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Schuele Lab Research Projects iPSC Detail

Patient-Specific iPSC for Disease Modeling

Overview:

The process of generating induced pluripotent stem cells (iPSCs) starts with the collection of patient tissue, i.e. fibroblast cells. The collected sample of tissue is cultured, expanded, characterized, and then stored in our cell bank. A sub-culture of these cells is used for nuclear reprogramming to generate the iPSC lines. The iPSC colonies that form are picked and once again cultured, expanded, characterized and then stored. These characterized iPSC lines are now ready to be turned into tissue types that are of interest to our PD study. Using directed neuronal differentiation to generate dopaminergic neurons an "authentic" model is generated. After characterization of these differentiated dopaminergic neurons, possible applications include the study of disease mechanisms, drug screening, and cell replacement therapy.

Generation of iPSCs:

The process of iPSC generation is briefly outlined in Figure 1. We are currently using two methods to generating iPSCs, both involve transducing collected tissue samples with a specific set of reprogramming factors. In the Takahashi, K. method (1), a four-factor retrovirus is used for reprogramming; alternately a four-factor excisable virus is used.

After the virus infection, there is a distinct morphological change in the newly formed iPSCs. Images of the skin cells before infection (Fig. 2a) and resultant iPSC colonies (Fig. 2b). see below.

Neuronal Differentiation:

The next step, after establishing and characterizing patient-specific iPSC lines, is to differentiate the cells into dopamine producing neurons that resemble the nerve cells dying in the brains of patients. The multistep process of neuronal differentiation begins with changing the iPSCs' media to a differentiation media with factors that mimic neuronal development in an embryo. Embroid bodies (Fig. 3b) eight to ten days after the intial media switch. The media is constantly adjusted throughout the differentiation period. Five to seven days after the embroid body formation, neuronal rosettes (Fig. 3c) will appear. The sand dollar like structures are a characteristic sign of neuronal differentiation. Figure 3d shows the neural precursor cells 10-14 days later.

Image - Neuronal Differentiation of iPSCs

Application and Next Steps:

IPSC-derived neurons can be used as a tool to study disease mechanisms in PD that previously could not be investigated. Also, the ability to model target tissues of interest in patient-specific samples with or without disease associated mutations will prove to be an invaluable tool.

The next step we wish to take is to further the characterization of PD specific characteristics to define a phenotype.

Some factors of interest include:

  • Lewy bodies
  • Post-transcriptional modification
  • Oxidative stress markers
  • Mitochondrial and lysosomal function
  • Disease progression in vitro (time point study)

References:

  1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131(5):861-72.
  2. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science. 318(5858):1917-20. Epub 2007 Nov 20.
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