Contributions
Abstract: PB2265
Type: Publication Only
Background
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an clonal myeloproliferation, bone marrow fibrosis and extramedular hematopoiesis. In PMF, driver somatic mutations occur in JAK2, MPL or CALR genes. Mutations in epigenetic regulators as TET2 and ASXL1 that could lead to loss-of-function were frequently identified. In this context, induced pluripotent stem cells (iPSC) could be used to study clonal heterogeneity and to recapitulate in vitro some hematological features of PMF. Tet2 deletion in mouse embryonic fibroblast was shown to reduce cellular reprograming efficiency. Therefore, we asked if it would be possible to obtain iPSC from a TET2 mutated PMF patient.
Aims
The main goal of this work was to assess the impact of somatic mutations in CALR and TET2 in both cellular reprogramming and hematopoietic differentiation using iPSC.
Methods
This work was approved by INCA Ethics committee and patients signed informed consent. We used next generation sequencing to screen a cohort of Brazilian PMF patients for myeloid somatic mutations. One of the patients granulocytes (P1) were shown by Sanger sequencing to harbor CALRins5 and TET2G898X mutations. CD34+ primary cells were isolated from P1 or a healthy donor control and erythroblasts were differentiated in vitro. We generated iPS cells from P1 erytroblasts (P1-iPS) or from the control erytroblasts (C1-iPS) using the Sendai virus system. The pluripotency was confirmed in iPSC colonies by the expression of embryonic stem cells markers (Oct-3/4, Sox-2, SSEA-4, and TRA-1-81). The capacity to form germ layers was evaluated by embryonic body formation and layer specific markers detection by immunohystochemistry. Hematopoietic differentiation was performed on feeder-free culture supplemented with cytokines and CD43+CD34+ progenitors sorted at day 10-14. Myeloid Colony-Forming Units (CFU) were quantified in methylcellulose assays, under microscopic evaluation on day 12. CFU-Megakaryocytes were scored in plasma clots assays, after 9 days of culture and labeling with anti-CD41a antibody and alkaline phosphatase staining. Granulocytes were differentiated from CD34+CD43+ progenitors in liquid culture during 20 days.
Results
After cellular reprogramming, 32 P1-iPSC clones were obtained, 20 CALRins5 /TET2wt and 12 CALRins5/TET2 G898X homozygous. The different genotypes observed for iPSC reflect the clonal diversity present in the PMF primary sample. We confirmed that TET2 WT as well as TET2 mutated iPSCs displayed pluripotency markers and were able to generate all three germ layers in EB assays, indicating that the G898X mutation did not impair cellular reprogramming. We next sought to study the role of these mutations in the hematopoietic differentiation. We observed that all type of myeloid colonies were generated in methylcellulose assays for P1-iPSC and C1-iPSC. Our preliminary results show that an increased number of granulocyte colonies derived from CD34+CD43+ progenitors of P1-TET2 mutated iPSC, when compared both with iPSC P1-TET2 WT and C1-iPSC. Using plasma clot assay, we observed higher numbers of large CFU-MK colonies derived from P1-iPSC versus C1-iPSC.
Conclusion
Our results suggest that the TET2G898X did not impair cellular reprogramming, since iPSC harboring this mutation displayed all the features of bona fide iPS and that mutations in CALR and TET2 have an impact on hematopoietic differentiation of iPSC.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Epigenetic, Mutation, Myelofibrosis
Abstract: PB2265
Type: Publication Only
Background
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an clonal myeloproliferation, bone marrow fibrosis and extramedular hematopoiesis. In PMF, driver somatic mutations occur in JAK2, MPL or CALR genes. Mutations in epigenetic regulators as TET2 and ASXL1 that could lead to loss-of-function were frequently identified. In this context, induced pluripotent stem cells (iPSC) could be used to study clonal heterogeneity and to recapitulate in vitro some hematological features of PMF. Tet2 deletion in mouse embryonic fibroblast was shown to reduce cellular reprograming efficiency. Therefore, we asked if it would be possible to obtain iPSC from a TET2 mutated PMF patient.
Aims
The main goal of this work was to assess the impact of somatic mutations in CALR and TET2 in both cellular reprogramming and hematopoietic differentiation using iPSC.
Methods
This work was approved by INCA Ethics committee and patients signed informed consent. We used next generation sequencing to screen a cohort of Brazilian PMF patients for myeloid somatic mutations. One of the patients granulocytes (P1) were shown by Sanger sequencing to harbor CALRins5 and TET2G898X mutations. CD34+ primary cells were isolated from P1 or a healthy donor control and erythroblasts were differentiated in vitro. We generated iPS cells from P1 erytroblasts (P1-iPS) or from the control erytroblasts (C1-iPS) using the Sendai virus system. The pluripotency was confirmed in iPSC colonies by the expression of embryonic stem cells markers (Oct-3/4, Sox-2, SSEA-4, and TRA-1-81). The capacity to form germ layers was evaluated by embryonic body formation and layer specific markers detection by immunohystochemistry. Hematopoietic differentiation was performed on feeder-free culture supplemented with cytokines and CD43+CD34+ progenitors sorted at day 10-14. Myeloid Colony-Forming Units (CFU) were quantified in methylcellulose assays, under microscopic evaluation on day 12. CFU-Megakaryocytes were scored in plasma clots assays, after 9 days of culture and labeling with anti-CD41a antibody and alkaline phosphatase staining. Granulocytes were differentiated from CD34+CD43+ progenitors in liquid culture during 20 days.
Results
After cellular reprogramming, 32 P1-iPSC clones were obtained, 20 CALRins5 /TET2wt and 12 CALRins5/TET2 G898X homozygous. The different genotypes observed for iPSC reflect the clonal diversity present in the PMF primary sample. We confirmed that TET2 WT as well as TET2 mutated iPSCs displayed pluripotency markers and were able to generate all three germ layers in EB assays, indicating that the G898X mutation did not impair cellular reprogramming. We next sought to study the role of these mutations in the hematopoietic differentiation. We observed that all type of myeloid colonies were generated in methylcellulose assays for P1-iPSC and C1-iPSC. Our preliminary results show that an increased number of granulocyte colonies derived from CD34+CD43+ progenitors of P1-TET2 mutated iPSC, when compared both with iPSC P1-TET2 WT and C1-iPSC. Using plasma clot assay, we observed higher numbers of large CFU-MK colonies derived from P1-iPSC versus C1-iPSC.
Conclusion
Our results suggest that the TET2G898X did not impair cellular reprogramming, since iPSC harboring this mutation displayed all the features of bona fide iPS and that mutations in CALR and TET2 have an impact on hematopoietic differentiation of iPSC.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Epigenetic, Mutation, Myelofibrosis