Derivation, enrichment and characterization of dopaminergic neurons from pluripotent stem cells

Parkinson s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons residing in the substantia nigra and innervating the straitum. Current medical interventions provide initial symptomatic relief, but unfortunately do not slow or reversing the disease course. Pluripotent stem cell (PSC) based replacement therapies are an attractive solution, since in theory PSC serve as an indefinite source capable of generating any somatic cell type. In recent years, derivation and preclinical transplantation of DA neurons from PSC have generated great enthusiasm in the field of regenerative medicine. Prior to clinical application, improved efficiency and a comprehensive understanding of molecular mechanisms governing DA differentiation is necessary. This thesis establishes and employs the NTera2 cell line as a PSC system to study, characterize and explore mechanisms and methods directing DA differentiation.

In paper I, the NTera2 cell line was established as a model system to examine DA neuron differentiation of hESC and expedite basic research. We showed that in addition to expression of PSC markers, undifferentiated NTera2 cells were similar to multiple hESC lines in overall gene expression profiles. Following co-culture with the stromal cell line PA6, NTera2 cells expressed DA and neuronal markers in a similar time frame and expression profile to what has been reported for hESC. We established important simplifications to the PA6 co-culture system including the use of PA6 conditioned medium (PA6 CM) to generate DA neurons. In a proof of principle approach, we used flow cytometry to select neuronal progenitors capable of generating functional DA neurons upon further differentiation.

In the following study (paper II), we designed, generated and validated a focused glial- DA array for the purpose of evaluating derived populations. Among the assessed populations, we examined both undifferentiated NTera2 cells and NTera2 derived neuronal progenitors directed towards DA neurons. Derivation techniques optimized in the NTera2 cell line were extrapolated to select neuronal progenitors from hESC differentiated towards DA neurons and examine their subsequent detailed genomic expression profiles (paper III). Interestingly, we observed activation of the 11.15p.5 chromosome, and an up-regulation of IGF2 and CDKN1C in both neuronal progenitors directed toward DA neurons and human substantia nigra DA neurons.

In paper IV, we identified several components of PA6 CM that were responsible for DA neuron differentiation. PA6 CM induced DA neuron differentiation in both NTera2 (hECSC line) and I6 cells (hESC line). We indentified candidate DA inducing factors through comparative microarray gene expression analysis and mass spectroscopy analysis of PA6 CM. Following the addition of candidate factors (SDF1α, sFRP1 and VEGFD) we observed an increase in DA neuron differentiation in both the NTera2 and I6 cell lines.

In paper V, we report that flow cytometry selection of neuronal progenitors resulted in a three-fold increase in the number of DA neurons generated in PA6 CM. However, this differentiation capacity was observed in PA6 CM and differentiation in defined medium resulted in a more than 10 fold reduction in the number of DA neurons. Global microarray gene expression allowed us to examine the characteristics of progenitors and their more mature progeny.

Taken together, our data provide important insight into the molecular mechanisms that promote the differentiation of DA neurons from PSC.

List of scientific papers

I. Schwartz CM, Spivak CE, Baker SC, McDaniel TK, Loring JF, Nguyen C, Chrest FJ, Wersto R, Arenas E, Zeng X, Freed WJ, Rao MS. (2005). "NTera2: a model system to study dopaminergic differentiation of human embryonic stem cells." Stem Cells Dev. 14(5): 517-34.
https://pubmed.ncbi.nlm.nih.gov/16305337

II. Luo Y, Schwartz C, Shin S, Zeng X, Chen N, Wang Y, Yu X, Rao MS (2006). "A focused microarray to assess dopaminergic and glial cell differentiation from fetal tissue or embryonic stem cells." Stem Cells. 24(4): 865-75.
https://pubmed.ncbi.nlm.nih.gov/16357341

III. Freed WJ, Chen J, Bäckman CM, Schwartz CM, Vazin T, Cai J, Spivak CE, Lupica CR, Rao MS, Zeng X (2008). "Gene expression profile of neuronal progenitor cells derived from hESCs: activation of chromosome 11p15.5 and comparison to human dopaminergic neurons." PLoS One. 3(1): e1422.
https://pubmed.ncbi.nlm.nih.gov/18183302

IV. Schwartz CM, Tavakoli T, Jamias C, Park S, Phillips TM, Yao PJ, Itoh K, Maudsley S, Ma W, Rao MS, Arenas E, Mattson MP (2010). "The identified stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells." [Manuscript]

V. Schwartz CM, Rao MS, Arenas E, Mattson MP (2010). "Characterization and maintenance of neural progenitors derived from human pluripotent stem cells differentiated towards dopaminergic neurons." [Manuscript]