Int J Stem Cells.  2015 May;8(1):106-114. 10.15283/ijsc.2015.8.1.106.

Direct Differentiation of Adult Ocular Progenitors into Striatal Dopaminergic Neurons

• Ahmad I
• Zhao X
• Parameswaran S
• Destache C
• Rodriguez-Sierra J
• Thoreson W
• Ahmad H
• Sorrentino J
• Balasubramanian S

• KMID: 2380804
• DOI: http://doi.org/10.15283/ijsc.2015.8.1.106

Abstract

Parkinson's disease, characterized by motor dysfunction due to the loss of nigrostriatal dopaminergic neurons, is one of the most prevalent age-related neurodegenerative disorders. Given there is no current cure, the stem cell approach has emerged as a viable therapeutic option to replace the dopaminergic neurons that are progressively lost to the disease. The success of the approach is likely to depend upon accessible, renewable, immune compatible, and non-tumorigenic sources of neural progenitors from which stable dopaminergic neurons can be generated efficaciously. Here, we demonstrate that neural progenitors derived from limbus, a regenerative and accessible ocular tissue, represent a safe source of dopaminergic neurons. When the limbus-derived neural progenitors were subjected to a well-established protocol of directed differentiation under the influence of Shh and FGF8, they acquired the biochemical and functional phenotype of dopaminergic neurons that included the ability to synthesize dopamine. Their intrastriatal transplantation in the rat model of hemi-Parkinsonism was associated with a reduction in the amphetamine-induced rotation. No tumor formation was observed 6 weeks post-transplantation. Together, these observations posit limbus-derived neural progenitors as an accessible and safe source of dopaminergic neurons for a potential autologous ex-vivo stem cell approach to Parkinson's disease.

Keyword

Parkinson's disease; Dopaminergic neurons; Directed differentiation; Stem cells

MeSH Terms

Adult*
Dopamine
Dopaminergic Neurons*
Humans
Models, Animal
Neurodegenerative Diseases
Parkinson Disease
Phenotype
Stem Cells
Dopamine

Figure

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  Fig. 1 Generation and differentiation of limbus-derived neural progenitors. A schematic representation of the differentiation protocol is provided in A. Cell dissociates from limbus when cultured in the presence of FGF2 and Noggin generated neurospheres with BrdU+ and Nestin+ cells (B; C, dissociated neurospheres). BrdU-tagged neurospheres, when co-cultured with E18 hippocampal cells spread out and cells acquired neuronal morphology, co-expressing neuronal markers β-tubulin/DAPI (D). Whole cell voltage clamped recordings of differentiated cells with neuronal morphology revealed fast inward currents and sustained outward currents (E). The current-voltage (I~V) curve (F) exhibited a typical I-V relationship of fast inward currents due to the voltage gated Na+ channels and sustained outward currents due to the outwardly rectifying K+ channels.

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  Fig. 2 Differentiation of limbus-derived neural progenitors along the dopaminergic cell lineage. Limbus-derived neurospheres were subjected to three stages directed dopaminergic neuron differentiation protocol, using Shh and FGF8 (A). Cell dissociates from GFP-rat limbus when cultured in the presence of FGF2 and Noggin generated neurospheres with GFP+ neural progenitors. Neurospheres spread under the influence of ITSFn (Stage1) and cells therein acquired neuronal morphology under the influence of Shh and FGF8 (Stage II) and increased in number with ascorbate (Stage III).

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  Fig. 3 Molecular and biochemical properties of limbus-derived dopaminergic neurons. RT-PCR analysis of cells in neurospheres (lane 1) and cells at the end of Stage III of differentiation (lane 2) demonstrated the induction of transcripts corresponding to dopaminergic neuron regulator (Nurr1 and Ptx3) and phenotypic and functional markers (Calbindin, TH, DAT, and AADC, compared to the former (A). Immunocytochemical analysis of BrdU-tagged Stage III cells revealed BrdU+ cells, expressing dopaminergic neuron markers, TH (B, C, F) and En1 (D, E, F).

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  Fig. 4 Functional analysis of limbus-derived dopaminergic neurons. The schematic representation of the functional analysis of non-cell autonomously derived dopaminergic neurons is given in A. the HPLC-EC analysis revealed that limbus-derived Stage III cells release DA and DOPAC in the culture medium in response to KCL (B). Un-induced cells were negative for DA and DOPAC. Transplantation of limbus-derived GFP+ stage III cells in the striatum of 6OHDA-lesioned rats reduced the amphetamine-induced rotations to sham-lesioned controls one-week post-translation (C). Retrospective immunohistochemical analysis of sections through the striatum of transplanted rats revealed GFP+ limbus-derived stage III cells expressing TH in the host’s striatum (D~H).

Reference

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