Direct conversion of human fibroblasts to dopaminergic neurons

Abstract

Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy.

Fig. 1.

Establishment of hEF cultures. (A) Fibroblasts isolated from human embryos were dissociated and plated under standard fibroblast conditions. The fibroblasts were passaged once, frozen, and subsequently used for experiments. (B) Immunostaining confirmed that the resulting cultures were composed of cells expressing collagen I and III, confirming their fibroblast identity, and TE-7 confirmed their mesodermal origin.

Fig. 2.

Conversion of neurons from hEFs. (A) Neurons expressing βIII-tubulin (red) obtained by direct conversion of hEFs at 12, 20, and 24 d after Ascl1, Brn2, and Myt1l expression (ABM). Blue indicates DAPI counterstain. (B and C) Conversion efficiency estimation of hiN cell formation. (n = 5 for B and n = 2–4 for C.) (D and E) Immunocytochemical staining for MAP2 and synaptophysin (SYP) on the hEF-derived hiN cells. (F) Representative traces of membrane potential changes induced by current injection steps from −20 pA to +50 pA in 10-pA increments before (Left) and after (Right) TTX. (G) Representative traces of whole-cell currents induced by 10-mV depolarizing voltage steps from −60 mV to +10 mV before (Left; with inward Na⁺ current) and after (Right; blocked Na⁺ current) TTX. Insets show respective traces on an expanded scale. Bars represent average conversion efficiency from three to six separate experiments ± SD. (Scale bars: A, 50 μm; D and E, 100 μm.)

Fig. 3.

Requirements for hiN conversion. (A and B) Removal of doxycycline at day 7 resulted in neurons expressing βIII (green) being formed with same efficiency and without affecting the morphological complexity of the resulting hiN cells (n = 37 for d3, n = 68 for d7, n = 91 for d0). (C) hiN cells expressing βIII-tubulin (green) obtained by conversion of hEFs in the presence of different combinations of Ascl1, Brn2, and Myt1l (ABM, A, B, M, AB, AM, and BM). (D) Conversion efficiency estimation of hiN cell formation using different combinations of conversion factors. Bars represent average conversion efficiency from three to six separate experiments ± SD.

Fig. 4.

Conversion of neurons from fetal lung fibroblasts. (A) Neurons expressing βIII-tubulin (green) and MAP2 (red) obtained by direct conversion of human fetal lung fibroblasts (HFL1) at 12 and 20 d after transduction with Ascl1, Brn2, and Myt1l (ABM). (B) Representative traces of membrane potential changes induced by current injection steps from −20 pA to +50 pA in 10-pA increments (Left) and representative traces of whole-cell currents induced by 10-mV depolarizing voltage steps from −60 mV to +10 mV (Right). (C) Representative traces of trains of action potentials induced by step injection of depolarizing current (Right shows traces on an expanded scale).

Fig. 5.

Conversion of neurons from postnatal fibroblasts. (A) Neurons expressing βIII-tubulin (green) and MAP2 (red) obtained by direct conversion of hFFs at 12 and 20 d after transduction with Ascl1, Brn2, and Myt1l. (B) Representative trace of an action potential induced by depolarizing current injection (Left) and representative traces of whole-cell currents elicited in voltage–clamp mode by step depolarization (Right). (C) Representative trace of trains of action potentials induced by step injection of depolarizing current (20 pA) of hFF-derived iN cells, before and after TTX. (Scale bars: 50 μm.)

Fig. 6.

Generation of dopamine neurons via direct conversion. (A and B) GABAergic and glutamatergic expressing hiN cells obtained by conversion with Ascl1, Brn2, and Myt1l. (C and D) hiN cells expressing TH (green) and βIII-tubulin (red) obtained by conversion of hEFs using Ascl1, Brn2, and Myt1l in combination with LentiDA, containing 10 genes involved in midbrain patterning and dopamine neuron differentiation (C) or in combination with Lmx1a and FoxA2 (LF) (D). (E) hiN cells expressing TH (green) and βIII-tubulin (red) obtained by conversion of HFL1 cells using Ascl1, Brn2, and Myt1l in combination with Lmx1a and Foxa2 (ABM + LF). (F) Quantification of dopaminergic neurons. Each symbol represents values obtained from separate biological replicates. Solid black symbols indicate data obtained when Lmx1a and FoxA2 were delivered 3 d after ABM; all other data points are from simultaneous delivery of all factors. (G) TH-expressing neurons (red) did not express peripherin (green). (H and I) hiN cells positive for TH (red) and βIII-tubulin (green) coexpress AADC (blue) and Nurr1 (blue; arrowhead). (J) TH (red) and AADC (green) expression in hiN cells obtained by conversion in the presence of Lmx1a and FoxA2 (LF; Top), Lmx1a alone (L; Middle), or FoxA2 alone (F; Bottom). (K) Example of an ABM + LF hEF iN cell exhibiting spontaneous, pacemaker-like action potentials that were gradually blocked by the addition of TTX to the bath solution. (L) Representative trace of an action potential induced by depolarizing current injection. (M) Example of an ABM + LF hEF iN cell exhibiting rebound depolarizations at the offset of brief membrane hyperpolarizations. Insets show respective traces on an expanded scale. (Scale bars: 50 μm.)