Limits...
In vitro generation of mature midbrain-type dopamine neurons by adjusting exogenous Nurr1 and Foxa2 expressions to their physiologic patterns

View Article: PubMed Central - PubMed

ABSTRACT

Developmental information aids stem cell biologists in producing tissue-specific cells. Recapitulation of the developmental profile of a specific cell type in an in vitro stem cell system provides a strategy for manipulating cell-fate choice during the differentiation process. Nurr1 and Foxa2 are potential candidates for genetic engineering to generate midbrain-type dopamine (DA) neurons for experimental and therapeutic applications in Parkinson's disease (PD), as forced expression of these genes in neural stem/precursor cells (NPCs) yields cells with a complete battery of midbrain DA neuron-specific genes. However, simple overexpression without considering their expression pattern in the developing midbrain tends to generate DA cells without adequate neuronal maturation and long-term maintenance of their phenotype in vitro and in vivo after transplantation. We here show that the physiological levels and timing of Nurr1 and Foxa2 expression can be replicated in NPCs by choosing the right vectors and promoters. Controlled expression combined with a strategy for transgene expression maintenance induced generation of fully mature midbrain-type DA neurons. These findings demonstrate the feasibility of cellular engineering for artificial cell-fate specification.

No MeSH data available.


Exogenous expression patterns derived from retroviral and lentiviral vectors with various promoters in NPCs during differentiation in vitro. The reporter GFP gene was engineered into retroviral and lentiviral vectors with various universal promoters. NPCs derived from rat embryonic cortices at E14 were transduced with each of the GFP-expressing vectors, and the transduced NPCs were differentiated into neurons from the day following transduction. (a–c) GFP expression by various expression systems at differentiation day 3 (D3). Representative GFP+ cell images (a). Viral transduction efficiency was estimated by % GFP+ cells of total DAPI+ cells (b) and GFP fluorescence intensity (MFI) of individual cells (c). (d–g) Expression of exogenous GFP during neuronal differentiation. Shown in d are representative GFP+/TuJ1+ cell images at D5 and D15. Insets, high-powered views of the boxed areas. Expression of GFP mRNA (e) and % of GFP+ cells out of TuJ1 cells (f) at D15 were compared with those at D5. Total numbers of TuJ1+ neurons expressing GFP at D15 (g). *Significantly different from D5 and the cultures transduced with Retro-pLTR at P<0.05 (n=20 microscopic fields). Scale bar, 20 μm. DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescence protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5382556&req=5

fig1: Exogenous expression patterns derived from retroviral and lentiviral vectors with various promoters in NPCs during differentiation in vitro. The reporter GFP gene was engineered into retroviral and lentiviral vectors with various universal promoters. NPCs derived from rat embryonic cortices at E14 were transduced with each of the GFP-expressing vectors, and the transduced NPCs were differentiated into neurons from the day following transduction. (a–c) GFP expression by various expression systems at differentiation day 3 (D3). Representative GFP+ cell images (a). Viral transduction efficiency was estimated by % GFP+ cells of total DAPI+ cells (b) and GFP fluorescence intensity (MFI) of individual cells (c). (d–g) Expression of exogenous GFP during neuronal differentiation. Shown in d are representative GFP+/TuJ1+ cell images at D5 and D15. Insets, high-powered views of the boxed areas. Expression of GFP mRNA (e) and % of GFP+ cells out of TuJ1 cells (f) at D15 were compared with those at D5. Total numbers of TuJ1+ neurons expressing GFP at D15 (g). *Significantly different from D5 and the cultures transduced with Retro-pLTR at P<0.05 (n=20 microscopic fields). Scale bar, 20 μm. DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescence protein.

Mentions: NPCs derived from rat embryonic cortices at E14 were transduced with identical titers of the viruses expressing GFP and then differentiated the next day of transduction. In the cultured NPCs, induction of the exogenous expression driven by the retroviral pLTR was the fastest and the most efficient. Prominent GFP expression was detected from the day following transduction (Figures 1a and b). At post-transduction day 4 (differentiation day 3, D3), strong GFP expression was detected in >70% of total cells in the cultures transduced with the GFP-retroviruses. Gene expressions induced by the lentiviral transductions in NPCs were, in general, less efficient than those with retro-pLTR during early post-transduction days (Figures 1a and b). GFP expression levels induced by PGK, EF1a and ubiquitin promoters (pPGK, pEF1a, pUb) were undetectable in the first 2 days of post transduction, but then gradually increased over the next 2–3 days. The intensity of GFP expression in individual cells (estimated by mean fluorescence intensity, MFI) driven by pEF1a, pUb or pPGK were much weaker than those driven by retroviral pLTR (Figure 1c). By contrast, exogenous gene expression levels driven by the CMV and CAG promoters (pCMV, pCAG) were relatively stronger among lentiviral transduction systems (Figure 1c).


In vitro generation of mature midbrain-type dopamine neurons by adjusting exogenous Nurr1 and Foxa2 expressions to their physiologic patterns
Exogenous expression patterns derived from retroviral and lentiviral vectors with various promoters in NPCs during differentiation in vitro. The reporter GFP gene was engineered into retroviral and lentiviral vectors with various universal promoters. NPCs derived from rat embryonic cortices at E14 were transduced with each of the GFP-expressing vectors, and the transduced NPCs were differentiated into neurons from the day following transduction. (a–c) GFP expression by various expression systems at differentiation day 3 (D3). Representative GFP+ cell images (a). Viral transduction efficiency was estimated by % GFP+ cells of total DAPI+ cells (b) and GFP fluorescence intensity (MFI) of individual cells (c). (d–g) Expression of exogenous GFP during neuronal differentiation. Shown in d are representative GFP+/TuJ1+ cell images at D5 and D15. Insets, high-powered views of the boxed areas. Expression of GFP mRNA (e) and % of GFP+ cells out of TuJ1 cells (f) at D15 were compared with those at D5. Total numbers of TuJ1+ neurons expressing GFP at D15 (g). *Significantly different from D5 and the cultures transduced with Retro-pLTR at P<0.05 (n=20 microscopic fields). Scale bar, 20 μm. DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescence protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382556&req=5

fig1: Exogenous expression patterns derived from retroviral and lentiviral vectors with various promoters in NPCs during differentiation in vitro. The reporter GFP gene was engineered into retroviral and lentiviral vectors with various universal promoters. NPCs derived from rat embryonic cortices at E14 were transduced with each of the GFP-expressing vectors, and the transduced NPCs were differentiated into neurons from the day following transduction. (a–c) GFP expression by various expression systems at differentiation day 3 (D3). Representative GFP+ cell images (a). Viral transduction efficiency was estimated by % GFP+ cells of total DAPI+ cells (b) and GFP fluorescence intensity (MFI) of individual cells (c). (d–g) Expression of exogenous GFP during neuronal differentiation. Shown in d are representative GFP+/TuJ1+ cell images at D5 and D15. Insets, high-powered views of the boxed areas. Expression of GFP mRNA (e) and % of GFP+ cells out of TuJ1 cells (f) at D15 were compared with those at D5. Total numbers of TuJ1+ neurons expressing GFP at D15 (g). *Significantly different from D5 and the cultures transduced with Retro-pLTR at P<0.05 (n=20 microscopic fields). Scale bar, 20 μm. DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescence protein.
Mentions: NPCs derived from rat embryonic cortices at E14 were transduced with identical titers of the viruses expressing GFP and then differentiated the next day of transduction. In the cultured NPCs, induction of the exogenous expression driven by the retroviral pLTR was the fastest and the most efficient. Prominent GFP expression was detected from the day following transduction (Figures 1a and b). At post-transduction day 4 (differentiation day 3, D3), strong GFP expression was detected in >70% of total cells in the cultures transduced with the GFP-retroviruses. Gene expressions induced by the lentiviral transductions in NPCs were, in general, less efficient than those with retro-pLTR during early post-transduction days (Figures 1a and b). GFP expression levels induced by PGK, EF1a and ubiquitin promoters (pPGK, pEF1a, pUb) were undetectable in the first 2 days of post transduction, but then gradually increased over the next 2–3 days. The intensity of GFP expression in individual cells (estimated by mean fluorescence intensity, MFI) driven by pEF1a, pUb or pPGK were much weaker than those driven by retroviral pLTR (Figure 1c). By contrast, exogenous gene expression levels driven by the CMV and CAG promoters (pCMV, pCAG) were relatively stronger among lentiviral transduction systems (Figure 1c).

View Article: PubMed Central - PubMed

ABSTRACT

Developmental information aids stem cell biologists in producing tissue-specific cells. Recapitulation of the developmental profile of a specific cell type in an in vitro stem cell system provides a strategy for manipulating cell-fate choice during the differentiation process. Nurr1 and Foxa2 are potential candidates for genetic engineering to generate midbrain-type dopamine (DA) neurons for experimental and therapeutic applications in Parkinson's disease (PD), as forced expression of these genes in neural stem/precursor cells (NPCs) yields cells with a complete battery of midbrain DA neuron-specific genes. However, simple overexpression without considering their expression pattern in the developing midbrain tends to generate DA cells without adequate neuronal maturation and long-term maintenance of their phenotype in vitro and in vivo after transplantation. We here show that the physiological levels and timing of Nurr1 and Foxa2 expression can be replicated in NPCs by choosing the right vectors and promoters. Controlled expression combined with a strategy for transgene expression maintenance induced generation of fully mature midbrain-type DA neurons. These findings demonstrate the feasibility of cellular engineering for artificial cell-fate specification.

No MeSH data available.