Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease
Yong-Hee Rhee, … , Kwang-Soo Kim, Sang-Hun Lee
Yong-Hee Rhee, … , Kwang-Soo Kim, Sang-Hun Lee
Published May 16, 2011
Citation Information: J Clin Invest. 2011;121(6):2326-2335. https://doi.org/10.1172/JCI45794.
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Research Article

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease

Abstract

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell–based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Authors

Yong-Hee Rhee, Ji-Yun Ko, Mi-Yoon Chang, Sang-Hoon Yi, Dohoon Kim, Chun-Hyung Kim, Jae-Won Shim, A-Young Jo, Byung-Woo Kim, Hyunsu Lee, Suk-Ho Lee, Wonhee Suh, Chang-Hwan Park, Hyun-Chul Koh, Yong-Sung Lee, Robert Lanza, Kwang-Soo Kim, Sang-Hun Lee

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Figure 1

Sequential induction of hiPSCs toward NPCs and DA neurons.

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Sequential induction of hiPSCs toward NPCs and DA neurons. (A) Experimen...
(A) Experimental procedures. (B–P) Representative phase-contrast (Pro-1; B–F) and immunostaining (G–P) images during in vitro differentiation. 2 hESC and 8 hiPSC lines were cocultured on MS5 feeder cells, then subcultured on MS5-SHH. Undifferentiated hiPSC colonies were sequentially transformed into those assembled with small and compactly arranged cells (B) and then primitive neuroepithelial rosette structures (C) abundantly expressing neural lineage markers such as Pax6, nestin, Sox2, and Cdh2 (G–I). Boxed regions are shown enlarged in the insets of B (×5) and C (×2). Primitive neuroepithelial colonies were harvested, then plated on FN-coated dishes. The majority of cells in the clusters were positive for nestin (J) and Ki67 (K). At the same time, extensive cellular processes emanating from the clusters were positive for TuJ1 (K) and TH (J), specific to neurons and DA neurons, respectively. To obtain uniform populations of NPCs, clusters were dissociated and cultured in the same media containing SHH, FGF8, and bFGF, resulting in morphologically and phenotypically uniform NPCs (E and L), some of which expressed embryonic midbrain markers Pax2 and Pax5 (M). Upon terminal differentiation, the majority of NPCs differentiated into neuronal cells extending neurites (F) and positive for TuJ1 with a few GFAP+ astrocytes (N). Among the TuJ1+ neuronal populations, TH+ neurons were the most abundant (O and P). Ser, serotonin. Image in G is merged with the respective DAPI-stained view (blue). Scale bars: 30 μm.