RESUMO
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.
Assuntos
Separação Celular/métodos , Neurônios Dopaminérgicos/citologia , Fator 3-beta Nuclear de Hepatócito/genética , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Western Blotting , Citometria de Fluxo , Imunofluorescência , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Lentivirus , Microscopia Confocal , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução GenéticaRESUMO
In a previous study, we unexpectedly found that tilapia growth hormone (tiGH) secreted to the culture media by transformed cells of the yeast Pichia pastoris lacks 46 amino acids from the C-terminal end. In the present study, we cloned the exact fragment that code for this truncated variant and demonstrated its growth promoting activity in goldfish when it's administered by immersion bath. Furthermore, a better characterization of this polypeptide was performed. Administration of the polypeptide derived from tiGH increased superoxide anion production and has a mitogenic effect on peripheral blood leukocytes. This molecule binds to liver membranes proteins in vitro in a saturable manner. Beside, we cloned and expressed tiGH and its truncated variant in mammalian cells using the signal peptide of this hormone and we observed that the secretion was drastically reduced in the truncated tiGH as compared to the intact molecule. Truncated tilapia growth hormone lacking the helix 4 and two disulfide loops is still a bioactive hormone, suggesting that the disulfide bonds and the helix 4 are not essential for the biological activities examined in this work. However, the growth hormone C-terminal portion seems to be essential for this hormone to be secreted by cultured cells in vitro.
