Modeling Megakaryopoiesis and Leukemogenesis Using Human and Murine Embryonic Stem Cells

Abstract 2502

Megakaryopoiesis involves the differentiation of progenitor cells into diploid megakaryocytes, which then undergo endomitosis and cytoplasmic maturation resulting in platelet release. Acute Megakaryoblastic Leukemia (AMKL) results when this process goes awry. Nearly one third of pediatric AMKL patients are infants who have the t (1;22) chromosomal translocation (t (1;22)-AMKL) resulting in fusion of the RNA Binding Motif 15 gene (RBM15) on chromosome 1 upstream of the transcriptional cofactor Megakaryoblastic Leukemia 1 gene (MKL1) on chromosome 22. Given that t (1;22)-AMKL primarily affects infants, the leukemia likely originates in utero when the hematopoietic system is in its embryonic stage.Our goal was to establish in vitro methods to study embryonic megakaryopoiesis and leukemogenesis using human and murine embryonic stem cells (hESCs and mESCs). Using the Invitrogen Gateway System, human MKL1 (hMKL1) and human RBM15-MKL1 (hRM) constructs were designed. H9-rtTA, a hESC line constitutively expressing the reverse transcriptional activator (rtTA) under the control of the Ubiquitin C promoter, were transduced with lentivirus harboring either hMKL1 or hRM with an IRES-GFP promoter under the control of the tetracycline responsive element (TRE) to derive doxycycline (dox) inducible cell lines. In the presence of dox, the rtTA binds to the TRE, causing transcriptional activation of the downstream transgene. hESC were co-cultured on murine OP-9 stromal cell layers with thrombopoietin to promote megakaryocytic differentiation. For construction of the inducible mESC lines, mESCs already containing the rtTA at the ROSA26 locus were electroporated with either murine MKL1 (mMKL1) or murine RBM15-MKL1 (mRM). Site-specific insertion of the transgenes using a cre recombinase and modified loxP sites placed mMKL1 or mRM under the control of a TRE. The inducible mMKL1 mESCs were differentiated into hematopoietic cells using embryoid body formation and subsequently co-cultured on OP-9s. The human and murine MKL1 and RM constructs were transiently transfected into 293FT cells and Western blotting was used to confirm their functionality. In the hESC studies, GFP expression was detected by flow cytometry 24 hrs after dox induction in both cell lines, with 54.7% (SD+3.1) GFP+ cells in hMKL1 transduced cells and 47.5% (SD+16.2) GFP+ cells in hRM transduced cells. hMKL1 protein was detected 24 hrs after dox induction; however, the hRM fusion protein was not detected in undifferentiated hESCs by Western blot or IP/IB after 6, 24, 48, 72, 96 hrs of dox exposure. Nearly one fourth of the cells transduced with hRM were GFP+ after only 6 hrs of doxycycline exposure. Interestingly, with continued dox exposure over six days, GFP expression in the undifferentiated hESCs declined, with a mean decrease in GFP expression of 14.5% in both cell lines. Successful incorporation of the vector carrying the hRM was confirmed by genomic PCR and resulted in dox-inducible expression of hRM mRNA detected by RT-PCR. Upon megakaryocytic differentiation, GFP and CD41 expression, detected by flow cytometry, occur in a mutually exclusive fashion in both MKL and RBM15-MKL1 transduced hESCs. In the mESC experiements, protein expression of mMKL1 in the undifferentiated cells was confirmed by Western blot 24, 48, and 72 hrs after dox addition. Expression of mRM protein in the undifferentiated mESCs was undetectable 6, 14, 24, 30, 48, or 72 hrs after dox addition. However, mRM mRNA was detected by qRT-PCR at 0, 6, 14, and 30 hours after dox treatment in the undifferentiated mESCs. Of note, transient transfection of undifferentiated mESCs with mRM resulted in protein expression by Western blot as early as 6 hrs after transfection. The fusion protein continued to be detected at 12 and 18 hrs after transfection, but by 24 hrs, it was no longer detectable. When differentiated, the mMKL1 mESC line no longer demonstrated inducible mMKL1 expression in the CD41+ (hematopoietic) population as determined by qRT-PCR. These findings confirm ongoing activity of the promoters in the undifferentiated hESC and mESC lines and indicate that the promoters may be silenced upon induction of hematopoietic differentiation of both hESC and mESC lines. In addition, the transient detection of RM after transfection into the mESCs suggests post-translational modification of the fusion protein likely results in its rapid degradation.