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 2

Residual expression of reprogramming genes in differentiated cells from hiPSCs.

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Residual expression of reprogramming genes in differentiated cells from ...
(A) Oct4 expression in hiPSC-NPCs. Substantial Oct4 expression was detected in NPCs derived from all 4 lentivirus-based hiPSCs. (B) Oct4 expression was also detected in nestin+ NPCs and in TuJ1+ and TH+ neurons from lentivirus-based hiPSCs before (left) and after (middle and right) terminal differentiation for 7 days. Scale bars: 30 μm. Boxed regions are shown enlarged in the insets (×3). (C) Upregulation of Oct4 expression by cAMP treatment. Lenti-3 NPCs were induced to differentiate by withdrawal of bFGF in the absence (No treat) or presence of BDNF, GDNF, or cAMP for 7 days. The percentage of Oct4-positive cells was counted (n = 4 coverslips per group). *P < 0.01 vs. untreated control, ANOVA with Tukey post-hoc analysis. (D) RT-PCR expression analyses for exogenous and endogenous genes. The indicated hiPSCs were cocultured and sequentially differentiated as in Figure 1A. RNAs were prepared from each step: undifferentiated, coculture with MS5, MS5-SHH, NPCs, and differentiated neurons (DA). Primers specific for exogenous (exo; proviral) and endogenous (endo; chromosomal) expression were used. GDF3, REX1, and DNMT3L were used as undifferentiated markers. N/D, not determined; N/A, not applicable (NANOG was not used for establishing the retrovirus-based iPSCs).