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Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells.

ABSTRACT: Human embryonic and induced pluripotent stem cells are potential cell sources for regenerative approaches in Parkinson disease. Inductive differentiation protocols can generate midbrain dopamine neurons but result in heterogeneous cell mixtures. Therefore, selection strategies are necessary to obtain uniform dopamine cell populations. Here, we developed a selection approach using lentivirus vectors to express green fluorescent protein under the promoter region of FOXA2, a transcription factor that is expressed in the floor plate domain that gives rise to dopamine neurons during embryogenesis. We first validated the specificity of the vectors in human cell lines against a promoterless construct. We then selected FOXA2-positive neural progenitors from several human pluripotent stem cell lines, which demonstrated a gene expression profile typical for the ventral domain of the midbrain and floor plate, but failed to enrich for dopamine neurons. To investigate whether this was due to the selection approach, we overexpressed FOXA2 in neural progenitors derived from human pluripotent stem cell lines. FOXA2 forced expression resulted in an increased expression of floor plate but not mature neuronal markers. Furthermore, selection of the FOXA2 overexpressing fraction also failed to enrich for dopamine neurons. Collectively, our results suggest that FOXA2 is not sufficient to induce a dopaminergic fate in this system. On the other hand, our study demonstrates that a combined approach of promoter activation and lentivirus vector technology can be used as a versatile tool for the selection of a defined cell population from a variety of human pluripotent stem cell lines.

SUBMITTER: Aguila JC

PROVIDER: S-EPMC4149300 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells.

Aguila Julio Cesar JC Blak Alexandra A van Arensbergen Joris J Sousa Amaia A Vázquez Nerea N Aduriz Ariane A Gayosso Mayela M Lopez Mato Maria Paz MP Lopez de Maturana Rakel R Hedlund Eva E Sonntag Kai-Christian KC Sanchez-Pernaute Rosario R

Stem cells translational medicine 20140714 9

Human embryonic and induced pluripotent stem cells are potential cell sources for regenerative approaches in Parkinson disease. Inductive differentiation protocols can generate midbrain dopamine neurons but result in heterogeneous cell mixtures. Therefore, selection strategies are necessary to obtain uniform dopamine cell populations. Here, we developed a selection approach using lentivirus vectors to express green fluorescent protein under the promoter region of FOXA2, a transcription factor th ...[more]

PMID: 25024431

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Project description:Importance:Schizophrenia candidate genes participate in common molecular pathways that are regulated by activity-dependent changes in neurons. One important next step is to further our understanding on the role of activity-dependent changes of gene expression in the etiopathogenesis of schizophrenia. Objective:To examine whether neuronal activity-dependent changes of gene expression are dysregulated in schizophrenia. Design, Setting, and Participants:Neurons differentiated from human-induced pluripotent stem cells derived from 4 individuals with schizophrenia and 4 unaffected control individuals were depolarized using potassium chloride. RNA was extracted followed by genome-wide profiling of the transcriptome. Neurons were planted on June 21, 2013, and harvested on August 2, 2013. Main Outcomes and Measures:We performed differential expression analysis and gene coexpression analysis to identify activity-dependent or disease-specific changes of the transcriptome. Gene expression differences were assessed with linear models. Furthermore, we used gene set analyses to identify coexpressed modules that are enriched for schizophrenia risk genes. Results:We identified 1669 genes that were significantly different in schizophrenia-associated vs control human-induced pluripotent stem cell-derived neurons and 1199 genes that are altered in these cells in response to depolarization (linear models at false discovery rate ?0.05). The effect of activity-dependent changes of gene expression in schizophrenia-associated neurons (59 significant genes at false discovery rate ?0.05) was attenuated compared with control samples (594 significant genes at false discovery rate ?0.05). Using gene coexpression analysis, we identified 2 modules (turquoise and brown) that were associated with diagnosis status and 2 modules (yellow and green) that were associated with depolarization at a false discovery rate of ?0.05. For 3 of the 4 modules, we found enrichment with schizophrenia-associated variants: brown (?2?=?20.68; P?=?.002), turquoise (?2?=?12.95; P?=?.04), and yellow (?2?=?15.34; P?=?.02). Conclusions and Relevance:In this analysis, candidate genes clustered within gene networks that were associated with a blunted effect of activity-dependent changes of gene expression in schizophrenia-associated neurons. Overall, these findings link schizophrenia candidate genes with specific molecular functions in neurons, which could be used to examine underlying mechanisms and therapeutic interventions related to schizophrenia.

Dataset Information

Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells.

Publications

Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells.

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