IntroductionAcute myeloid leukemia (AML) refers to a genetically and biologically heterogeneous group of diseases characterized by an abnormal increase of myeloblasts in the bone marrow (BM) and peripheral blood (PB) circulation. Chemotherapy and hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment, but these modalities have reached an impasse with an overall cure rate of only 30%-40%. 1 An attractive strategy is to target specific genetic and biochemical alterations in AML, thereby providing an alternative treatment modality that may improve patient outcome. 2,3 FLT3 (fms-like tyrosine kinase-3) is a receptor tyrosine kinase (RTK) that is highly expressed in hematopoietic stem and progenitor cells. It includes an extracellular domain (ECD), a transmembrane domain (TMD), a juxtamembrane domain (JMD), and 2 tyrosine kinase domains (TKDs) separated by a kinase insert. 4 On binding with FLT3 ligand secreted by BM stromal cells, FLT3 undergoes dimerization, phosphorylation, and TKD activation. The FLT3 gene is mutated in approximately 30% of AMLs, particularly those with normal karyotypes, t(6;9), t(15;17), or trisomy 8. 5,6 The most common mutation is an internal tandem duplication (ITD) up to a few hundred base-pairs within the JMD. Single-base mutations have also been described, most commonly resulting in a substitution of aspartic acid with tyrosine or less commonly a histidine at residue 835 in the TKD. 7,8 At a molecular level, these mutations result in constitutive activation of the FLT3 receptor and hence downstream PI3K/AKT/mTOR, Ras/Raf/MEK/ERK, and JAK/ STAT pathways. 9,10 The biologic consequences are enhanced proliferation and reduced apoptosis of the myeloblasts, which contribute to leukemogenesis. [11][12][13][14][15] Patients with FLT3-ITD respond poorly to conventional chemotherapy and have an inferior prognosis, 16,17 particularly in those with a high FLT3-ITD ϩ cell burden, 18 long ITD sequences, 19 and multiple FLT3-ITD ϩ subclones, 20 underscoring a pathogenetic role of FLT3-ITD in human AML. In mice, knock-in of a heterozygous FLT3-ITD resulted in a preleukemic model of a myeloproliferative disease, providing an in vivo demonstration of the important role of FLT3 in leukemia initiation. 21 We 22 and others 23,24 have shown that the FLT3-ITD allele can be found in leukemia initiating cells (LICs), as distinguished by their capability of regenerating leukemic progeny in transplanted immunodeficient mice. Therefore, targeting FLT3-ITD might provide a novel approach to therapeutic intervention.Several clinical trials on multi-TK inhibitors with different FLT3 specificities and in vitro efficacies have been reported, including the use of midostaurin, 25 lestautinib, 26 tandutinib, 27 sunitinib, 28 and sorafenib. 29 In most studies, clinical efficacy was restricted to FLT3-ITD ϩ AML and correlated with inactivation of FLT3 phosphorylation. 25,[30][31][32] Complete remission was rare and limited to anecdotal reports in relapsed AML after allogeneic HSCT. 33 Furthermore, after ...

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.

Key Points• Zebrafish flt3 plays an important role in the initiation of definitive hematopoietic stem cells.

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