Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model

Abstract

Activating mutations affecting the JAK-STAT signal transduction is the genetic driver of myeloproliferative neoplasms (MPNs) which comprise polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. The JAK2p.V617F mutation can produce both erythrocytosis in PV and thrombocytosis in ET, while JAK2 exon 12 mutations cause only erythrocytosis. We hypothesized that these two mutations activated different intracellular signals. In this study, the induced pluripotent stem cells (iPSCs) were used to model JAK2-mutated MPNs. Normal iPSCs underwent lentiviral transduction to overexpress JAK2p.V617F or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs were differentiated into erythroid cells. Compared with JAK2V617F-iPSCs, JAK2exon12-iPSCs yielded more total CD71⁺GlycophorinA⁺ erythroid cells, displayed more mature morphology and expressed more adult hemoglobin after doxycycline induction. Capillary Western immunoassay revealed significantly higher phospho-STAT1 but lower phospho-STAT3 and lower Phospho-AKT in JAK2exon12-iPSCs compared with those of JAK2V617F-iPSCs in response to erythropoietin. Furthermore, interferon alpha and arsenic trioxide were tested on these modified iPSCs to explore their potentials for MPN therapy. Both agents preferentially inhibited proliferation and promoted apoptosis of the iPSCs expressing mutant JAK2 compared with those without doxycycline induction. In conclusion, the modified iPSC model can be used to investigate the mechanisms and search for new therapy of MPNs.

Figure 1

Verification of JAK2 gene mutations and expression in the modified induced pluripotent stem cells (iPSCs). (A) Conventional polymerase chain reaction (PCR) using transgene-specific primers showed exogenous JAK2 genes in the two modified iPSC lines. The normal iPSC line was used as a negative control. The full gel is presented in the Supplementary Fig. S1A. (B) DNA sequencing confirmed the point mutation p.V617F in exon 14 and the p.N542_E543del in exon 12 of JAK2 gene in the respective iPSC lines. (C) Exogenous JAK2 gene expression levels in iPSCs after transfection comparing normal, JAK2V617F and JAK2 exon 12 mutation with and without doxycycline (DOX) induction for 24 h and analyzed by real-time quantitative RT-PCR. Data are presented as means ± standard deviations (SD) from three independent experiments. The asterisks (*) and (**) denoted p < 0.05 and p < 0.01.

Figure 2

Characteristics of modified induced pluripotent stem cells (iPSCs). (A) Karyotyping of the genetically modified iPSCs: JAK2V617F-iPSCs and JAK2N542_E543del-iPSCs (JAK2exon12-iPSCs). (B) The expression of NANOG, OCT4, SOX2, KLF4 and MYC of JAK2V617F-iPSCs and JAK2exon12-iPSCs using reverse transcriptase polymerase chain reaction (RT-PCR). The full gels are presented in the Supplementary Fig. S1B. (C) Immunofluorescence of differentiated cells from JAK2V617F-iPSCs and JAK2exon12-iPSCs. Embryoid bodies were transferred onto 0.1% gelatin coverslips and cultured for 14 days for differentiation. Cells were stained with antibodies specific to ectoderm (red and green), mesoderm (green), endoderm (green) layers and DAPI (blue) for nuclei (×400 magnification). The images were captured by Axio Observer fluorescence microscopy.

Figure 3

Erythroid cell differentiation from modified induced pluripotent stem cells (iPSCs) via ES-Sacs. (A) ES-derived sacs containing hematopoietic progenitor cells were generated from modified iPSCs on day 14 at ×100 and ×400 magnifications. (B) The percentages of CD34⁺ cells derived from JAK2V617F-iPSCs and JAK2exon12-iPSCs were similar with or without doxycycline. (C) Erythroid cells in a culture plate and red blood cell pellets after centrifugation. (D) Expression mRNA levels of exogenous JAK2 in hematopoietic progenitor cells determined by real-time quantitative RT-PCR. (E) Expression mRNA levels of exogenous JAK2 in erythroid cells. (F) The capillary Western immunoassay showed total JAK2 proteins from JAK2V617F-iPSCs and JAK2exon12-iPSCs at the induced pluripotent stem cell, hematopoietic progenitor cell (HPC) and erythroid cell (RBC) stages in the conditions with vs. without doxycycline. Each band was electrophoresed in a separate capillary tube. The full blot is presented in the Supplementary Fig. S2A.

Figure 4

Characteristics of modified induced pluripotent stem cells (iPSC)-derived erythroid cells. (A) Flow cytometry of erythroid-specific surface molecules on iPSC-derived erythroid cells without vs. with doxycycline. Cells were stained with PE-conjugated anti-human CD71 and FITC-conjugated anti-human Glycophorin A (GPA) antibodies. (B) Total CD71⁺GPA⁺ erythroid cell numbers from erythroid differentiation via ES-Sacs with vs. without doxycycline. (C) Five stages of erythroid series were classified by Wright-Giemsa stain consisting of proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts and erythrocytes. The images were captured by Leica DM 1000 microscopy using LAS49 software and the scale bars represented 10 µm for all panels. (D) The percentage of erythroid cell differentiation stages. (E) Chromatograms from the ion exchange high performance liquid chromatography (HPLC) Bio-Rad Variant II showed mainly embryonic hemoglobin in modified iPSC-derived erythroid cells with and without doxycycline. (F) The relative expression of beta-similar globin genes which were epsilon, gamma and beta in iPSC-derived erythroid cells comparing overexpression of JAK2V617F vs. JAK2 exon 12 mutants and analyzed by real-time quantitative RT-PCR. Data were presented as mean ± SD from three independent experiments. The asterisks (**) denoted p < 0.01.

Figure 5

Signal transduction and drug treatment of modified induced pluripotent stem cells (iPSCs). (A) The capillary Western immunoassay of phosphorylated and total signaling proteins which were JAK2, STAT1, STAT3, STAT5, ERK1/2 and AKT in the absent and presence of doxycycline in JAK2V617F-iPSCs and JAK2exon12-iPSCs. Each band was electrophoresed in a separate capillary tube. The full blot is presented in the Supplementary Fig. S2B. (B) The relative changes of phosphorylated signaling proteins after doxycycline induction in JAK2V617F-iPSCs and JAK2exon12-iPSCs compared with those without doxycycline. The levels of phosphoproteins were corrected by the amounts of respective total proteins. (C) Erythroid cell numbers after incubations without (untreated) vs. with arsenic trioxide, interferon alpha and the combination of both drugs. (D) The relative increases in numbers of apoptotic cells in modified iPSCs in the presence of arsenic trioxide, interferon alpha and the combination of both drugs. Data were presented as mean ± SD from three independent experiments. The asterisks (*), (**) and (***) denoted p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 6

Schematic diagrams of in vitro differentiation protocols for erythrocytes production via ES-Sacs formation. VEGF Vascular endothelium growth factor, TPO thrombopoietin, SCF stem cell factor, EPO erythropoietin, HPCs hematopoietic progenitor cells.