Dysregulated Expression of miRNAs in Polycythemia Vera Erythroid Progenitors.

Abstract Polycythemia vera (PV), the most common myeloproliferative disorder, arises due to somatic mutation(s) of a single hematopoietic stem cell leading to clonal hematopoiesis. A somatic JAK2 V617F point mutation is found in over 80% of PV patients; however, it is not clear if the JAK2 V617F is the disease initiating mutation, sincethere are PV JAK2 V617F negative patients who have monoclonal hematopoiesis and erythropoietin independent erythropoiesis;in individual PV families, there are PV subjects with and without the JAK2 V617F mutation; andanalysis of clonal PV populations reveals the presence of <50 and >50% mutated JAK2 cells (Nussenzweig’ abstract this mtg), suggesting a mixed population of cells with regard to JAK2 status.In order to search for possible PV contributing molecular defect(s), we studied microRNAs (miRNAs) in a homogeneous population of in vitro expanded erythroid progenitors. MiRNAs are non-coding, small RNAs that regulate gene expression at the posttranscriptional level by direct mRNA cleavage, by translational repression, or by mRNA decay mediated by deadenylation. MiRNAs play an important regulatory role in various biological processes including human hematopoiesis. In vitro expanded erythroid progenitors were obtained from peripheral blood mononuclear cells of 5 PV patients (JAK2 V617F heterozygotes) and from 2 healthy donor controls. The cells were cultured in an erythroid-expansion medium for 21 days resulting in 70–80% homogenous erythroid cell population of identical differentiation stage. Gene expression profiling of miRNAs (Thomson, Nature Methods, 1:1, 2004) was performed using a custom microarray (Combimatrix) with 326 miRNA probes. Data were normalized by the global median method. The miRNAs with expression ratios greater than 1.5 or less than 0.5 were considered to be abnormal. Comparative analyses of controls versus PV samples revealed up-regulated expression of miR-let7c/f, miR-16, miR-451, miR-21, miR-27a, miR-26b and miR-320 and down-regulation of miR-150, miR-339 and miR-346 in PV. In addition, miR-27a, miR-26b and miR-320 were expressed only in PV. The putative targets of these miRNAs were predicted by TargetScan prediction algorithm. Up-regulated miR-let-7, miR-16 and miR-26b may modulate cyclin D2, which has an important role in G1/S transition and can be a target in the JAK2/STAT5 pathway (Walz, JBC, 281:18177, 2006). One of the putative targets of up-regulated miR-27a is EDRF1 (erythroid terminal differentiation related factor1), a positive regulator of erythroid differentiation. The BCL-6 gene is predicted to be the target of miR-339 and miR-346, and its activation blocks cellular differentiation. MiR-16 is known to be down-regulated in CLL, where it targets anti-apoptotic BCL-2; in contrast, we show that miR-16 is up-regulated in PV erythroid cells. We identified differentially expressed miRNAs in PV which target genes involved in the JAK/STAT pathway or genes that are modulated by JAK2 downstream molecules. This study indicates that miRNA dysregulation may play an important role in erythropoietic differentiation and proliferation in PV. Expression analyses of these miRNAs in a larger set of PV samples, using quantitative Real-Time-PCR, are in progress. Further, earlier erythroid and pluripotent hematopoietic progenitors are also being analyzed.

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In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽

10.1182/blood.v106.11.3506.3506 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3506-3506 ◽

Cited By ~ 1

Author(s):

Josef T. Prchal ◽

Ko-Tung Chang ◽

Jaroslav Jelinek ◽

Yongli Guan ◽

Amos Gaikwad ◽

...

Keyword(s):

Tyrosine Kinase ◽

Polycythemia Vera ◽

Peripheral Blood ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Erythroid Progenitors ◽

Erythroid Precursors ◽

In Vitro Expansion ◽

V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G>T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G>T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G>T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G>T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

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Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

PLoS ONE ◽

10.1371/journal.pone.0247858 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0247858

Author(s):

Ron Baik ◽

Stacia K. Wyman ◽

Shaheen Kabir ◽

Jacob E. Corn

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Cytokine Signaling ◽

Competitive Growth ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Myeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop “scarless” Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

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Pure Heterozygous and Mixed JAK2 V617F Genotypes Result in Distinct Mechanisms of Erythrocytosis in Polycythemia Vera.

Blood ◽

10.1182/blood.v108.11.662.662 ◽

2006 ◽

Vol 108 (11) ◽

pp. 662-662

Author(s):

Sabrina Dupont ◽

Aline Masse ◽

Chloe James ◽

Nicole Casadevall ◽

William Vainchenker ◽

...

Keyword(s):

Polycythemia Vera ◽

Progenitor Cells ◽

Mononuclear Cells ◽

Fold Increase ◽

Jak2 V617f ◽

Molecular Event ◽

Erythroid Progenitors ◽

Low Frequencies

Abstract The JAK2 V617F mutation is present in most patients with polycythemia vera (PV) and half with essential thrombocythemia (ET). Using real-time quantitative PCR, we analyzed the levels of JAK2 V617F in granulocytes and/or bone marrow mononuclear cells from 159 PV and 149 ET patients. High JAK2 V617F levels were correlated with higher leukocyte, granulocyte, hemoglobin values and higher endogenous erythroid colony formation. Thus, the phenotype of PV and ET may be closely linked to the JAK2 V617F level, which may reflect the clonal genotypic pattern of hematopoietic progenitor cells. It is thought that the occurrence of the mitotic recombination, which generates homozygous JAK2 V671F clones, is a key molecular event for the onset of PV. In this work, we aimed to study the consequences of the clonal JAK2 V617F genotype on the amplification properties and erythropoietin (EPO) hypersensitivity of PV (n=14) and ET (n=6) progenitors. Analysis of clonal genotypic patterns shows that ET patients harbor a mix of heterozygous and normal progenitors. Interestingly, we distinguish pure heterozygous PV profiles (3/14 patients) with no homozygous progenitors from homozygous PV profiles (11/14 patients) with normal, heterozygous and homozygous progenitors. Similar low frequencies of mutated immature progenitors, comprising long-term culture-initiating cells and lympho-myeloid progenitors, are found in ET and PV. In contrast, PV patients with pure heterozygous PV profiles have striking higher proportions (>90%) of mutated committed progenitors than other PV and ET patients. This result suggests a selective amplification of heterozygous cells in the early phases of hematopoiesis. Furthermore, by using increasing concentrations of EPO, homozygous mutated erythroid progenitors are demonstrated to be more sensitive to EPO than heterozygous ones, a majority of the former (69,5%) being EPO independent. Moreover, we demonstrate a two to three fold increase in in vitro amplification of ET and PV progenitor cells when compared to normal ones in serum free liquid culture containing IL3, Stem Cell Factor, Dexamethasone and 1 IU/mL EPO. In addition, the quantification of the mutated allele in immature CD34+CD38- cells, in CD34+CD38+ committed progenitor cells, in mature erythroblasts (GPA+) and in granulocytes shows a marked in vivo selective advantage of mutated cells in late stages of hematopoiesis. These results suggest that in PV, erythrocytosis results from two distinct mechanisms: a terminal erythroid amplification advantage triggered by homozygosity or a two-step process including the upstream amplification of heterozygous cells that may involve additional molecular event(s).

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FOG1 requires NuRD to promote hematopoiesis and maintain lineage fidelity within the megakaryocytic-erythroid compartment

Blood ◽

10.1182/blood-2009-10-251280 ◽

2010 ◽

Vol 115 (11) ◽

pp. 2156-2166 ◽

Cited By ~ 30

Author(s):

Gregory D. Gregory ◽

Annarita Miccio ◽

Alexey Bersenev ◽

Yuhuan Wang ◽

Wei Hong ◽

...

Keyword(s):

Gene Expression ◽

Cell Lineage ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Gata Factors ◽

Chromatin Remodeling Complex ◽

Nuclear Factors ◽

Continuous Presence ◽

Inappropriate Expression

Abstract Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. The cofactor FOG1 interacts with transcription factors GATA1 and GATA2 to control erythroid and megakaryocyte (MK) differentiation. In contrast, FOG1 antagonizes the ability of GATA factors to promote mast cell (MC) development. Normal FOG1 function in late-stage erythroid cells and MK requires interaction with the chromatin remodeling complex NuRD. Here, we report that mice in which the FOG1/NuRD interaction is disrupted (Fogki/ki) produce MK-erythroid progenitors that give rise to significantly fewer and less mature MK and erythroid colonies in vitro while retaining multilineage capacity, capable of generating MCs and other myeloid lineage cells. Gene expression profiling of Fogki/ki MK-erythroid progenitors revealed inappropriate expression of several MC-specific genes. Strikingly, aberrant MC gene expression persisted in mature Fogki/ki MK and erythroid progeny. Using a GATA1-dependent committed erythroid cell line, select MC genes were found to be occupied by NuRD, suggesting a direct mechanism of repression. Together, these observations suggest that a simple heritable silencing mechanism is insufficient to permanently repress MC genes. Instead, the continuous presence of GATA1, FOG1, and NuRD is required to maintain lineage fidelity throughout MK-erythroid ontogeny.

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Differential Expression of CD49f Discriminates the Independently Emerged Hematopoietic Stem Cells and Erythroid-Biased Progenitors

Blood ◽

10.1182/blood-2019-130429 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3700-3700

Author(s):

Ai-Ning Xu ◽

Dan Liu ◽

Yu-Ting Dai ◽

Fan Zhang ◽

Jian Shen ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Endothelial Cells ◽

Differential Expression ◽

Regulatory Mechanism ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

In Vitro System

While hematopoietic stem cells (HSCs) can sustain the production of all types of mature blood cells throughout the life, there also exists HSC-independent hematopoiesis, which partially supports embryonic hematopoiesis and generation of specific types of adult hematopoietic cells (e.g., macrophages). Examples of the HSC-independent hematopoiesis include (i) the primitive wave of hematopoiesis that produces unipotent progenitors for erythrocytes, megakaryocytes or macrophages, and (ii) the "pro-definitive" hematopoiesis that produces multipotent erythro-myeloid progenitors (EMPs). Given that HSCs and HSC-independent progenitors are both derived from endothelial cells in distinct or overlapping hematopoietic sites, tracing their developmental origins and clarifying the regulatory mechanism will enhance our understanding of the profound difference between them and may improve in vitro generation of HSCs. Human HSCs have been refined based on the expression of CD49f (ITGA6). In combination with other HSC markers (CD34+CD38-CD45RA-CD43+CD90+), high expression of CD49f identifies long-term multilineage engrafting HSCs, whereas the cells with low CD49f represent a subtype of hematopoietic progenitor cells (HPCs) that possess transient engrafting activity. Meanwhile, CD49f has also been shown to be heterogeneously expressed in hemogenic endothelial cells (HECs), which give rise to both HSCs and EMPs via endothelial-to-hematopoietic transition (EHT). Thus, determining the changes (i.e., persistence, gain or loss) of CD49f expression during EHT is a key step in tracing the origins of HSCs and HSC-independent HPCs. In this study, using an in vitro system of HSC differentiation from human embryonic stem cells (hESCs), we observed that, while CD49f is highly expressed in all hESCs, only a portion of HECs express CD49f. Importantly, live cell imaging analysis revealed that CD49f expression persists during EHT, which is accompanied by initiating CD43 expression. To test whether the differential CD49f expression is associated with HSC versus HPC functions, we sorted the CD49fhigh and CD49flow cells and performed colony forming assay and gene expression profiling. The results showed that the CD49fhigh cells have multilineage potential, whereas the CD49flow cells lack lymphoid potential but show a strong erythroid preference. Gene expression analysis confirmed that the CD49fhigh and CD49flow cells represent HSCs and erythroid-biased HPCs, respectively, and that the Wnt and Notch signaling pathways may play a role in their functions. Collectively, these observations suggest that the CD49fhigh and the CD49flow cells are concurrently derived from the CD49fhigh and CD49flow HECs, thus modeling the in vivo generation of HSCs and HSC-independent HPCs. Based on the in vitro observations, we proposed that CD49f in vivo may also specify the distinct HSPCs emerged at different developmental stages/sites. To test this hypothesis, we isolated mouse primitive HPCs, EMPs and definitive HSCs, as well as their parental HECs, from yolk sac, embryo, and aorta-gonad-mesonephros (AGM) of different embryonic stages and determined their CD49f expression. The results showed that the primitive erythroid progenitors have lowest, whereas the definitive AGM HSCs have highest, CD49f levels; this trend was also observed in the related HECs isolated from various stages/sites. Thus, it is likely that the embryonic hematopoiesis is recapitulated, at least partially, by the in vitro system in terms of the sequential emergence of HSPCs ranging from unipotent erythroid progenitors to multipotent definitive HSCs, and this may also underlie the situation that EMPs and HSCs can be produced at the same stage/site but independently from different HECs. In summary, using the in vitro HSC differentiation system, we found that the differential expression of CD49f discriminates HSCs and HSC-independent progenitors, which are concurrently emerged from HECs. The persistent CD49f expression during EHT suggests that the fates of HSCs and HSC-independent HPCs are pre-defined in their parental HECs. Combining our in vivo data, the differential expression of CD49f also provide a possible regulatory mechanism for the multi-wave hematopoiesis. Further exploring the function and mechanism of CD49f in these regulations should be important for fully understanding the precisely regulated HSC generation and activities. Disclosures No relevant conflicts of interest to declare.

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Seven Cases of JAK2 Mutation Negative Chronic Polycythemia Patients with Elevated IGF-1R

Blood ◽

10.1182/blood-2019-123006 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5389-5389

Author(s):

Guanfang Shi ◽

Rewais Morcus ◽

Maksim Liaukovich ◽

Ching Wong ◽

Vladimir Gotlieb ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Polycythemia Vera ◽

Mononuclear Cells ◽

Jak2 V617f ◽

Driver Mutations ◽

Erythroid Progenitors ◽

Jak2 Mutation ◽

Minor Criteria

We previously reported that assaying blood MNC (Mononuclear cells) IGF-1 levels by Flow cytometry, will be helpful in differentiating Polycythemia vera (PV) from secondarypolycythemia (1). Patients with chronic polycythemia who are negative for JAK2 V617F or exon 12 mutation and who lack the typical bone marrow findings of polycythemia vera will remain a diagnostic enigma. We collected 7 cases of patients who had persistent chronic erythrocytosis ranging from 1-15 years with negative driver mutations, lacking the typical PV bone marrow findings and absence of secondary causes such as smoking or malignancies. Blood mononuclear cells (MNC ) were collected as well as blood DNA extracted for 237 genes including EPO-R, PHD2, VHL or HIF-1-alpha (Familial erythrocytosis genes)with Next generation sequence, performed by Genoptyx lab (Carisbad, CA) and assayed for IGF-1R by flow cytometry as described previously (1). The results are shown in Table 1. Conclusion. All these 7 patients with elevated IGF-1R who had no evidence of familial erythrocytosis gene mutation nor had any secondary cause for erythrocytosis, likely carried the diagnosis of PV. It was shown that EEC formation in PV is due to IGF-1 hypersensitivity (2), andsecondary polycythemia do not show significantly elevated IGF -1R (1). Hence the elevated IGF-1R in these 7 patients strongly suggests the diagnosis of PV, re-affirming our proposal that simple procedures to assay blood MNC cells for IGF-1R by flow cytometry will be helpful in the diagnosis of PV and to be added as one of the minor criteria in the diagnosis of PV. References 1. Wang JC, et al . Quantification of IGF-1 Receptor May Be Useful in Diagnosing Polycythemia Vera-Suggestion to Be Added to Be One of the Minor Criterion.PLoS One. 2016 Nov 3;11(11):e0165299. doi: 10.1371/journal.pone.0165299. 2. Correa PN, Eskinazi, D and Axelrad AA .Circulating Erythroid Progenitors in Polycythemia Vera Are Hypersensitive to Insulin-like Growth Factor-l In Vitro: Studies in an Improved Serum-Free Medium Blood, Vol83, No 1 (January l), 1994: pp 99-1 Disclosures Wang: Incyt: Research Funding.

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Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

10.1101/855452 ◽

2019 ◽

Author(s):

Ron Baik ◽

Stacia K. Wyman ◽

Shaheen Kabir ◽

Jacob E. Corn

Keyword(s):

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Cytokine Signaling ◽

Competitive Growth ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

AbstractMyeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop “scarless” Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

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Will Imitanib Be Useful for Patients with Polycythemia Vera?.

Blood ◽

10.1182/blood.v106.11.2601.2601 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2601-2601 ◽

Cited By ~ 3

Author(s):

Amos S. Gaikwad ◽

Srdan Verstovsek ◽

Ko-Tung Chang ◽

Donghoon Yoon ◽

William Vainchenker ◽

...

Keyword(s):

Tyrosine Kinase ◽

Glucose Uptake ◽

Polycythemia Vera ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Jak2 V617f ◽

Erythroid Progenitors ◽

Jak2 Mutation

Abstract Polycythemia vera (PV) is an acquired myeloproliferative, clonal stem cell disorder characterized by cytokine hypersensitivity. Several groups reported a clonal mutation in the pseudo kinase domain of the Janus kinase 2 (Jak2) protein, substituting phenylalanine at position 617 with valine (V617F) that causes constitutive activation of the JAK/STAT pathway in PV patients. Chronic myelogenous leukemia is another myeloproliferative disorder associated with aberrant tyrosine kinase (bcr-abl) that arises from t(9:22) translocation. Constitutive bcr-abl tyrosine kinase activity suppresses apoptosis leading to survival and proliferation of cells at low cytokine concentrations. Imitanib has been developed to inhibit the bcr-abl kinase activity and has had impressive therapeutic efficacy. However, imitanib also inhibits other tyrosine kinases. Clinical benefits of imitanib in PV have been reported. Mouse FDCP cells over-expressing either the wild-type Jak2 (JAK/W) or two cell lines with the V617F mutation (V617F), one with cytokine hypersensitivity and the other cytokine independent, were created. We examined the effect of imitanib by MTT proliferation assay and propidium iodide staining analysis. No appreciable changes in the proliferation and DNA content were observed in all three cell-lines after imitanib treatment at ~1μM (the concentration effective for the bcr-abl expressing cells and achievable in vivo in imitanib treated patients). However, after 72h of exposure, the cells expressing JAK2 V617F mutant showed 50% inhibition of growth at ~6μM imitanib with no significant effect on the growth of cells expressing JAK/W. To further understand the mechanism of growth inhibition of V617F cells by imitanib, we examined the metabolism of these cells since the constitutively active tyrosine kinases has been demonstrated to change the glucose metabolism. Imitanib treatment (5μM) for 72h caused about 30% decrease in the glucose uptake in V617F cells with only a marginal (~5%) decrease in glucose uptake was observed in the JAK/W cells. We then examined the effect of imitanib on in vitro expanded native human erythroid progenitors (CD71 and CD235a positive cells) from three normal and four PV individuals who expressed variable proportion of mutant JAK2 V617F alleles. In this small study, between 18–30% decrease in the proliferation of the cells from PV patients was seen with 1–2μM of imitanib compared to 8% seen with normal erythroid progenitors; however, the in vitro expansion was accompanied by a decrease of proportion of cells with JAK2 mutation (see abstract Prchal et. Al. at this meeting). Further, in ongoing clinical studies, one of 5 tested PV patients who showed an excellent clinical response to imitanib therapy had decrease of V617F mutation from 58 to 19% in circulating granulocytes. We conclude that high concentration of imitanib is required to achieve the cytotoxic effects in reporter cells transfected with JAK2 mutation that are not readily achievable in vivo; however, lower doses (but still significantly higher than those needed for bcr-abl expressing cells) are effective in native PV progenitors. These data do not exclude possible useful therapeutic effect of imitanib in PV either alone or in combination with other drugs. Amos Gaikwad, Ph.D. and Srdan Verstovsek, M.D. contributed equally to this study.

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In Vitro Erythroid Cell Expansion Analysis in Polycythemia Vera.

Blood ◽

10.1182/blood.v108.11.4916.4916 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4916-4916 ◽

Cited By ~ 1

Author(s):

Donghoon Yoon ◽

Hana Bruchova ◽

Archana M. Agarwal ◽

Josef T. Prchal ◽

Jaroslav F. Prchal

Keyword(s):

Polycythemia Vera ◽

Molecular Mechanism ◽

Mononuclear Cells ◽

Biological Behavior ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Peripheral Blood Mononuclear ◽

Expansion Protocol ◽

Differentiation And Proliferation

Abstract Polycythemia vera (PV) is an acquired myeloproliferative clonal stem cell disorder characterized by cytokine hypersensitivity. The erythroid colony forming assay has been useful in PV diagnosis. However, studies aiming to elucidate molecular mechanism in PV pathogenesis often require large numbers of cells from a specific erythroid stage. For this purpose, we had adapted the mouse expansion assay described by Karur et al (Blood, 2006 in E-Pub) and differential display method described by Zhang et al (Blood, 2003, 102; 3938) to develop a human erythroid expansion protocol. In this study, we used peripheral blood mononuclear cells (PB-MNCs) from PV patients and healthy donors, and expanded them along erythroid lineage in 21 day culture. Through the culture, we took samples at 8 timepoints (days 1, 7, 9, 11, 14, 16, 19, 21) to evaluate differentiation patterns. We had generated 5 different regions using CD71 (transferrin receptor) and CD235a (glycophorin A) and characterized these regions by standard morphology analysis. This protocol allowed differentiation of PB-MNCs to all erythroid stages ending with late normoblast, reticulocyte, and mature erythrocytes (which do not survive well in this culture environment). The erythroid differentiation and proliferation patterns were different between PV and normal. In the first week of culture, there was no significant proliferation difference observed between PV and normal. In contrast, the differentiation progressed more rapidly in PV, likely reflecting the differentiation of Epo independent PV population (since the first week culture contains no Epo). In the second week of culture (Epo present), there was a markedly increased expansion of PV erythroid cells. However, the differentiation pattern of PV resembled that of normal. In conclusion, we demonstrated that our in vitro expansion method allows expansion of PB-MNCs along erythroid lineage with 60~80% stage homogeneity as to the stage of differentiation in both PV and normal. The differences of differentiation and proliferation pattern between PV and normal reflected the expected biological behavior of erythroid progenitors. Thus, the expansion protocol is useful to study molecular mechanism of PV pathogenesis, which requires large number of erythroid progenitors of various stages.

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Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro

Blood ◽

10.1182/blood.v92.10.3658 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3658-3668 ◽

Cited By ~ 128

Author(s):

Birgit Panzenböck ◽

Petr Bartunek ◽

Markus Y. Mapara ◽

Martin Zenke

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Stem Cell Factor ◽

Large Cell ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Peripheral Blood Stem Cells ◽

Erythroid Progenitor ◽

Cell Numbers

Abstract Stem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62dok that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.

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