Functional integration of dopaminergic neurons directly converted from mouse fibroblasts

Abstract

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of one somatic cell type to another by defined factors. However, it is not clear to what extent this type of reprogramming is able to generate fully functional differentiated cells. In addition, the activity of the reprogrammed cells in cell transplantation assays, such as those envisaged for cell-based therapy of Parkinson's disease (PD), remains to be determined. Here we show that ectopic expression of defined transcription factors in mouse tail tip fibroblasts is sufficient to induce Pitx3+ neurons that closely resemble midbrain dopaminergic (DA) neurons. In addition, transplantation of these induced DA (iDA) neurons alleviates symptoms in a mouse model of PD. Thus, iDA neurons generated from abundant somatic fibroblasts by direct lineage reprogramming hold promise for modeling neurodegenerative disease and for cell-based therapies of PD.

Figure 1. Direct conversion of fibroblasts into functional DA neurons

(a) Strategy for lineage reprogramming of iDA neurons from Pitx3-eGFP TTF. TTFs were transduced with lentiviral pools encoding 11 transcription factors followed by culture for 10 days in dox-containing N3 media. (b) Morphology and immunofluorescence for TH positive DA neuron like cells (red) in fibroblasts transduced 11 transcription factors (top left panel). No TH+/GFP+ signal in control fibroblasts lacking M2rtTA (top right panel). TH positive DA neuron like cells were detected in 11 factor infected Pitx3-eGFP fibroblasts (middle left panels), which are double-labeled with GFP (bottom panels). TH positive DA neuron like cells (red) were detected from TTFs derived from wild type mice (middle right panel). Scale bars=100μm. (c) Flow cytometry analysis for induction of eGFP+ cells from Pitx3-eGFP TTFs transduced 11 transcription factors (bottom panel). Control infection (top panel). (d) Induction of eGFP+ cells from Pitx3-eGFP TTFs by the ectopic expression of only two factors, Acsl1 and Pitx3. (e) Quantitative RT-PCR of the expression of DA-neuron marker genes on FACS purified Ascl1/Pitx3 induced eGFP+ and eGFP-cells. 10days after infection, the expression of DA neuron specific genes were significantly upregulated in eGFP+ cells. Data represent mean ± SEM; three independent experiments were performed; ANOVA test, *P < 0.05. (f) Immunostaining of iDA neurons for the mature neuronal and DA neuronal markers Tuj1, MAP2, DAT and AADC. Scale bars=100μm (g) FACS analysis for eGFP induction from Pitx3-eGFP TTFs transduced with 6 reprogramming factors after 4, 8, 12 and 18 days.

Figure 2. Functional characterizations of iDA neurons

(a) Gene expression profiling using quantitative RT-PCR analysis of neuronal, DA neuronal, ESC, and fibroblast marker gene expression in fibroblasts, NSC, 2- and 6- factor induced DA neurons and primary embryonic and adult midbrain DA neurons. Rows represent the evaluated genes and heat map represents the relative expression of genes as indicated. (b) Detection of dopamine from iDA neurons by reverse phase HPLC. The 6 factor infected cell cultures 15 days after viral transduction were analyzed and significant amount of DA and the DA derivative, 3,4dihydroxyphenylactic acid (DOPAC) were detected in the 6 factor induced DA neurons. (c-e) Electrophysiological properties of iDA neurons. (c) Representative recording of action potentials recorded from an iDA neuron. Bottom traces represent current injections (−20pA to +120pA), whereas top traces indicate voltage recordings. (d) Voltage-dependent membrane currents, depolarizing voltage steps elicited fast inward sodium currents (bottom traces, magnified inset) and slow inactivating outward potassium currents (top traces). (e) Effect of tetrodotoxin (TTX) on action potential of iDA neurons. Top panel: iDA neuron before TTX application. Bottom panel: same neuron after treatment with TTX. Depolarizing current injections ranged from −100 pA to +200pA in 10 mV steps. TTX completely inhibited the action potential evoked by depolarization current injections in iDA neurons. (f-h) Quantification of membrane properties in iDA neurons at 15days after infection. Numbers in the bars represent the numbers of recorded cells. Data are presented as mean ± s.e.m. RMP, resting membrane potentials; AP, action potential; Rin, membrane input resistances. (i) Amphetamine-induced (4mg/kg) rotational behaviors for 90 minutes in 6OHDA lesioned mice before the cell transplantation as well as 4 and 8 weeks after the transplantation of Pitx3-eGFP+ cells (about 50,000 cells) and control fibroblasts (sham controls) and primary embryonic midbrain Pitx3-eGFP+cells into the lesioned striatum. Transplantation of reprogrammed Pitx3-eGFP+ cells and primary embryonic Pitx3-eGFP+ cells led to a significant reduction in amphetamine-induced rotation scores in 6OHDA lesioned mice 8 weeks after transplantation. None of the intact controls (6OHDA lesioned, but did not receive cell transplants) or sham experiments (control fibroblasts) showed reduced rotation (n=12). Data represent mean ± SEM, ANOVA test, *P < 0.05. (j) Statistical analysis of amphetamine induced rotational behaviors 8 weeks after transplantation. Data represent mean ± SEM, ANOVA test, *P < 0.05. (k) Substantial graft-derived reinnervation of the lesioned striatum 8weeks after transplantation. FACS purified Pitx3-eGFP+ cells were sorted and transplanted into the striatum of 6-OHDA lesioned adult mice. The boxed area in k is shown at larger magnification left. Partial rescue of TH+ cells and fibers in 6-OHDA lesioned striatum is shown, and most of the TH+ neurons show a large size and elongated shape typical of midbrain DA neurons. (l) The grafted GFP+ cells co-expressed TH and other DA neurons maker, AADC. Scale bars=100μm (m) Total TH+ cells in the graft (n=5). 5 brain slices with 50um thickness around the lesioned site were counted. Data represent mean ± SEM, ANOVA test, *P < 0.05. (n) Summary of HPLC quantification of dopamine levels in both iDA neuron transplanted and control striatum. Data represent mean ± SEM, (n=5), ANOVA test, *P < 0.05.