The donor-dependent supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). This approach involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) from PSC-derived hematopoietic progenitors through the overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. The resulting imMKCLs can be expanded in culture over extended periods (4-5 months), even after cryopreservation. Halting the overexpression of c-MYC, BMI1, and BCL-XL in growing imMKCLs led to the production of CD42b(+) platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo. The combination of robust expansion capacity and efficient platelet production means that appropriately selected imMKCL clones represent a potentially inexhaustible source of hPSC-derived platelets for clinical application.

Notch signaling is of crucial importance in normal T-cell development and Notch 1 is frequently mutated in T-cell acute lymphoblastic leukemias (T-ALL), leading to aberrantly high Notch signaling. In this report, we determine whether T-ALL mutations occur not only in Notch1 but also in the F-box protein hCdc4 (Sel-10, Ago, or Fbxw7), a negative regulator of Notch1. We show that the hCDC4 gene is mutated in leukemic cells from more than 30% of patients with pediatric T-ALL and derived cell lines. Most hCDC4 mutations found were missense substitutions at critical arginine residues (Arg 465 , Arg 479 , and Arg 505 ) localized in the substrate-binding region of hCdc4. Cells inactivated for hCdc4 and T-ALL cells containing hCDC4 mutations exhibited an increased Notch1 protein half-life, consistent with the proposed role of hCdc4 in ubiquitindependent proteolysis of Notch1. Furthermore, restoration of wild-type but not mutant hCdc4 in HCT 116 hCDC4-negative cells led to an increased Notch1 ubiquitylation and decreased Notch1 signaling. These results show that hCdc4 mutations interfere with normal Notch1 regulation in vivo. Finally, we found that mutations in hCDC4 and NOTCH1 can occur in the same cancers and that patients carrying hCDC4 and/or NOTCH1 mutations have a favorable overall survival. Collectively, these data show that mutation of hCDC4 is a frequent event in T-ALL and suggest that hCDC4 mutations and gainof-function mutations in NOTCH1 might synergize in contributing to the development of pediatric T-ALL leukemogenesis.

Induced pluripotent stem cell (iPSC) technology enables us to investigate various potential iPSC-based therapies. Although the safety of iPSC derivation has not been completely validated, anucleate cells, such as platelets or erythrocytes, derived from iPSCs are promising targets. However, the efficiency of in vitro platelet generation from megakaryocytes (MKs) under static culture conditions is lower than is seen in vivo. In this study, we demonstrate the proof of concept by a two-dimensional flow culture system that enabled us to increase platelet yield from human embryonic stem cell or iPSC-derived MKs using a biomimetic artificial blood vessel system. The bioreactor was composed of biodegradable scaffolds with ordered arrays of pores made to mimic in vivo bone marrow through salt leaching. Within the system, two flows in different directions in which the angle between the directions of flow is 60 degrees but not 90 degrees contributed to suitable pressure and shear stress applied to MKs to promote platelet generation. Generated platelets derived from human embryonic stem cells or human induced pluripotent stem cells through the bioreactor with a 60-degree angle revealed intact integrin αIIbβ3 activation after agonist stimulation. Collectively, our findings indicate that two flows in different directions of two-dimensional flow culture may be a feasible system for in vitro generation of platelets from pluripotent stem cells (i.e., iPSC-derived MKs) in numbers sufficient for transfusion therapy.

No abstract

Notch signaling is a highly conserved signaling pathway, which is critical for many cell fate decisions. Ligand activation of Notch leads to cleavage of the Notch receptor and liberation of the Notch intracellular domain (ICD) from the membrane-tethered receptor. After translocation to the nucleus, the Notch ICD interacts with the DNA-binding protein CSL to activate gene transcription. To better understand the temporal and spatial aspects of Notch signaling, we here describe a fluorescent protein-based reporter assay that allows Notch activation to be followed in real time in individual cells. We have generated a reporter construct composed of 12 CSL-binding motifs linked to fluorescent proteins with different half-lives: a stabler red fluorescent protein (DsRedExpressDR) and a destabilized form of green fluorescent protein (d1EGFP). The fluorescent reporters reflect the activation status of Notch signaling with single-cell resolution. The reporters rapidly respond to various forms of Notch activation, including ligand activation of full-length Notch receptors. Finally, we use this assay to gain insights into the level of Notch signaling in CNS progenitor cells in culture and in vivo.

Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptormediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl -/-mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34 + multipotent hematopoietic progenitor (MPP) population and development of the CD41 + GPA + megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34 + hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC-derived CD34 + HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT.

The chromatin remodeling complex SWI/SNF is known to regulate the transcription of several genes by controlling chromatin structure in an ATP-dependent manner. SWI/SNF contains the Swi2p/Snf2p like ATPases BRG1 or BRM exclusively. We found that the expression of BRM gradually increases and that of BRG1 decreases as liver cells differentiate. Chromatin immunoprecipitation assays revealed that the ATPase subunits of SWI/SNF and tumor suppressor retinoblastoma (RB) family proteins bind to the promoter region of the albumin gene in hepatocytes, and that the replacement of BRG1 with BRM and pRB with p130 at this site occurs over the course of differentiation. Small interfering RNA experiments showed that blocking the expression of BRG1 and BRM in fetal and adult hepatocytes, respectively, causes a reduction in albumin expression. In luciferase reporter assays with a pREP4-based reporter plasmid that forms a chromatin structure, BRG1 showed activity stimulating the expression of the albumin promoter mediated by CCAAT/enhancer-binding protein alpha (C/EBPalpha). This enhancement was facilitated by the RB family members pRB and p130. ATPase assays showed that both pRB and C/EBPalpha proteins directly stimulate the ATPase activity of BRG1. Our findings suggest that the mechanism by which the activity of transcription factors is enhanced by RB family members and SWI/SNF includes an increase in the ATPase activity of the chromatin remodeling complex.

Transcriptional coactivators, CREB-binding protein (CBP) and p300, exhibit high homology in structure and similar functions. In the present study, we analyzed the function of CBP and p300 proteins as transcriptional coactivators in the expression of albumin in hepatocytes. The expression levels of CBP and p300 were high in fetal hepatocytes, but low in adult ones. Immunoprecipitation assays showed that both CBP and p300 interacted with hepatocyte nuclear factor-1alpha (HNF-1alpha) in primary hepatocytes. Furthermore, CBP and p300 were co-precipitated without HNF-1alpha. Chromatin immunoprecipitation (ChIP) assays revealed that both CBP and p300 are located in the albumin promoter region in hepatocytes. These results suggested that HNF-1alpha, CBP and p300 were incorporated into a preinitiation complex of RNA polymerase II at the albumin promoter. Luciferase reporter assays showed that CBP and p300 cooperatively triggered HNF-1alpha-mediated transcription of the albumin promoter. In addition, inhibition of CBP or p300 using small interfering RNAs (siRNAs) resulted in a reduction in albumin expression. These results suggest that both CBP and p300 are required for enhanced expression of albumin.