Evidence of Distinct Tumour-Propagating Cell Populations with Different Properties in Primary Human Hepatocellular Carcinoma

Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice.The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features.Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution

Building Pluripotency Identity in the Early Embryo and Derived Stem Cells

The fusion of two highly differentiated cells, an oocyte with a spermatozoon, gives rise to the zygote, a single totipotent cell, which has the capability to develop into a complete, fully functional organism. Then, as development proceeds, a series of programmed cell divisions occur whereby the arising cells progressively acquire their own cellular and molecular identity, and totipotency narrows until when pluripotency is achieved. The path towards pluripotency involves transcriptome modulation, remodeling of the chromatin epigenetic landscape to which external modulators contribute. Both human and mouse embryos are a source of different types of pluripotent stem cells whose characteristics can be captured and maintained in vitro. The main aim of this review is to address the cellular properties and the molecular signature of the emerging cells during mouse and human early development, highlighting similarities and differences between the two species and between the embryos and their cognate stem cells

Chromosome instability in stem cells

Stem cells (SCs) are able to differentiate into many different cell types of the body during early life and growth. They are unspecialized cells capable of self-renewal and, when chal-lenged, they can be induced to differentiate into specific and mature cell types. SCs can be classified into two different types: somatic stem cells (SSCs) and embryonic stem cells (ESCs). One major problem concerning in vitro SC culture is the difficulty to maintain their genetic and karyotype integrity during long-term culture. This chapter describes the different types of abnormalities accumulated by ESCs, derived from different species, and SSCs, of different somatic origin, during long-term culture in vitro. It outlines the main processes that are implicated in the onset of chromosome abnormalities in cells during culture. Among these processes, only one has been so far described in SCs

New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining

The non-homologous end-joining (NHEJ) pathway is a mechanism to repair DNA double strand breaks, which can introduce mutations at repair sites. We constructed new cellular systems to specifically analyze sequence modifications occurring at the repair site. In particular, we looked for the presence of telomeric repeats at the repair junctions, since our previous work indicated that telomeric sequences could be inserted at break sites in germ-line cells during primate evolution. To induce specific DNA breaks, we used the I-SceI system of Saccharomyces cerevisiae or digestion with restriction enzymes. We isolated human and hamster cell lines containing the I-SceI target site integrated in a single chromosomal locus and we exposed the cells to a continuous expression of the I-SceI endonuclease gene. Additionally, we isolated human cell lines that expressed constitutively the I-SceI endonuclease and we introduced the target site on an episomal plasmid stably transfected into the cells. These strategies allowed us to recover repair junctions in which the I-SceI target site was modified at high frequency (100% in hamster cells and about 70% in human cells). Finally, we analyzed junctions produced on an episomal plasmid linearized by restriction enzymes. In all the systems studied, sequence analysis of individual repair junctions showed that deletions were the most frequent modifications, being present in more than 80% of the junctions. On the episomal plasmids, the average deletion length was greater than at intrachromosomal sites. Insertions of nucleotides or deletions associated with insertions were rare events. Junction organization suggested different mechanisms of formation. To check for the insertion of telomeric sequences, we screened plasmid libraries representing about 3.5 x 10(5) junctions with a telomeric repeat probe. No positive clones were detected, suggesting that the addition of telomeric sequences during double strand break repair in somatic cells in culture is either a very rare event or does not occur at all