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 4

Protein-based hiPSCs generate functional midbrain-like DA neurons in vitro.

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Protein-based hiPSCs generate functional midbrain-like DA neurons in vit...
(A–C) TuJ1+ cells differentiated from protein-based hiPSC-NPCs were positive for hNCAM (A), HuC/D (B), and MAP2 (C). (D) TuJ1+ cells differentiated from protein-based hiPSC-NPCs were electrophysiologically active neurons. Exemplary membrane potential response upon depolarizing current steps (30, 60, and 90 pA for 300 ms) was abolished by 500 nM tetrodotoxin. (E–M) Midbrain marker expression in protein-based hiPSC–derived DA neurons. TH+ cells from differentiated protein-based hiPSC-NPC cultures were immunoreactive for markers specific for neuronal (hNCAM, HuC/D, and MAP2; E–G) and for DA homeostasis (VMAT2 and DAT; H and I). Markers specific for midbrain (EN1 and Nurr1; J and K), A9 midbrain (GIRK2; L), and A10 midbrain (calbindin 1, 28 kDa; M) colocalized in subpopulations of TH+ cells. (N–P) Presynaptic DA neuronal functions, such as DA release (N and O) and DA uptake (P). (N) Typical HPLC chromatograms for DA released in the absence and presence of 56 mM KCl for 30 minutes compared with that of standard mixture (NE, norepinephrine; E, epinephrine). IS, internal standard (3,4-dihydroxybenzylamine) used for quantification of DA concentrations. (O and P) DA release and uptake values, respectively, of 4 independent cultures. In P, DAT-mediated specific DA uptake was calculated by subtracting nonspecific uptake (with nomifensine) from total uptake. *P < 0.01, **P < 0.05 vs. respective control, 2-tailed Student’s t test (O, left, and P) and 2-tailed paired t test (O, right).