stromal cell types and human growth factor-supplemented medium. To elucidate the intervening transitional steps we then used multiplexed flow cytometry to compare the progeny generated in this culture system over a 2-week time course from previously defined lymphoid progenitor-enriched (P-L), NM progenitor-enriched (P-NM) and less restricted P-mix cord blood (CB) subsets. The results suggested that gain of CD45RA (RA) expression and loss of CLEC12A (C) expression appeared to accompany the sequential restriction of early CD34+ progenitors first to cells with dual NM+B-lineage potential and then just to B-lineage potential.Subsequent tracking of the lineage outputs of CD34+ RA-C- cells initially produced in larger numbers from unfractionated CD34+ CB cells either in vitro or in xenografted immunodeficient mice, confirmed the CD34+ RA-C- subset to be highly enriched in cells with dual NM+B potential. In contrast, co-generated CD45RA+ (RA+C-) and RA+CLEC12A+ (RA+C+) phenotypes displayed separate B- and NM lineage-restricted activity, respectively, as indicated by their largely exclusive clonal outputs of CD19+ pre-B and CD14+/CD15+ NM precursors. In agreement with these phenotypically established separate NM and B-lineage outputs, RA+C- cells were found to contain higher levels of B-lineage-associated gene transcripts (e.g., DNTT, CD79A, and EBF1), whereas RA+C+ cells contained higher levels of the NM transcription factor mRNAs encoded by SPI1 and CEBPA. In a further optimized stroma-free liquid culture system, the RA-C- cells could be shown to produce continuously RA+C- and RA+C+ progeny after another 3-4 days, and also RA-C- progeny which are not produced from more restricted RA+C- and RA+C+ cells, suggesting that the acquired expression of RA precedes the separation of NM and B-lineage potential that is then marked by the differential activation of C expression in RA+ cells.To examine more precisely how the process of B+NM restriction to a single lineage might be related to successive cell cycles, we labeled RA-C- cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) to enable the phenotypes and growth potential of the the progeny obtained after 6 days to be directly related to their prior division histories. This revealed extensive heterogeneity in the overall distribution of the initial progeny cell cycle times but with a clear segregation in the outputs of the faster and slower dividing cells. Notably, the faster dividers produced ultimately small M+B or uni-lineage clones whereas the initially slow dividers subsequently produced larger clones, 25% of which still contained CD34+ cells or N+M+B progeny.Taken together, these findings identify new hallmark phenotypic changes that identify a critical step in the lineage restriction of primitive human hematopoietic cells with dual NM+B-lineage potential and a previously unknown association of this process with a shortening of their cell cycle transit time. DisclosuresNo relevant conflicts of interest to declare.

Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel

The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.

PF573 SINGLE CELL RNA-SEQ REVEALED A RARE ITGA2 EXPRESSING CELL SUBPOPULATION WITH DISTINCT CANCER STEM CELL MARKER SIGNATURE IN MULTIPLE MYELOMA

Background: Multiple myeloma (MM) stem cells (MMSC) are a rare cell population capable of self-renewal and differentiation into the predominating mature myeloma cells. Specific targeting this cell population could effectively eradicate the tumors in the patients. Previous studies reported CD138 non-expressing myeloma cells as putative MMSC. However, conflicting results by subsequent studies demonstrated the tumor-initiating and clonogenic cells expressed CD138 and apoptotic features in CD138 non-expressing cells. Aims: In this study, we aimed at identification of MMSC by examination of the transcriptome of myeloma cells at single cell level. Methods: Single cell RNA-Seq with flow-based SMART-Seq2 protocol was employed to determine the transcriptome of 768 myeloma cells from MM1 s and NCIH929 cell lines (384 cells each line) and 68 cells from a clinical MM bone marrow sample. The percentages of myeloma cells expressing the candidate biomarker(s) protein were determined by flow cytometry. Results: Results of quality control analyses indicated ∼400K read counts per cell with 15–20% of multiple mapping representing ribosomal RNAs and 12–15% mitochondrial RNAs and with 6700–8600 variable genes for transcriptome clustering analyses. By unsupervised analysis using t-distributed stochastic neighbor embedding (t-SNE), myeloma cells were clustered according to their cell cycle statuses. After controlling the cell cycle effects by regression, two clusters with similar cell numbers were observed in each cell line. Interestingly, the results of supervised analysis with t-SNE on 24 cancer stem cell markers expression revealed a rare subpopulation of myeloma cells (MM1 s: 2.6%; NCIH929: 3.6%) that were signified by Integrin alpha 2 (ITGA2) expression but no expression of OCT4 and NANOG stem cell markers. Flow cytometry using anti-ITGA2 antibody confirmed the presence of this rare cell population (∼0.3%) in MM cells. For the cells from the clinical sample, 3 clusters were observed by unsupervised analysis after controlling cell cycle effects. Studies of the signature genes for each cluster indicated two clusters were derived from erythroid cells and lymphocytes; while the 3rd cluster (n = 49) from myeloma cells, within which one ITGA2-expressing cell was idenitifed. ITGA2 encodes a cell surface protein CD49b integrin receptor as a marker for primitive human hematopoietic stem cells. In leiomyoma, CD49b marked the side population cells and the stemness of CD49b-expressing cells was also demonstrated. Recently, increased NOTCH1 and its downstream targets expression implying high activity of NOTCH signaling, which are involved in stem cell self-renewal and differentiation, were reported in CD49b-expressing trophoblast progenitor cells in human placenta. All these findings suggested the link of ITGA2 and stem cell properties. Functional studies of the ITGA2 positive myeloma cells are currently in progress regarding their clonogenic potentials and self-renewal capacity. Summary/Conclusion: Our study revealed a rare subpopulation of ITGA2-expressing cells with distinct cancer stem cell markers signature in MM cell lines and a clinical sample, potentially representing MMSC.

Distinct signaling programs control human hematopoietic stem cell survival and proliferation

AbstractSeveral growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38−CD45RA−CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90−CD49f− cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

Analysis of parameters that affect human hematopoietic cell outputs in mutant c-kit-immunodeficient mice.

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo. However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34+ cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1−/−-IL2Rγc−/− (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD-Prkdcscid/scid-IL2Rγc−/−(NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W41/W41 derivatives showed superior chimerism in the W41-deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice.

The Functional Significance of the MPL Expression in the Primitive Human Hematopoietic Stem Cell Compartment

The Functional Significance of the MPL Expression in the Primitive Human Hematopoietic Stem Cell Compartment

Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D3 to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC50–0.3 nM): ~50-fold more is required for activation of RARα (EC50–16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

GPI-80 Defines Primitive Human Cord Blood-Derived CD34-Positive and Negative Hematopoietic Stem Cells

Background. We identified very primitive CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) in human cord blood (CB) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). These CD34- SRCs possess myeloid-biased differentiation potential, which suggests that they are a distinct class of primitive hematopoietic stem cell (HSC) and that they reside at the apex of the human HSC hierarchy (Blood Cancer J 2015:5;e290). We recently developed high-resolution purification methods for CD34- SRCs using 18 Lineage (18Lin)-specific antibodies, which can enrich CD34- SRC at the 1/1,000 level in 18Lin- CD34- fractions (Exp Hematol 2011:39:203). In addition, we previously identified CD133 as a positive marker for CD34- SRCs as well as for CD34+ SRCs (Leukemia 2014:28;1308). The results showed that CD34+/- SRCs were enriched to approximately 1/100 and 1/140 in 18Lin- CD34+/- CD133+ fractions, respectively.Aim. In order to further elucidate the details of the characteristics of human CD34+/- HSCs, we aimed to identify additional positive markers for the high-level purification of CB-derived CD34+/- SRCs.Materials and Methods. First, weextensively analyzed the candidate positive markers, including cell adhesion molecules and homing receptors that are expressed on 18Lin- CD34+ CD38- and 18Lin- CD34- cells by multicolor FACS. Finally, we discovered that glycosylphosphatidylinositol-anchored protein GPI-80, which has recently been reported as a marker for human fetal liver hematopoietic stem/progenitor cells (HSPCs) (Cell Stem Cell 2015:16;80), was also expressed on human full-term CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- cells. Next, we sorted 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells from human CB. The HSPC characteristics of the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were assessed as follows: (1) the in vitro maintenance/production capacities of CD34+ and CD34+ CD38- cells were examined in co-cultures with mesenchymal stroma cells (MSCs) established from human bone marrow-derived CD45- Lin- CD271+ SSEA-4+ cells (Stem Cells 2015:33;1554); (2) an SRC assay was performed using NOD/Shi-scid/IL-2Rγcnull (NOG) mice; (3) limiting dilution analyses (LDAs) were performed to determine the SRC frequencies in these four fractions of cells.Results. In the CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- fractions, 10.1% and 14.4% of cells expressed GPI-80, respectively. The 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were then co-cultured with human MSCs for 7 days in the presence of SCF and TPO. As a result, the 18Lin- CD34+ CD38- GPI-80+ cells maintained significantly higher percentages of CD34+ (86.4%) and CD34+ CD38- cells (24.8%) in comparison to 18Lin- CD34+ CD38- GPI-80- cells (78.7% and 14.3%, respectively). However, 18Lin- CD34- GPI-80+/- cells produced comparable levels of CD34+ (50.3% and 50.8%) and CD34+ CD38- cells (4.4% and 5.3%). These four fractions of cells were then transplanted into NOG mice using the IBMI method. All of these four fractions of cells showed long-term repopulating SRC activities with multi-lineage differentiation potential in mouse bone marrow. However, LDAs demonstrated that the frequencies of SRC in the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- fractions were 1/21, 1/35 and 1/28, 1/874, respectively. These data clearly demonstrate that both CD34+/- SRCs are enriched in GPI-80+ fractions. Surprisingly, a number of mice received a limited number of 18Lin- CD34+ CD38- GPI-80+ (2 cells) and 18Lin- CD34- GPI-80+ cells (10 cells), and thus also showed multi-lineage long-term human hematopoietic cell repopulation.Conclusion. These observations clearly demonstrated that GPI-80 defines CB-derived human primitive HSCs. Furthermore, these results indicate that GPI-80 is a useful marker for the high-level purification of human CB-derived CD34+/- SRCs (HSCs). DisclosuresNo relevant conflicts of interest to declare.

Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo.

Developmental downregulation of LIN28 and HMGA2 expression in primitive normal human hematopoietic cells

Developmental downregulation of LIN28 and HMGA2 expression in primitive normal human hematopoietic cells

CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells

The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.

Mapping of MN1 Sequences Necessary for Myeloid Transformation

The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1’s in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12–228 (Δ12–228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12–228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12–228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.

Expression of Integrin α2 Receptor in Human Cord Blood CD34+CD38−CD90+ Stem Cells Engrafting Long-Term in NOD/SCID-IL2Rγcnull Mice

Human hematopoietic stem cells reside in the CD34+CD38-CD90+ population in cord blood and bone marrow. However, this cell fraction is heterogeneous, and the phenotype of the rare primitive stem cells remains poorly defined. We here report that primitive cord blood CD34+CD38-CD90+ stem cells, with the ability to reconstitute NOD/SCID-IL2Rγ(c) null (NSG) mice long-term, at 24 weeks after transplantation, can be prospectively isolated at an increased purity by using integrin α2 receptor as an additional stem cell marker. Using a limiting dilution transplantation assay, we found a highly significant enrichment of multilineage reconstituting stem cells in the CD34+CD38-CD90+ cell fraction expressing the integrin α2 receptor, with a frequency of 1/29 cells, as compared to a frequency of 1/157 in the corresponding integrin α2- cells. In line with this, long-term reconstituting stem cells within the cord blood CD34+CD38- cell population were significantly enriched in the integrin α2+ fraction, while stem cells and progenitors reconstituting short-term, at 8-12 weeks, were heterogeneous in integrin α2 expression. Global gene expression profiling revealed that the lineage-marker negative (Lin-) CD34+CD38-CD90+CD45RA- integrin α2+ cell population was molecularly distinct from the integrin α2- cell population and the more mature Lin-CD34+CD38-CD90-CD45RA- cell population. Our findings identify integrin α2 as a novel stem cell marker, which improves prospective isolation of the primitive human hematopoietic stem cells within the CD34+CD38-CD90+ cell population for experimental and therapeutic stem cell applications.

Characterization of activin A in the culture of primitive human umbilical cord blood hematopoietic cells

Transforming growth factor-β (TGF-β) superfamily controls many physiological processes such as cell proliferation and differentiation, immune responses, wound repair and various endocrine activities. As a member of TGF-β, activin A can maintain the pluripotency of embryonic stem cells. We report here that activin A exhibited cell type-dependent function of expanding the human primitive hematopoietic cells isolated from umbilical cord blood (UCB). However, the multipotency of the cells pretreated with activin A was exhausted in the sequential dilution culture. In conclusion, activin A may not be a key factor, but a regulator, in the multipotency maintenance of primitive hematopoietic cells and the application of activin A in the hematopoietic stem/progenitor cells (HS/PCs) culture expansion remains a significant challenge.

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML chronic phase samples, about 70% of CML accelerated phase samples, and >80% CML blast crisis phase samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p < 0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage cell apoptosis, while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, and P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G(2)/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.

Detection of a Stem Cell Population with Hemangioblastic Characteristics in Primary Myelofibrosis (PMF),

Abstract 3860Primary myelofibrosis (PMF) comprises a myeloproliferative neoplasia accompanied by imbalance of various tissues of the mesoderm, let alone the hematopoietic tissue. Involvement of multiple hematopoietic lineages during disease progression suggests the clonality of myelofibrosis that can be attributed to an initial stem cell defect at the very early stage of the stem cell hierarchy. Hematopoietic and endothelial phenotypes of circulating multipotent stem cells in patient peripheral blood, along with the increased microvascular density in the bone marrow, leads to the hypothesis that the critical event in PMF involves malignant transformation of a stem cell with hemangioblastic potential.Former studies have provided functional evidence that activated JAK2 signalling in primitive human hematopoietic cells is sufficient to drive key processes involved in the pathogenesis of the disease. In this study, the functionality and differentiation potential of circulating primitive JAK2V617F+ stem cells from primary myelofibrosis patients is assessed.Primitive stem cells were isolated from peripheral blood of 25 patients. All patients participating in the study were diagnosed with primary myelofibrosis, have been untreated, and were found positive for JAK2V617F mutation. Isolated stem cells were analysed for purity and assessed for the expression of markers characteristic for the hemangioblast phenotype (CD34, CD133, CD45, VEGFR2, VE-Cadherin, E-Cadherin, CD31) with flow cytometry. Genomic DNA was isolated from various stem cell populations to determine the mutational status by PCR. Our results indicate that long term repopulating stem cells circulating in peripheral blood bear the JAK2V617F mutation. Hemangioblast resembling populations within the isolated prime stem cells were also found positive for the mutation. Long term repopulating stem cells bearing different allele burden for JAK2V617F mutation from PMF patient peripheral blood were expanded for up to 4 months. Various colonies formed after seeding in semisolid media were characterised by morphological features (CFU-GEMM, CFU-GM, CFU-E, CFU-M, CFU-Endo) and expressing genes by quantitative PCR. Moreover, allele burden determination for various progenitors of both hematopoietic and endothelial lineages was performed. JAK2V617F allele burden varied within individual progeny phenotypes, indicating the acquisition of the mutation that boosts the outgrowth of the malignant clone within the hemangioblast compartment of the bone marrow. Endothelial and macrophage progenitors appear heterozygotic while all rest progenitors of various hematopoietic lineages can be either heterozygotic or homozygotic. This indicates high genomic instability of the JAK2V617F+ malignant clone as it is driven into hematopoietic differentiation.Our results indicate the existence of a malignant clone with hemangioblast phenotype in PMF which can differentiate into hematopoietic and/or endothelial progenitors in vitro. Our experiments shed light to the pathogenesis of PMF by characterising the potential of the defective stem cell subpopulation responsible for the disease. Disclosures:No relevant conflicts of interest to declare.

Effects of LMO2 on Human T-Cell Development Are Modulated by Notch Signaling

Abstract 2470LMO2 is overexpressed in a significant percentage of human T cell acute lymphoblastic leukemia (T-ALL) and its locus has been the target of insertional mutagenesis in gene therapy trials. In the past years, 4 X-linked severe combined immunodeficiency (X-linked SCID) and one Wiskott-Aldrich syndrome (WAS) patients who were treated by retrovirus-mediated gene therapy developed T-ALL as a result of retroviral integration in the LMO2 locus. In these patients, leukemia developed 2 to 3 years after gene therapy without prior significant haematological abnormalities. However, both the latency of disease and the finding of additional somatic mutations and/or translocations in these leukemias suggest that the overexpression of LMO2 alone is insufficient to generate leukemia, a notion that has been supported by studies in mouse. Though LMO2 is typically recognized as a T-cell oncogene, reports have shown that it is also aberrantly expressed in acute myeloid leukemias (AML), chronic myeloid leukemia (CML), B-ALL and some non-hodgkin B cell lymphomas.In order to study the impact of LMO2 overexpression on human hematopoietic stem/progenitor cells, a lentiviral vector was used to express this oncogene together with EGFP in lineage-depleted umbilical cord blood. In myeloid-promoting cultures, LMO2 had no effect on either differentiation or proliferation. Moreover, the expression of LMO2 did not modify the frequency or lineage distribution of colony forming progenitors compared to controls. However, significant differences were noted when transduced cells were assayed on OP9-Delta-Like 1 (DL1) stroma, an in vitro system that promotes T cell proliferation and differentiation. Cells overexpressing LMO2 were blocked at the double negative stage (CD4-CD8-) of differentiation and proliferated 50 to 100 times more than control cells. However, these cells were not immortalized as they proliferated for a median of 75 days, versus 50 days for controls.Immunodeficient mice transplanted with primitive human hematopoietic cells expressing LMO2 (hereafter referred as LMO2 mice) had bone marrow engraftment levels comparable to controls at 20–24 weeks post-transplant. Neither B-lymphoid nor myeloid development were affected by LMO2 overexpression. Strikingly, in the thymus, the percentage of EGFP+ cells was significantly increased in LMO2 mice compared to controls (mean of 47.7% versus 8.8%, p=0.0001), clearly indicating that expression of this oncogene enhances thymic T-cell engraftment. We next analyzed the phenotype of LMO2-expressing T cells in the thymus and peripheral blood of these mice. Surprisingly, unlike our in vitro studies, there was no evidence of a block at the DN-stage of differentiation. Instead, there were significantly less EGFP+ DN cells in the thymi of LMO2 mice compared to controls (mean of 7.5% vs 14.5%, p=0.035). These results clearly demonstrate that unlike what was observed in OP9-DL1 co-cultures, LMO2 overexpression does not induce a block in T-cell differentiation in our in vivo system. One possible explanation for this difference is the constitutive NOTCH signaling provided via DL1 on stroma compared to the in vivo setting where LMO2-expressing cells would encounter different levels and forms of NOTCH signaling throughout development. To test this hypothesis, LMO2 cells were cultured on OP9-DL1 stroma for 50 days then switched onto OP9 stroma lacking NOTCH ligand. Upon transfer, the DN cells promptly stopped proliferating and differentiated into DP (CD4+CD8+) cells expressing CD3 and TCRαβ.Thus, our results suggest that in the in vivo setting, as cells migrate through the thymus and face a decrease in NOTCH signaling, LMO2 overexpression alone can promote proliferation, but is not sufficient to maintain a differentiation block. However, constitutive NOTCH signaling can cooperate with LMO2 overexpression to block T cell differentiation at a proliferative DN stage. Thus, one can postulate that LMO2 exerts a proliferative effect on developing T-cells in thymic regions with high levels of NOTCH signaling, potentially providing a setting for the development of secondary leukemogenic events. NOTCH mutations are common in human T-ALL and can therefore allow for LMO2 overexpressing cells to become independent of the stromal niche. Taken together, our results suggest cooperation between LMO2 overexpression and NOTCH signaling in human T-cell leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Abstract 4301: Deletion-mapping identifies the regions within MN1 that regulates its transforming activity in hematopoietic cells

Abstract The oncogene MN1 is deregulated in human acute myeloid leukemias and its overexpression is known to induce proliferation and repress myeloid differentiation of primitive human and murine hematopoietic cells, which causes myeloid leukemia in mouse models. To study the role of different regions within MN1 in MN1-induced leukemia, we generated in-frame deletion mutants of the protein. Using retroviral transduction assays, we found that the regions between amino acids 12 to 228 and 570 to 1273 are required for its in vivo transforming activity, although the 12-228-mutant was still capable of repressing myeloid differentiation and inducing self-renewal in vitro. Both MN1 and the 12-228-mutant increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparison of purified CMPs identified Hoxa9, Meis1, Myb, Cebpa, Cebpb and Cebpd among many other differentially expressed transcripts as potential candidates that mediate MN1-induced leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2011-4301

Inhibition of Grb2 expression demonstrates an important role in BCR–ABL-mediated MAPK activation and transformation of primary human hematopoietic cells

Chronic myeloid leukemia (CML) results from expression of the BCR/ABL oncogene in a primitive hematopoietic cell. However BCR/ABL-activated signaling mechanisms are dependent on the cellular context in which it is expressed, and mechanisms underlying primitive human hematopoietic cell transformation by BCR-ABL are not well understood. Our previous studies have shown that BCR/ABL-Y177 plays an essential role in Ras activation and human hematopoietic progenitor transformation in CML. The adapter protein growth factor receptor binding protein-2 (Grb2) can bind phosphorylated BCR/ABL-Y177, induce Grb2-SoS complex formation, and activate Ras signaling. We investigated the role of Grb2 in CML progenitor transformation by co-transducing human CD34+ cells with lentivirus vectors expressing shRNA to Grb2 and retrovirus vectors expressing BCR/ABL. We show that Grb2 knockdown significantly inhibits proliferation and survival of BCR-ABL-expressing CD34+ cells, but not control CD34+ cells. Grb2 knockdown reduced MAPK activity in BCR-Abl-expressing hematopoietic cells. We conclude that inhibition of Grb2 expression demonstrates an important role in BCR-ABL mediated MAPK activation and transformation of primary human hematopoietic cells. These results support further investigation of downstream effectors of Grb2-mediated signals and targeting of Grb2 interactions in the treatment of CML.

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.