Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Neural differentiation of embryonic stem (ES) cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming.</p> <p>Results</p> <p>Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of <it>Oct4 </it>and <it>NANOG </it>and increased expression of <it>nestin</it>. ES cells containing a GFP gene under the control of the <it>Sox1 </it>regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents.</p> <p>Conclusion</p> <p>Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.</p

Synaptically-Competent Neurons Derived from Canine Embryonic Stem Cells by Lineage Selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABAA- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model

Expression of CoAA in embryoid body of P19 cells and in brain of mouse embryo

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> () Evaluation of anti-RRM and anti-CoAA antibodies using western blot analysis by overexpression of CoAA and CoAM in 293 cells. () Light microscopy of P19 cells at indicated differentiation stages (×400). () Embryoid body at Day 4 was paraffin-embedded, sectioned and stained with anti-RRM (against both CoAA and CoAM), anti-CoAA (against CoAA only) and anti-active caspase-3 (against cleaved caspase-3) antibodies. Two representative views are shown (×400). Enlarged views are shown below. The results show CoAM and active caspase-3 staining in the EB cavity. () Western blot analysis of CoAA, CoAM and CoAR at indicated differentiation stages of P19 cells. () Immunohistochemistry analyses of mouse embryonic brain at gestation stages of E12.5 and E15.5. The sagittal sections were stained with affinity-purified anti-CoAA antibody (1:200) and counterstained with hematoxylin (×400)

Overexpression of CoAM and treatment with CoAA siRNA induce Sox6 expression in the absence of retinoic acid

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> P19 cells were transfected with CoAM or with CoAA-specific siRNA (25 nM) at the EC stage, and then were cultured in suspension for 4 days in the absence of RA induction. The cells were trypsinized, plated and further cultured for 3 days. The untreated and RA-induced (for 4 days) P19 cells were similarly cultured as controls. RT-PCR analyses were carried out with indicated marker genes at each indicated stage

Tentative model of CoAA alternative splicing regulation in the embryoid body of stem cells

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> CoAA is produced by alternative splicing with inclusion of the second exon. A competitive 5′ alternative splicing event excluding a portion of the second exon (2b) produces a dominant negative variant, CoAM, which lacks the activation domain. The basal promoter of the CoAA gene, shown as an open box, promotes splicing of the CoAA form, which is expressed in the outer layer of the embryoid body (gray). The upstream -regulating sequence, shown as a filled box, promotes alternative splicing of CoAM, which is expressed in the cavity of EB (white). The loss of upstream -regulating element in cancer may prevent the alternative splicing switch and disrupt stem cell differentiation

Alternative splicing switch from CoAA to CoAM during P19 embryonal carcinoma cell differentiation

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> () Undifferentiated P19 cells (EC) were induced with 500 nM retinoic acid (RA) in suspension culture up to 4 days to form embryoid bodies (EB2–EB4) which were trypsinized and further differentiated in the tissue culture dish for an additional 12 days in the absence of RA (D3–D12). Total RNA was isolated and normalized at each stage and analyzed using gene-specific primers as indicated by RT-PCR. GAPDH was the control. () CoAA and CoAM were analyzed by RT-PCR using shared primers. () The relative quantity of CoAA and CoAM was analyzed by quantitative real-time PCR. () Mouse ES cells were induced with 1 μM RA for 6 days. The EB was undisrupted in continuous culture for 15 days in the absence of RA. RT-PCR was carried out using RNA isolated at each stage