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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.


Endogenous Nurr1 and Foxa2 expression patterns during development are recapitulated in NPC cultures by exogenous expression driven by pUb in lentiviral transduction and pLTR in retroviral transduction. (a, b) Nurr1 and Foxa2 expression patterns in the developing VMs of mouse embryos at E10.5 and E12 (a) and SN in adult midbrain (b). Proliferating cells in VZ were labeled with PCNA (a) and DA neurons were labeled with TH (a, b). Shown in the bottom row of b are individual and merged images of Nurr1, Foxa2 and TH staining of the boxed area. Scale bar; 100 μm. (c, d) Exogenous Nurr1 and Foxa2 expression levels were induced in NPCs derived from the mouse embryonic cortex using transduction with Retro-pLTR vector (middle row) or Lenti-pUb vector (last row). Exogenous expression patterns were compared with those of endogenous Nurr1 and Foxa2 expressions during in vitro differentiation of NPCs derived from rat embryonic VM at E12 (upper row). Data in graphs are means±s.e.m.'s of % immunoreactive cells (n=10 microscopic fields). Scale bar, 20 μm. DA, dopamine; LTR, long terminal repeat; PCNA, proliferating cell nuclear antigen; SN, substantia nigra; TH, tyrosine hydroxylase.
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fig2: Endogenous Nurr1 and Foxa2 expression patterns during development are recapitulated in NPC cultures by exogenous expression driven by pUb in lentiviral transduction and pLTR in retroviral transduction. (a, b) Nurr1 and Foxa2 expression patterns in the developing VMs of mouse embryos at E10.5 and E12 (a) and SN in adult midbrain (b). Proliferating cells in VZ were labeled with PCNA (a) and DA neurons were labeled with TH (a, b). Shown in the bottom row of b are individual and merged images of Nurr1, Foxa2 and TH staining of the boxed area. Scale bar; 100 μm. (c, d) Exogenous Nurr1 and Foxa2 expression levels were induced in NPCs derived from the mouse embryonic cortex using transduction with Retro-pLTR vector (middle row) or Lenti-pUb vector (last row). Exogenous expression patterns were compared with those of endogenous Nurr1 and Foxa2 expressions during in vitro differentiation of NPCs derived from rat embryonic VM at E12 (upper row). Data in graphs are means±s.e.m.'s of % immunoreactive cells (n=10 microscopic fields). Scale bar, 20 μm. DA, dopamine; LTR, long terminal repeat; PCNA, proliferating cell nuclear antigen; SN, substantia nigra; TH, tyrosine hydroxylase.

Mentions: We next examined the expression patterns of Nurr1 and Foxa2 in the developing VM. Nurr1 expression was barely detected in mouse VM at early embryonic days (data not shown and Figure 2a).13 In VM at E12, Nurr1 expression was identified in the intermediate and mantle zones (IZ/MZ), but not in the ventricular zone (VZ), indicating commencement of Nurr1 expression from the late precursor cell stage. In contrast, Foxa2, one of the earliest genes to be expressed in the floor plate of VM,32, 33 was detected in the VM at E10.5 (Figure 2a). The expression domain of Foxa2 in the VM at E12 was wider than that of Nurr1 and included the alar regions lateral to the Nurr1-expressing domain (Figure 2a). Notably Foxa2 expression was detected in the proliferating VZ of E12 VM (Figure 2a). Foxa2 expression was co-localized in Nurr1-expressing cells in the IZ/MZ, and portions of Nurr1/Foxa2-expressing cells in the MZ were positive for the DA neuronal marker, TH. In the adult midbrain, Nurr1 and Foxa2 expression were continued and co-localized in TH+ DA neurons (Figure 2b).23


In vitro generation of mature midbrain-type dopamine neurons by adjusting exogenous Nurr1 and Foxa2 expressions to their physiologic patterns
Endogenous Nurr1 and Foxa2 expression patterns during development are recapitulated in NPC cultures by exogenous expression driven by pUb in lentiviral transduction and pLTR in retroviral transduction. (a, b) Nurr1 and Foxa2 expression patterns in the developing VMs of mouse embryos at E10.5 and E12 (a) and SN in adult midbrain (b). Proliferating cells in VZ were labeled with PCNA (a) and DA neurons were labeled with TH (a, b). Shown in the bottom row of b are individual and merged images of Nurr1, Foxa2 and TH staining of the boxed area. Scale bar; 100 μm. (c, d) Exogenous Nurr1 and Foxa2 expression levels were induced in NPCs derived from the mouse embryonic cortex using transduction with Retro-pLTR vector (middle row) or Lenti-pUb vector (last row). Exogenous expression patterns were compared with those of endogenous Nurr1 and Foxa2 expressions during in vitro differentiation of NPCs derived from rat embryonic VM at E12 (upper row). Data in graphs are means±s.e.m.'s of % immunoreactive cells (n=10 microscopic fields). Scale bar, 20 μm. DA, dopamine; LTR, long terminal repeat; PCNA, proliferating cell nuclear antigen; SN, substantia nigra; TH, tyrosine hydroxylase.
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fig2: Endogenous Nurr1 and Foxa2 expression patterns during development are recapitulated in NPC cultures by exogenous expression driven by pUb in lentiviral transduction and pLTR in retroviral transduction. (a, b) Nurr1 and Foxa2 expression patterns in the developing VMs of mouse embryos at E10.5 and E12 (a) and SN in adult midbrain (b). Proliferating cells in VZ were labeled with PCNA (a) and DA neurons were labeled with TH (a, b). Shown in the bottom row of b are individual and merged images of Nurr1, Foxa2 and TH staining of the boxed area. Scale bar; 100 μm. (c, d) Exogenous Nurr1 and Foxa2 expression levels were induced in NPCs derived from the mouse embryonic cortex using transduction with Retro-pLTR vector (middle row) or Lenti-pUb vector (last row). Exogenous expression patterns were compared with those of endogenous Nurr1 and Foxa2 expressions during in vitro differentiation of NPCs derived from rat embryonic VM at E12 (upper row). Data in graphs are means±s.e.m.'s of % immunoreactive cells (n=10 microscopic fields). Scale bar, 20 μm. DA, dopamine; LTR, long terminal repeat; PCNA, proliferating cell nuclear antigen; SN, substantia nigra; TH, tyrosine hydroxylase.
Mentions: We next examined the expression patterns of Nurr1 and Foxa2 in the developing VM. Nurr1 expression was barely detected in mouse VM at early embryonic days (data not shown and Figure 2a).13 In VM at E12, Nurr1 expression was identified in the intermediate and mantle zones (IZ/MZ), but not in the ventricular zone (VZ), indicating commencement of Nurr1 expression from the late precursor cell stage. In contrast, Foxa2, one of the earliest genes to be expressed in the floor plate of VM,32, 33 was detected in the VM at E10.5 (Figure 2a). The expression domain of Foxa2 in the VM at E12 was wider than that of Nurr1 and included the alar regions lateral to the Nurr1-expressing domain (Figure 2a). Notably Foxa2 expression was detected in the proliferating VZ of E12 VM (Figure 2a). Foxa2 expression was co-localized in Nurr1-expressing cells in the IZ/MZ, and portions of Nurr1/Foxa2-expressing cells in the MZ were positive for the DA neuronal marker, TH. In the adult midbrain, Nurr1 and Foxa2 expression were continued and co-localized in TH+ DA neurons (Figure 2b).23

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.