Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

Abstract

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

Figure 1

Successful conversion of human fibroblast into hiDA cells. (A) A schematic illustration of the protocol that we used to transform human fibroblasts into hiDA cells. (B) Top panel: Representative micrographs of hiDA cells stained with various DA neuron-specific (TH, DDC, DAT), general (Tuj1), and other types of neuron (serotonin and ChAT) antibodies. DAPI staining was used to identify individual cells. Lower panel: fraction of surviving cells (stained by DAPI) that stained positive for DDC. The scale bars represent 200 μm. The error bars represent SEM, n = 5. M: Mash1; MS: Mash1 + Sox2; MN: Mash1 + Ngn2; MSN: Mash1 + Sox2 + Ngn2; MSNN: Mash1 + Sox2 + Ngn2 + Nurr1; MSNNP: Mash1 + Sox2 + Ngn2 + Nurr1 + Pitx3. (C) Lack of proliferation in IMR90 fibroblast cells transduced with five factors. Top panel, growth curve of 5F-transduced (IMR90 + 5F) cells; Middle panel, fraction of cells labeled with BrdU when it was added at different times after transduction of 5F; Lower panel: photomicrographs of BrdU staining at day 0 and day 3 after gene transduction. The scale bars represent 200 μm. (D) Q-PCR analysis of key DA-neuron-specific gene expression (left panel). The right panel shows semi-quantitative PCR analysis of the gene. PCR products were electrophoresed in an agarose gel and stained with EtBr. Error bars represent SEM, n = 3.

Figure 2

Dopamine release and uptake of fibroblast-derived DA neuron-like cells. (A) Dopamine uptake in putative DA-neuron cells. Cells in 12-well plates were incubated for 10 min with 50 nM [³H]Dopamin in the absence and presence of 0.1 mM cocaine. Cells were then washed in ice-cold KRH buffer, lysed, and radioactivity was determined by a scintillation counter. Specific uptake of dopamine was calculated as the difference between measurement with and without 0.1 mM cocaine. Error bars represent SEM, n = 3. (B) Dopamine production from putative DA-neuron cells. Left panel are representative HPLC chromatograms showing the peaks that were detected for 10 nM DHBA and DA standard solutions (a), DHBA internal standard and endogenous DA in neurons (b), and only DHBA internal standard in fibroblasts (c). Samples were injected at time 0, and retention times of the standards were used to identify DHBA and DA in the tissue samples. Right panel shows quantitative results of HPLC chromatography experiments measuring DA from DA-neuron like cells. Error bars represent SEM, n = 5.

Figure 3

Electrophysiological profiles of putative hiDA neurons. (A) Whole-cell membrane currents from a representative DIV12 cell held at an initial holding potential of −70 mV was first hyperpolarized to −100 mV before successive 10 mV steps were delivered (average of 5 repetitions at a rate of 1 Hz). Fast, rapidly inactivating inward current, detailed in inset, was followed by a non-inactivating outward current. Fast inward currents were quantified from another cell (DIV11), isolated with cesium-based electrode solution to demonstrate expected E_Na+ of +83.5 mV and were completely blocked by TTX (1 μM). Symbols ± SEM bars represent the mean of y-axis currents (pA) in the presence and absence of TTX. (B) Current clamp recording in which current injections maintained a starting voltage of −70 mV, followed by a hyperpolarizing current step (−40 pA) and successive 30 pA steps. In response, a typical DIV13 cell demonstrated fast, rapidly inactivating voltage spikes upon depolarizing current injections, which were also sensitive to TTX (1 μM). These are typical of DA neuron action potentials. (C) Overlay of two consecutive whole-cell recording events shown in a representative DIV21 cell before and after application of the D2-receptor selective antagonist (raclopride, 1 μM). Action potentials resulted after a hyperpolarizing step (−40 pA) in the presence of the antagonist. (D) In the every cell assessed, the membrane potential was significantly depolarized in the presence of raclopride (5 cells, mean ± SEM (mV) *P < 0.05, **P < 0.001, paired Student's t-test) compared to baseline control. The input resistance similarly increased in the presence of raclopride for each cell, suggesting D2-autoreceptor antagonism (5 cells, mean ± SEM (MΩ) *P < 0.05, **P < 0.001, paired Student's t-test, input resistance baseline control versus raclopride for each cell).

Figure 4

In vivo cell transplantation studies with reprogrammed DA neuron-like cells. (A) About 3 × 10⁵ reprogrammed cells (12 days post infection) were injected into the middle of striatum of rats to the side with 6-OHDA-induced lesions with a Hamilton syringe. Right before and at different times after cell transplant, rats were evaluated for amphetamine-induced rotational behavior with a rotometer. Error bars represent SEM, n = 8. (B) Rotational behaviors of four 6-OHDA-lesioned rats injected with hiDA cells. (C) Brain tissues from the two rats that showed consistent correction of amphetamine-induced rotational behaviors were examined for evidence of transplanted cells through immunoflurescenece. HN: staining for human nuclear protein, which is an indicator of injected human cells. Scale bars represent 200 μm.