Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation

Lineage tracing of Pf4-Cre marks hematopoietic stem cells and their progeny

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15-60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage

Differentiation of embryonic stem cells into functional neurons in vitro

Embryonic stem (ES) cells are pluripotent cells that are derived from the inner cell mass of blastocysts. We produced neuronal cells by directed differentiation of a newly established murine ES cell line in vitro (Royan B1). Immunohistochemistry, scanning electron microscopy, Campenot's assay and electrophysiology were used to evaluate the differentiated neurons. Antibodies against microtubule-associated protein (MAP2), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) antigens showed that the cells had GABAergic and dopaminergic characteristics. The Campenot's assay revealed there were a number of peripheral nerveous system related neurons but they were rare. The cells also produced action potentials and responded to electrical stimulation by expressing calcium channels in the processes. We showed therefore that functional neurons can be generated by directed differentiation from embryonic stem cells in vitro

Stress induces neurogenesis in non-neuronal cell cultures of adult olfactory epithelium

Among the basal cells of the olfactory epithelium is a stem cell which divides and whose progeny differentiate into new sensory neurons throughout adult life. Olfactory neurogenesis is highly regulated, for example it is stimulated by epithelial damage. Previous reports implicate several growth factors in progenitor cell proliferation and neuronal differentiation in vitro but these studies differ in growth conditions and age of donors making it difficult to determine precisely the roles of neurogenic stimuli and their sites of action. The aims of the present study were to develop purified basal cell cultures from adult olfactory epithelium and to stimulate neurogenesis in defined growth conditions in order to elucidate the cellular mechanisms by which neurogenesis is stimulated after epithelial damage. We show here that differentiated olfactory sensory neurons arise after biochemical or mechanical stress of rat and mouse olfactory epithelial cell cultures in the absence of growth factors, complex media (e.g., serum, conditioned media, pituitary and hypothalamic extracts), or other cells (e.g., explants, feeder layers of glia, or other non-epithelial cells). Prior to the stress, these cultures contained basal cells and supporting cells but not neurons. After the stress, some cells differentiated into bipolar neurons expressing a number of neuronal proteins including olfactory marker protein. Bromodeoxyuridine experiments show that the differentiated neurons arose from recently divided cells which did not divide again before differentiating. We conclude that stress disrupts cell surface contacts to induce the immediate neuronal precursors to undergo final differentiation into olfactory sensory neurons. This may be a mechanism for enhanced neurogenesis after epithelial damage.No Full Tex