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Stem Cells Transl Med. 2014 Sep;3(9):1032-42. doi: 10.5966/sctm.2014-0011. Epub 2014 Jul 14.

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

Author information

1
Laboratory of Stem Cells and Neural Repair and Cytometry and Advanced Optical Microscopy Facility, Inbiomed, San Sebastian, Spain; STEMCELL Technologies, Inc., Vancouver, British Columbia, Canada; Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands; Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA.
2
Laboratory of Stem Cells and Neural Repair and Cytometry and Advanced Optical Microscopy Facility, Inbiomed, San Sebastian, Spain; STEMCELL Technologies, Inc., Vancouver, British Columbia, Canada; Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands; Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA rpernaute@inbiomed.org.

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.

KEYWORDS:

Cell selection; Dopamine; FACS; Floor plate; Neurogenesis; Promoter

PMID:
25024431
PMCID:
PMC4149300
DOI:
10.5966/sctm.2014-0011
[Indexed for MEDLINE]
Free PMC Article

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