Pluripotent Hematopoietic Cells Research Articles

Expression of RUNX1-JAK2 in Human Induced Pluripotent Stem Cell-Derived Hematopoietic Cells Activates the JAK-STAT and MYC Pathways.

A heterogeneous genetic subtype of B-cell precursor acute lymphoblastic leukemia is driven by constitutive kinase-activation, including patients with JAK2 fusions. In our study, we model the impact of a novel JAK2 fusion protein on hematopoietic development in human induced pluripotent stem cells (hiPSCs). We insert the RUNX1-JAK2 fusion into one endogenous RUNX1 allele through employing in trans paired nicking genome editing. Tagging of the fusion with a degron facilitates protein depletion using the heterobifunctional compound dTAG-13. Throughout in vitro hematopoietic differentiation, the expression of RUNX1-JAK2 is driven by endogenous RUNX1 regulatory elements at physiological levels. Functional analysis reveals that RUNX1-JAK2 knock-in cell lines yield fewer hematopoietic progenitors, due to RUNX1 haploinsufficiency. Nevertheless, these progenitors further differentiate toward myeloid lineages to a similar extent as wild-type cells. The expression of the RUNX1-JAK2 fusion protein only elicits subtle effects on myeloid differentiation, and is unable to transform early hematopoietic progenitors. However, phosphoprotein and transcriptome analyses reveal that RUNX1-JAK2 constitutively activates JAK-STAT signaling in differentiating hiPSCs and at the same time upregulates MYC targets—confirming the interaction between these pathways. This proof-of-principle study indicates that conditional expression of oncogenic fusion proteins in combination with hematopoietic differentiation of hiPSCs may be applicable to leukemia-relevant disease modeling.

Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay.

Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible. PLB-985 cells, which are pluripotent hematopoietic cells that can easily be differentiated to neutrophil-like cells, are amenable to genetic manipulations, including the expression of fluorescently tagged proteins-of-interest (POI) and gene editing using the CRISPR/CAS9 system to delete genes-of-interest (GOI). The use of PLB-985 cells can therefore greatly facilitate our understanding of the molecular mechanisms governing neutrophil biology during chemotaxis and serve as a good system to complement results gained from pharmacological inhibition of primary neutrophils. To better study the role and localization of proteins during chemotaxis, the underagarose assay has become a widely used and quantitative assay for measuring several aspects of chemotaxis. The objective of this chapter is to provide protocols for (1) the generation of genetically altered PLB-985 cell lines, (2) the set-up of an underagarose chemotaxis assay, and (3) the analysis of cell movement in chemotactic gradients from an underagarose experiment.

Viral programming of progenitor cell commitment.

Human cytomegalovirus (HCMV) infects pluripotent haematopoietic progenitor cells, yet it is latent in monocyte/myeloid-lineage cells. A new study reveals that HCMV achieves this by actively reprogramming the infected progenitor cell into a unique monocyte subset, enabling the successful lifelong persistence of HCMV in its host.

Comparative Analysis of Infused “Static”, Ex Vivo-Generated Platelets Vs. Infused Megakaryocytes-Generated Platelets: A Cautionary Tale

Ex vivo-generated (EV) platelets beginning with embryonic stem cells or induced pluripotent stem cells (iPSCs) or hematopoietic progenitors cells (HPCs) may have clinical utility over donor-derived platelets, and efforts to produce such EV-platelets have been pursued in several laboratories under static megakaryocyte (Meg) culture conditions. Success in generating these has been reported, even demonstrating EV-platelet incorporation into growing thrombi in murine models. We have pursued an alternative strategy for thrombopoiesis using EV-Megs, grown from either human adult HPCs or from iPSCs or fetal livers, and directly infusing them into NOD-SCID gamma-interferon-deficient (NSG) mice. These studies were based on our prior observation that infused murine EV-Megs into wildtype mice are entrapped in the pulmonary bed and over the subsequent 1-4 hours release a wave of functional platelets at a significant level. We now show that infusion of human EV-Megs do the same in NSG mice, but resulting in two different pools of derived platelets: (1) A pool of young (as determined by thiazole orange staining) platelets having the same bell-shaped size distribution was seen as after infusion of human donor-derived platelets in these mice. These platelets take several hours to appear, but then have the same half-life as donor-derived platelets. These platelets are derived from the infused EV-Megs and were termed in vivo-generated (IV)-platelets. (2) A second pool of mostly older platelets was present that originated during the static growth of the EV-Megs, and these EV-platelets varied widely in size and age. Initially, these platelets accounted for a third of all the human platelets seen. Unlike IV-platelets, EV-platelets are immediately present and circulate with a markedly short half-life of 2-3 hours unless the recipient NSG mice were pre-treated with clodronate-ladened liposomes to delete their macrophage pools. Rapid removal of EV-platelets by macrophages is due to their being preactivated as determined by surface P-selectin expression in whole mice blood. These EV-platelets also had very limited further responsiveness to convulxin activation. On the other hand, human IV-platelets were quiescent prior to agonist stimulation in whole mice blood and responded strongly to agonist, similar to human donor-derived platelets infused into NSG mice. The IV-platelets were also selectively incorporated into cremaster arteriole laser injury thrombi over EV-platelets. Finally, directly harvested “platelets” from EV static-grown Megs were isolated and analyzed both in vitro and in vivo. Only a third of these particles are CD41+/CD42+ platelets and approximately half are actually CD41-/CD42-. Both pools showed the same wide size distribution in vitro and in vivo after infusion into mice. The CD41+/CD42+ fraction behaved just as the EV-platelets, but the CD41-/CD42- fraction half-life was unaffected by pre-treatment with clodronate-ladened liposomes. In summary, infused human Megs grown under static growth conditions released platelets in a recipient mouse’s lung with features characteristic of donor-derived platelets. On the other hand, “platelets” harvested from the same Megs were predominantly not even platelets as measured using CD41/CD42 markers. The portion that were CD41+/CD42+ platelets were preactivated, poorly responsive to agonists, and cleared rapidly. These findings set a standard on how to judge the potential clinical value of platelets derived from EV-Megs and also raise concerns whether direct visual imaging of “platelet release” in static culture is biologically meaningful given that most particles released were not CD41+/CD42+ platelets, and the ones that were CD41+/CD42+ were mis-sized and functionally limited. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1456: MicroRNA improves the efficacy of imatinib on the treatment of chronic myeloid leukemia

Abstract Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells that is caused by the deregulated activity of the tyrosine kinase that is encoded by the chimeric bcr-abl oncogene. Given that CML cells are usually highly resistant to chemotherapy, novel and more efficacious strategies against this disease are urgently needed. MicroRNA (miRNA) is a set of newly discovered 20-22 nucleotides RNA molecules, and they broadly repress their cognate target genes at post-transcriptional/trans-lational level. To date, there are approximately 2,500 human miRNA transcripts have been characterized, which are estimated to modulate more than 30% human genes by being involved in many essential normal biological and pathological processes including differentiation, proliferation, apoptosis, and tumorigenesis as well. In this study, we studied let-7a, a miRNA with tumor suppressive signature, for its therapeutic potential in CML. When treating CML K562 and KU812 cell by imatinib, let-7a can be significantly induced. Overexpression of let-7a can sensitize K562 and KU812 cells to imatinib, while knock-down of let-7a can increase the resistance of these cells towards this drug. The functional studies found that let-7a was able to promote apoptosis of CML cells, inhibit the tumor cell proliferation, and lead to a cell cycle arrest in the G0/G1 phase. These results demonstrate that let-7a is a potential target for treating CML by imatinib and/or other anti-cancer drugs. The pro-apoptosis and anti-proliferation roles of let-7a in CML cells may provide novel insights into discovery and development of more efficacious drugs for treating this malignant disease. Citation Format: Bin Yi, Ruixia Ma, Xiangling Feng, Zhiwei Xing, Xiaoguo Zhang, Gary Piazza, Yaguang Xi. MicroRNA improves the efficacy of imatinib on the treatment of chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2014-1456

Growth of human mast cells from bone marrow and peripheral blood-derived CD34(+) pluripotent hematopoietic cells.

Human mast cells (HuMCs) are derived from CD34(+) pluripotent hematopoietic cells which are KIT (CD117)(+) and FcεRI(-), and lack lineage-specific surface markers. Bone marrow and peripheral blood are the two readily available sources for obtaining CD34(+) cells from which HuMCs can be cultured. CD34(+) cells are isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34(+) cells may be immediately cultured in serum-free culture media containing recombinant human (rh) stem cell factor (SCF), rhIL-6, and rhIL-3 (added only during the first week). Weekly hemidepletions and removal of adherent cells and/or debris enables the investigator to obtain HuMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 weeks.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Are mast cells the real culprit in atherosclerosis?

This editorial refers to ‘Mast cells in human carotid atherosclerotic plaques are associated with intraplaque microvessel density and the occurrence of future cardiovascular events’, by S. Willems et al. , doi:10.1093/eurheartj/eht186 Atherosclerosis in the presence of hyperlipidaemia is considered today as an inflammatory disease of the arterial wall. The dominant inflammatory cell is the macrophage; however, interaction with other inflammatory cells and red cells may be critical in the outcome of the plaque, i.e. whether plaques remain stable or become unstable. It has been repeatedly shown that macrophage interaction with T cells and dendritic cells is critical for the progression of plaques and for maintaining and sustaining inflammation. The role of mast cells in atherosclerosis has only been studied sporadically, and that too mostly in the aortic and coronary beds. Willems et al .1 now show for the first time not only that severe carotid atherosclerotic disease is associated with a high number of mast cells but that the instability of the plaque may be influenced by the number of mast cells and their state of activation, which probably contribute to angiogenesis and plaque haemorrhage. Also, patients with high intraplaque mast cells had significantly greater cardiovascular events during a 3-year follow-up. However, the basic mechanisms involved will need further study in both animal models and man. Paul Ehrlich's doctoral thesis was a milestone in the study of mast cells, and he noted that they had similar staining characteristics to basophils, demonstrating metachromatic staining with aniline dyes.2 Mast cell development depends on the expression of KIT ligand, also known as mast/stem cell growth factor (SCF), receptor from the pluripotent haematopoietic progenitor cells (HPCs) that express CD34 + , CD117 + , CD13 + , FcɛRI – , maturing in the target tissues, such as skin, mucosal surfaces, and probably also …

Abstract 4051: Loss of nuclear beta catenin, following chibby enforced expression, activates endoplasmic reticulum stress in cells expressing the BCR-ABL1 fusion gene of chronic myeloid leukemia.

Abstract CML is a myeloproliferative disease originated from a pluripotent hematopoietic cell, the bona fide leukemic stem cell (LSC), where reciprocal translocation t(9;22) generates the BCR-ABL rearranged gene. This single genetic lesion drives the clonal expansion of leukemic hematopoiesis through the constitutively activated tyrosine kinase (TK) of its protein. The majority of CML patients undergoes complete hematologic remission in response to TK inhibitor Imatinib (IM). However, BCR-ABL+ LSC are neither dependent on BCR-ABL TK for proliferation and survival nor killed by IM and second generation inhibitors, providing a putative source of drug-resistance. β catenin is a central component of BCR-ABL+ LSC self-renewal. Moreover, it may have a role in the stemness recovery of committed granulocyte/macrophage progenitors at the outcome of blast crisis. Consequently, beta catenin subcellular distribution is a central component of signaling activity and its nuclear-cytoplasmatic shuttling is tightly regulated by multiple carrier protein, including 14-3-3. Chibby (CBY) is a protein that directly interacts with beta catenin and competes with TCF/LEF factors for beta catenin binding, repressing its transcriptional activity. Moreover, Chibby cooperates with 14-3-3 to regulate beta catenin subcellular distribution and signaling activity. We have recently found a significant CBY reduction associated with BCR-ABL TK with a consequent increment of active beta catenin. This findings suggested a CBY putative role in the proliferative advantage of CML hematopoiesis and addressed our further investigation towards the CBY impact on proliferation and survival of BCR-ABL+ cells. To this purpose we stably transduced the BCR-ABL+ K562 cell line with a construct containing C22orf2. The cytoplasmatic accumulation of beta catenin triggered the endoplasmic reticulum (ER)-associated stress pathway known as the unfolded protein response (UPR). The UPR is primarily a survival response which activates a series of complementary adaptive mechanisms to resolve the dysregulation of protein folding. However, under prolonged and irreversible ER stress conditions UPR triggers apoptosis through caspase activation and modulation of ER Ca2+ signaling. The ER stress elicited by beta catenin cytoplasmatic accumulation in consequence of CBY overexpression doomed K562 cells to apoptotic death induced by a significant increase of CHOP, which amplifies the pro-apoptotic signal by transcriptional activation of BIM and the release of PKR-like eIF2 kinase (PERK) and inositol-requiring enzyme (IRE1), all mediators of ER stress. Our results suggest that enforced CBY expression significantly reduced K562 proliferation through events encompassing the beta catenin prominent relocation to the cytoplasm and its transcriptional silencing. Citation Format: Manuela Mancini, Elisa Leo, Enrica Borsi, Fausto Castagnetti, Michelangelo Fiorentino, Ilaria Iacobucci, Michele Cavo, Maria Alessandra Santucci, Giovanni Martinelli. Loss of nuclear beta catenin, following chibby enforced expression, activates endoplasmic reticulum stress in cells expressing the BCR-ABL1 fusion gene of chronic myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4051. doi:10.1158/1538-7445.AM2013-4051

Abstract 1848: Let-7 brings new insights into chronic myeloid leukemia therapy.

Abstract Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells that is caused by the deregulated activity of the tyrosine kinase that is encoded by the chimeric bcr-abl oncogene. Given that CML cells are usually highly resistant to chemotherapy, novel and more efficacious strategies against this disease are urgently needed. MicroRNA (miRNA) is a set of newly discovered 20-22 nucleotides RNA molecules, and they broadly repress their cognate target genes at post-transcriptional/translational level. Up to date, there are approximately 2,000 human miRNA transcripts have been characterized, which are estimated to modulate more than 30% human genes and are widely involved in many essential biological processes including differentiation, proliferation, apoptosis, and also tumorigenesis. In this study, we studied let-7, a miRNA with tumor suppressive signature, for its therapeutic potential in CML. When treating CML K562 and KU812 cell by imatinib, let-7 can be significantly induced. Overexpression of let-7 can sensitize K562 and KU812 cells to imatinib, while knock-down of let-7 can increase the drug resistance of these cells. The mechanistic studies found that let-7 was able to promote apoptosis of CML cells, inhibit the tumor cell proliferation, and lead to a cell cycle arrest in the G0/G1 phase. These results demonstrate that let-7 is a potential target for treating CML by imatinib and/or other anti-cancer druges. The pro-apoptosis and anti-proliferation roles of let-7 in CML cells may provide novel insights into discovery and development novel drugs for conquering this malignant disease. Citation Format: Bin Yi, Xiaobo Li, Wenjun Du, Gary A. Piazza, Yaguang Xi. Let-7 brings new insights into chronic myeloid leukemia therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1848. doi:10.1158/1538-7445.AM2013-1848 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Effectiveness and safety of lenalidomide in myelofibrosis patients: a case series from the Spanish compassionate use program

Myelofibrosis (MF) is a chronic myeloproliferative neoplasm (CMPN) characterized by clonal proliferation of the pluripotent hematopoietic germinal cell, fibrosis, and bone marrow angiogenesis, and with extra medullary hematopoiesis. Lenalidomide is an immunomodulatory and antiangiogenic agent that has shown clinical benefit in MF patients in several phase II clinical trials. In this work, we present the results of the retrospective assessment of a series of 32 patients diagnosed with MF that received treatment with lenalidomide within the Spanish program of hospital compassionate use. Multicenter, retrospective study of a case series of MF patients receiving compassionate treatment with lenalidomide. We gathered information on 32 MF patients that were treated with lenalidomide at 17 Spanish hospitals. The mean age was 68 years (range, 50-83), 72% males. Twenty-six (81%) patients had intermediate-high risk according to Dupriez prognostic score. In 16 patients, the daily dose scheduled was 10 mg/day for 21 days in 28-day cycles; the remaining patients received lower doses or other regimes. In 29 out of 32 patients, response assessment was available. Two patients had complete clinical and hematologic response and 14 had partial response. The most common adverse events observed were neutropenia (56%), thrombocytopenia (50%), and anemia (40%). Continuous treatment with lenalidomide is active in MF patients pretreated heavily. Lenalidomide-induced myelosuppression and other associated toxicities are manageable with dose adjustments.

Inability of Human Induced Pluripotent Stem Cell-Hematopoietic Derivatives to Downregulate MicroRNAs In Vivo Reveals a Block in Xenograft Hematopoietic Regeneration

Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here we show that, unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources.

Sphincter abnormality in polytransfused patient due to paroxysmal nocturnal hemoglobinuria (PNH): initial manifestation of tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM)

Paroxysmal nocturnal hemoglobinuria (PNH) is a clinical entity resulting from specific change to pluripotent hematopoietic cells and subsequent clonal proliferation, with the capacity to affect the entire line1. The erythrocytic membrane has greater sensitivity to lithic action of the complement, for CD59 deficiency2. Allogenic bone marrow transplantation is the only support treatment available. According to the clinical manifestations of the disease, folic acid and iron, corticosteroids in low doses, androgens, immunosuppressants and blood component transfusion may be administered1 […] Sphincter [...]

ChemInform Abstract: Aldehyde Dehydrogenase-Mediated Cellular Relative Insensitivity to the Oxazaphosphorines

As judged by findings in preclinical models, determinants of cellular sensitivity to cyclophosphamide and other oxazaphosphorines include two cytosolic aldehyde dehydrogenases, viz., ALDH1A1 and ALDH3A1. Each catalyzes the detoxification of the oxazaphosphorines; thus, cellular sensitivity to these agents decreases as cellular levels of ALDH1A1 and/or ALDH3A1 increase. Of particular clinical relevance may be that stable sublines, relatively insensitive to the oxazaphosphorines due to elevated ALDH1A1 or ALDH3A1 levels, emerged when cultured human tumor cells were exposed only once to a high concentration of one of these agents for 30 to 60 minutes. Whether differences in cellular levels of either enzyme accounts for the clinically-encountered uneven therapeutic effectiveness of the oxazaphosphorines remains to be determined. However, it has already been established that measurable levels of these enzymes are found in some, but not all, tumor types, and that in those tumor types where measurable levels are present, e.g., infiltrating ductal carcinomas of the breast, they vary widely from patient to patient. Potentially useful clinical strategies that might be pursued if it turns out that ALDH1A1 and/or ALDH3A1 are, indeed, clinically operative determinants of cellular sensitivity to the oxazaphosphorines include 1) individualizing cancer chemotherapeutic regimens based, at least in part, on the levels of these enzymes in the malignancy of interest, and 2) sensitizing tumor cells that express relatively large amounts of ALDH1A1 and/or ALDH3A1 to the oxazaphosphorines by preventing the synthesis of these enzymes, e.g., with antisense RNA, or by introducing an agent that directly inhibits the catalytic action of the operative enzyme. Further, the fact that ALDH1A1 and ALDH3A1 are determinants of cellular sensitivity to the oxazaphosphorines provides the rationale for the investigation of two additional strategies with clinical potential, viz., decreasing the sensitivity of vulnerable and essential normal cells, e.g., pluripotent hematopoietic cells, to the oxazaphosphorines by selectively transferring into them the genetic information that encodes 1) ALDH1A1 or ALDH3A1, or 2) a signaling factor, the presence of which would directly or indirectly, stably upregulate the expression of these enzymes.

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

AbstractHematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150+ subset of the EPCR+CD48−CD45+ fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150− subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.

Transformation of E2A-Deficient Pluripotent Progenitors by BCR-ABL Generates Imatinib-Resistant Leukemic Stem Cells.

Chronic myeloid leukemia (CML) is effectively treated with imatinib (imatinib mesylate, IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors. However, it is still unclear whether IM-therapy is able to eradicate BCR-ABL-positive HSC and progenitors. By transforming murine E2A-deficient pluripotent hematopoietic cells (Ikawa, et al., Immunity 04) with p210BCR-ABL, we determined that as few as 50 granulocyte macrophage progenitors (GMP)-like cells were sufficient to induce a transplantable CML-like disease in congenic mice, and that the leukemogenic GMP displayed higher levels of β-catenin activity than either the non-transformed GMP or the transformed nonGMP (most of which were myeloid differentiated cells), both in culture and in transplanted mouse bone marrow (Proc Natl Acad Sci USA 08, in press). The initiation of transformation required BCR-ABL kinase activity; however, whereas transformed nonGMP were sensitive to IM-treatment, expansion and survival of the leukemogenic progenitors in ex vivo-culture or in mice were not completely inhibited by IM-treatment. The drug resistance did not correlate with higher levels of BCR-ABL, mutations at ABL-kinase domain, induction of quiescence, stromal support or drug efflux. Additionally, the differential responses between the transformed progenitors and differentiated cells to IM-treatment were not affected by the restoration of E2A-function. These results imply that leukemic progenitors possess innate resistance to IM and that eradication of these cells with other drugs is required to cure CML. We are also investigating BCR-ABL-positive residual disease in HSC and progenitors of chronic phase CML patients on IM-therapy.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1

Specific deletion of Notch1 and RBPjkappa in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

Substances from the medicinal mushroom Daedalea gibbosa inhibit kinase activity of native and T315I mutated Bcr-Abl

Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells characterized by a distinctive cytogenetic abnormality resulting in the creation of a p210 Bcr-Abl fusion protein with abnormal tyrosine kinase activity. Recently, a selective Abl kinase inhibitor, Imatinib mesylate, was introduced as a first line therapy for CML. Despite the initial response, CML patients develop a resistantance to Imatinib, which is mediated mainly by point mutations within the Abl protein. Herein, we describe the identification of mycelium organic extracts of Daedalea gibbosa with selective anti-proliferating and apoptosis-inducing activities against K562 cells and other laboratory model of CML. Using activity-guided purification, we isolated an active fraction, F6, which inhibits in vitro kinase activity of recombinant Abl. The active fraction significantly inhibits the autophosphorylation of native and mutated Bcr-Abl, which are resistant to Imatinib treatment including the T315I mutation. Using a colony-forming assay, we demon-strated that the active fraction is effective in inhibiting the colony formation of the Ba/F3 cell line harboring either native Bcr-Abl or its mutations, including the T315I mutation. Our data illustrated the potential of natural products in cancer therapeutics.

A NUP98-HOX Fusion Gene Containing the Homeodomain of HOXA10 Promotes Significant Expansion of Primitive Human Hematopoietic Cells in Extended Cultures.

Expanding human hematopoietic stem cells (HSCs) in vitro is a major goal in clinical hematology but remains a major challenge due to the potent differentiating activity of known cytokines. We recently demonstrated that a NUP98-HOX fusion gene containing only the homeodomain (hd) of HOXA10 (NUP98-HOXA10hd) is a powerful stimulator of murine HSC expansion in vitro - causing >1000-fold net HSC increases in 10 days (Sekulovic et al, ISEH 2005). To investigate the proliferative effect of NUP98-HOXA10hd on primitive human hematopoietic cells, highly enriched CD34+ cord blood cells were prestimulated overnight and exposed to self-inactivating MNDUSNUP98-HOXA10hd pgkGFP or control pgkGFP lentiviruses for 6h. The gene transfer efficiency into CD34+ cells determined 4 days after infection was 56 ± 5% for NUP98-HOXA10hd and 66 ± 5% for the GFP control. GFP+ cells were sorted on day 5 and then maintained for another 5 days in serum-free cultures containing Flt3-ligand, Steel factor, IL-3, IL-6 and G-CSF. An aliquot of each was then plated into “primary” colony-forming cell (CFC) assay cultures. No difference was detected in either the numbers or the types of colonies generated in these primary CFC assays of the 10-day cultured cells from the NUP98-HOXA10hd and control arms. However, when these primary CFC assays were replated into secondary CFC assays, the number of colonies obtained from the NUP98-HOXA10hd-transduced cells was 5-fold higher as compared to the GFP-control transduced cells and, upon replating into tertiary CFC assays, this difference increased to over a 100-fold. To determine the effect of NUP98-HOXA10hd on more primitive hematopoietic cells, 104 day-10 GFP+ cells were co-cultured on mouse fibroblast feeders engineered to produce human SF, IL-3 and G-SCF. At the end of 6 weeks, 13-fold more cells were recovered from the cultures initiated with NUP98-HOXA10hd-transduced cells than from the control cultures (474,000 ± 190,000 vs 37,000 ± 16,000, 3 experiments). CFC outputs were also greatly enhanced (21-fold more CFC than in the controls cultures, range=20–80, 3 experiments). Moreover, the proportion of progenitors in the assays of the cultures initiated with NUP98-HOXA10hd cells that were multi-lineage (CFU-GEMM) was >10-fold higher as compared to the CFCs obtained from the control cultures (8 ± 3% vs 0.7 ± 0.7%). When this experiment was repeated using limiting dilutions of initial day-10 cells, the frequency of NUP98-HOXA10hd-transduced cells able to generate CFCs another 6 weeks later was 10-fold higher as compared to the day-10 GFP control-transduced cells. These findings document an unprecedented potency of NUP98-HOXA10hd for stimulating the ex-vivo expansion of very primitive pluripotent human hematopoietic cells.

Unexpected and Sustained Symmetry in the Repopulation Programs Displayed by Clonally Amplified Hematopoietic Stem Cells.

Heterogeneity in progeny output by individual pluripotent hematopoietic cells is a well documented but poorly understood paradigm. Importantly, the extent to which this functional heterogeneity is pre-determined by intrinsic mechanisms that specify distinct programs, as opposed to conditions that result in a series of stochastic events, is still debated. The prospective isolation of phenotypically defined subpopulations with more restricted behaviors has lent recent support to the concept of predetermined hierarchies with preset, but alternative pathways of lineage restriction and differentiated cell output. Here we have used highly purified starting populations to compare the long-term cell output dynamics of individual multipotent repopulating cells in sublethally irradiated W41/W41mice transplanted with single Ly-5 congenic CD45midlin−Rho−SP adult mouse bone marrow cells (158 mice) or their clonal progeny generated after 4 days in vitro in 300 ng/ml SF, 20 ng/ml IL-11 and 1 ng/ml Flt3-L (194 mice). WBC samples collected 4, 8, 12, 16, and 24 weeks post-transplant were analyzed for donor contributions to the myeloid (GM) and lymphoid (B and T) lineages. In 49 of the 158 mice (31%) and 44 of the 194 mice (23%), the cells produced in vivo contributed ≥1% of all the WBCs present at ≥16 weeks. The overall and lineage-specific contributions to the WBCs in each recipient mouse varied widely both over time post-transplant and between mice. However, examination of the ratio of the donor contributions to the myeloid and lymphoid lineages (GM:B+T) in each mouse at 16 weeks post-transplant allowed 4 patterns to be readily identified: α and β with GM:B+T ratios of ≥2 and 0.25–2, respectively; γ, with a GM:B+T ratio of <0.25 including a ≥1% contribution to both lymphoid and myeloid lineages at 16 weeks; and δ, also with a GM:B+T ratio of <0.25, but with contribution only to the lymphoid lineages at this time. Secondary transplants performed after 24 weeks showed long-term repopulation (≥16 weeks) of most recipients of type α and β progeny (10/11 and 11/12, respectively) but none of the recipients of type γ and δ progeny were repopulated (0/6 and 0/17, respectively). Interestingly, the variation over time in both the overall and lineage-specific contributions was remarkably similar in pairs of secondary recipients injected with cells from the same primary donor. In addition, the lineage contribution ratios seen in the secondary recipients tended to recapitulate that of the primary donors (i.e., α or β), and these trends remained obvious when tertiary transplants were performed. Preservation of stem cell programming was also evident from sequential analyses of multiple mice injected with aliquots of the same clones generated in vitro after 10 days from single CD45midlin−Rho−SP cells. Very similar patterns of total and lineage-specific contributions were again observed amongst the different recipients of cells from the same clones. Collectively, these findings indicate that by early adult life hematopoietic stem cells have acquired intrinsically fixed patterns of lineage specification that can be stably transmitted through many self-renewal generations.