Unknown,Transcriptomics,Genomics,Proteomics

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Immunomagnetic purification of mES derived neuronal precursors increases neuronal migration and differentiation in vivo

ABSTRACT: All established protocols for differentiation of mouse and human pluripotent stem cells into specific neural subpopulations generate a considerable cellular heterogeneity that hampers experimental and clinical progress. In order to obtain a homogenous population of neuronal precursor cells and to streamline the differentiation of embryonic stem cells (ESCs), we assessed PSA-NCAM, a surface glycoprotein that is specifically expressed on immature neurons. We developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from differentiated ESC cultures and characterized their neuronal differentiation potential in vitro. PSA-NCAM enrichment at an early step of neural differentiation increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. The in vivo potential of in vitro derived PSA-NCAM+ enriched precursors was functionally characterized by grafting into the forebrain of adult mice. Analysis for several neuronal and glia markers at 10 or 40 days post graft showed a distinct differentiation pattern. While unsorted control cells gave rise to a mixed population composed of immature precursors, early postmitotic neurons or glial cells, the majority of PSA-NCAM+ enriched cells differentiated into NeuN positive neurons. Furthermore, when in contact with the rostral migratory stream, higher numbers of cells integrated into the stream and migrated towards the olfactory bulb when the PSA-NCAM enriched population was grafted. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells. Two conditions (step 4, step 5), each represented by three biological replicates of control and enriched cells (Cy5); mESC was used as common reference (Cy3)

ORGANISM(S): Mus musculus

SUBMITTER: Stefan Tomiuk

PROVIDER: E-GEOD-35125 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors.

Barral Serena S Ecklebe Josephine J Tomiuk Stefan S Tiveron Marie-Catherine MC Desoeuvre Angélique A Eckardt Dominik D Cremer Harold H Bosio Andreas A

Stem cell research 20121102 2

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derive ...[more]

PMID: 23237958

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Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs. Individual transcription factors (TFs) (Ascl1, Smad7, and Nr2f1) were induced in mouse ESCs to facilitate neural differentiation. Expression of transgenic TFs was repressed by doxycycline (Dox); thus, TFs were induced in Dox- conditions, whereas Dox+ conditions represent control cells with no expression of a transgene. For neural differentiation, cells were cultured 3 days in alpha-MEM medium and then in NeuroCult neural differentiation medium. Cells marked as PSANCAM+ were enriched by magnetic microbeads with anty-PSA-NCAM antibody (Miltenyi Biotec) on day 6 of culturing, whereas remaining cells are marked as PSANCAM-. Both PSANCAM+ and PSANCAM- cells were then cultured for another 8 days (total 14 days in differentiation). A time course experiment with Ascl1 induction did not include cell enrichment procedure. RNA was extracted with either Trizol (invitrogen) or mirVana kit (Thermo Fisher Scientific).

Dataset Information

Immunomagnetic purification of mES derived neuronal precursors increases neuronal migration and differentiation in vivo

Publications

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors.

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