Emergence of human angiohematopoietic cells in normal development and from cultured embryonic stem cells.

Human hematopoiesis proceeds transiently in the extraembryonic yolk sac and embryonic, then fetal liver before being stabilized in the bone marrow during the third month of gestation. In addition to this classic developmental sequence, we have previously shown that the aorta-gonad-mesonephros (AGM) embryonic territory produces stem cells for definitive hematopoiesis from 27 to 40 days of human development, through an intermediate blood-forming endothelium stage. These studies have relied on the use of traditional markers of human hematopoietic and endothelial cells. In addition, we have recently identified and characterized a novel surface molecule, BB9, which typifies the earliest founders of the human angiohematopoietic system. BB9, which was initially identified with a monoclonal antibody raised to Stro-1(+) bone marrow stromal cells, recognizes in the adult the most primitive Thy-1(+) CD133(+) Lin(-), non-obese diabetic--severe combined immunodeficiency disease (NOD-SCID) mouse engrating hematopoietic stem cells (HSCs). In the 3- to 4-week embryo, BB9 expression typifies a subset of splanchnopleural mesodermal cells that migrate dorsally and colonize the ventral aspect of the aorta where they establish a population of hemogenic endothelial cells. We have indeed confirmed that hematopoietic potential in the human embryo, as assessed by long-term culture-initiating cell (LTC-IC) and SCID mouse reconstituting cell (SRC) activities, is confined to BB9-expressing cells. We have further validated these results in the model of human embryonic stem cells (hESCs) in which we have modeled, through the development of hematopoietic embryoid bodies (EBs), primitive and definitive hematopoieses. In this setting, we have documented the emergence of BB9(+) hemangioblast-like clonogenic angiohematopoietic progenitors that currently represent the earliest known founders of the human vascular and blood systems.

Mutation analysis of EP300 in colon, breast and ovarian carcinomas.

The putative tumour suppressor gene EP300 is located on chromosome 22q13 which is a region showing frequent loss of heterozygosity (LOH) in colon, breast and ovarian cancers. We analysed 203 human breast, colon and ovarian primary tumours and cell lines for somatic mutations in EP300. LOH across the EP300 locus was detected in 38% of colon, 36% of breast, and 49% of ovarian primary tumours but no somatic mutations in EP300 were identified in any primary tumour. Analysis of 17 colon, 11 breast, and 11 ovarian cancer cell lines identified truncating mutations in 4 colon cancer cell lines (HCT116, HT29, LIM2405 and LIM2412). We confirmed the presence of a previously reported frameshift mutation in HCT116 at codon 1699 and identified a second frameshift mutation at codon 1468. Bi-allelic inactivation of EP300 was also detected in LIM2405 that harbours an insC mutation at codon 927 as well an insA mutation at codon 1468. An insA mutation at codon 1468 was identified in HT29 and a CGA>TGA mutation at codon 86 was identified in LIM2412. Both these lines were heterozygous across the EP300 locus and western blot analysis confirmed the presence of an apparently wild-type protein. Our study has established that genetic inactivation of EP300 is rare in primary colorectal, breast and ovarian cancers. In contrast, mutations are common among colorectal cancer cell lines with 4/17 harbouring homozygous or heterozygous mutations. The rarity of EP300 mutations among these tumour types that show a high frequency of LOH across 22q13 may indicate that another gene is the target of the loss. It is possible that bi-allelic inactivation of EP300 is not necessary and that haploinsufficiency is sufficient to promote tumorigenesis. Alternatively, silencing of EP300 may be achieved by epigenetic mechanisms such as promoter methylation.