Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

Nuclear migration is regulated by the LIS1 protein, which is the regulatory subunit of platelet activating factor (PAF) acetyl-hydrolase, an enzyme complex that inactivates the lipid mediator PAF. Among other functions, PAF modulates cell proliferation, but its effects upon mechanisms of the cell cycle are unknown. Here we show that PAF inhibited interkinetic nuclear migration (IKNM) in retinal proliferating progenitors. The lipid did not, however, affect the velocity of nuclear migration in cells that escaped IKNM blockade. The effect depended on the PAF receptor, Erk and p38 pathways and Chk1. PAF induced no cell death, nor a reduction in nucleotide incorporation, which rules out an intra-S checkpoint. Notwithstanding, the expected increase in cyclin B1 content during G2-phase was prevented in the proliferating cells. We conclude that PAF blocks interkinetic nuclear migration in retinal progenitor cells through an unusual arrest of the cell cycle at the transition from S to G2 phases. These data suggest the operation, in the developing retina, of a checkpoint that monitors the transition from S to G2 phases of the cell cycle

Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naive cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)[E26/111.556/2008]Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)[E26/103.081/2008]Conselho Nacional para o Desenvolvimento Cientifico e Tecnologico (CNPq)[573.975/2008-6]Conselho Nacional para o Desenvolvimento Cientifico e Tecnologico (CNPq)[573476/08-0]Conselho Nacional para o Desenvolvimento Cientifico e Tecnologico (CNPq)[MCT/CNPq 01/2005 - Institutos do Milenio]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2006/61285-9

Neural differentiation in ES and iPS cells after RA treatment.

<p>(A) Schematic representation of protocol. (B) Immunofluorescence quantification confirmed commitment into neural progenitors (nestin) and young neurons (βIII-tubulin). The images correspond to ES-differentiated cells. The bars indicate mean ± S.E.M. of two independent assays, *p<0.05. ES, embryonic stem cells; iPS, induced pluripotent stem cells; RA, <i>all-trans</i> retinoic acid.</p

During neurogenesis, stem cells are prone to chromosome loss and micronuclei formation, which correlates with survivin downregulation.

<p>Neural aneuploidy may contribute to cell diversity within the CNS. CNS, central nervous system.</p

Chromosomal instability increases after induction of neural phenotype by RA in EC cells.

<p>(A) The rate of chromosomal instability presented a 2-fold increase. The bars indicate mean ± S.E.M. of two independent assays, *p<0.05. (B) Analysis of relative DNA content demonstrated that neural cells (RA) presented less DNA than cells incubated with vehicle. RA, <i>all-trans</i> retinoic acid.</p

Hypoploidy is the main kind of aneuploidy in RA-NPCs.

<p>Upon (A) ES cells and (B) iPS cells differentiation with RA, the hypoploidy level increased, whereas hyperploidy level was not altered. The data refer to mean ± S.E.M. of three independent experiments, *p<0.05. ES, embryonic stem cells; iPS, induced pluripotent stem cells; RA-NPCs, neural progenitor cells derived from <i>all-trans</i> retinoic acid treatment.</p

RA induces aneuploidy increase in pluripotent stem cells but not in somatic cells.

<p>(A) Metaphase spreads of RA-NPCs derived from ES cells stained with DAPI. After RA-neural differentiation, the aneuploidy level increased in (B) ES and (C) iPS cells, while (D) mouse embryonic fibroblasts (MEF) exposed to RA did not present aneuploidy increase. The data refer to mean ± S.E.M. of three independent experiments, *p<0.05. ES, embryonic stem cells; iPS, induced pluripotent stem cells; NPCs, neural progenitor cells; RA, <i>all-trans</i> retinoic acid.</p

Reduced survivin expression in RA-treated pluripotent stem cells.

<p>(A) Survivin mRNA and (B) survivin protein were significantly reduced after RA treatment. For quantitative PCR, relative mRNA levels represent the amount of survivin mRNA compared to β-actin mRNA; for western blotting, relative survivin levels are compared to α-tubulin. Bars indicate mean ± S.E.M. of three independent assays, * p<0.1. Data were expressed as the ratio of the mean of RA to the mean of vehicle.</p

RA-treated stem cells do not necessarily undergo apoptosis.

<p>No difference in apoptosis between RA-NPCs and cells incubated with vehicle was observed in (A) EC cells or (B) ES cells. The bars indicate mean ± S.E.M. of three independent assays, *p<0.05. RA-NPCs, neural progenitor cells derived from <i>all-trans</i> retinoic acid treatment; EC, embryonal carcinoma cells; ES, embryonic stem cells.</p

Micronuclei formation increase in pluripotent ES and iPS cells after RA exposure.

<p>(A) Photomicrography of two daughter cells nuclei (arrowhead) and a micronucleus (arrow) detected by DAPI staining. (B) RA increased micronuclei formation in ES and iPS cells. Data was expressed as the ratio of the mean of RA to the mean of vehicle. Bars indicate mean ± S.E.M. of three independent assays, *p<0.05, #p<0.1. ES, embryonic stem cells; iPS, induced pluripotent stem cells; RA, <i>all-trans</i> retinoic acid.</p