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1 In the format provided by the authors and unedited. Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson s disease therapy Junsang Yoo 1, Euiyeon Lee 2, Hee young Kim 3, Dong-ho Youn 5, Junghyun Jung 4, Hongwon Kim 1, Yujung Chang 1, Wonwoong Lee 6, Jaein Shin 1, Soonbong Baek 1, Wonhee Jang 4, Won Jun 4, Soochan Kim 7, Jongki Hong 6, Hi-Joon Park 8, Christopher J. Lengner 9, Sang Hyun Moh 10, Youngeun Kwon 2, Jongpil Kim 1 *. NATURE NANOTECHNOLOGY 1

2 Supplementary Figures. Supplementary Figure 1. (a) Schematic illustrations showing fabrication of RGD AuNPs for magnetization by EMF exposure. RGD AuNPs were prepared via sequential ligand exchange of the mpeg350-sh and RGD peptide. (b) Analysis of the NATURE NANOTECHNOLOGY 2

3 RGD peptide. Thiolated RGD peptide (*) was analyzed by reverse phase HPLC and electron spray ionization mass spectrometry. (c) The expected mass was Da and the observed mass was Da. (d) The UV-vis spectrum of 20-nm AuNPs was observed before (black) and after (blue) conjugation to the RGD peptide. AuNPs were conjugated to the RGD peptide, forming RGD AuNPs via sequential ligand exchange of the mpeg350-sh thiol ligand and RGD peptide. (e) The size distribution of 20-nm AuNPs before (black) and after (blue) ligand exchange was monitored by dynamic light scattering (DLS). The hydrodynamic size of AuNPs increased from 23.3 ± 3.2 nm to 35.6 ± 8.8 nm by the addition of the RGD peptide. (f) Intensities of EMF induced by a controllable EMF generator in the cell culture incubator. (g) Calculation of magnetic flux images under the EMF generator. The red color indicates a specific strength of magnetic flux. (h) Picture of the EMF generator in the cell culture incubator. EMF intensities and frequencies are controllable through the main computer. (i) Immunofluorescence of GFP expression by transient transfection in control fibroblast and EMF-exposed AuNPs magnetization condition (Scale bar = 50 μm). (j) Quantification of (I). Three independent experiments with three replicates each were performed. Data presented as mean ± SEM. (k) qrt-pcr analysis of exogenously expressed reprogramming factor Ascl1 in control mouse fibroblasts and Ascl1- transfected fibroblasts with and without EMF induced magnetized AuNPs condition. Data presented as mean ± SEM. (l) Magnetization experiments of gold nanoparticles without EMF exposure by the VSM magnetometer. NATURE NANOTECHNOLOGY 3

4 Supplementary Figure 2. (a) Cell viability test by MTT assay at different frequency and intensity of EMF exposure. Each bar represents different frequencies of EMF at the tested intensities. Data presented as mean ± SEM. (b) Immunofluorescence of DA NATURE NANOTECHNOLOGY 4

5 neuronal markers including TH and TuJ1 at the different intensities of EMF after APLN transfection (Scale bar = 50 μm). (c) qrt-pcr analysis of neuronal markers including Tuj1, Map2, Gad67, and synapsin at different EMF settings. Data presented as mean ± SEM; Student s t-test, *p < Three independent experiments with three replicates each were performed. (d) Representative trace of action potentials in EMF induced ida neurons. (e) qrt-pcr analysis of marker genes for liver, muscle, osteoblasts, and neurons in APLN-transfected fibroblasts following EMF exposure. Data presented as mean ± SEM; Student s t-test, *p < 0.05, **p < Three independent experiments with three replicates each were performed. (f) Heat map representing the global gene expression pattern of control fibroblasts, APLNtransfected fibroblasts (Control), APLN-transfected fibroblasts on magnetized AuNPs with EMF exposure (EMF + AuNPs), and primary dopaminergic neurons. NATURE NANOTECHNOLOGY 5

6 Supplementary Figure 3. (a) Representative voltage-dependent membrane currents of control APLN transiently transfected cultures (b) Quantification of membrane properties of primary DA neurons and ida neurons in EMF induced magnetized AuNPs condition (Data represent mean ± SEM, *p < 0.05, n = 12 for each). AP, action potential; RMP, resting membrane potential; Rin, input resistance. (c) qrt-pcr analysis of the DA neuronal markers Synapsin and Vmat2 at 10 days after transfection NATURE NANOTECHNOLOGY 6

7 on EMF induced magnetized AuNPs substrates. Expression levels were normalized to Gapdh. Data represent mean ± SEM; ANOVA, **p < (d) qrt-pcr analysis of neuronal markers, Map2 and synapsin, in ins 10 days after direct lineage reprogramming with one transcription factor or the small molecule combination CFV under the magnetized AuNPs condition. Expression levels were normalized to Gapdh. Data presented as mean ± SEM; ANOVA test, **p < (e) Immunofluorescence staining for expression of the mature neuronal markers TuJ1, Map2, TH and DAT in induced neurons (ins) from fibroblasts transfected with only one factor, Ascl1, Pitx3 or treated with three small molecules: CHIR99021, forskolin, and valproic acid (CFV) on EMF induced magnetized AuNPs substrates (Scale bars = 50μm). (f) Number of TuJ1+ and Map2+ ins generated by EMF-induced magnetized AuNPs. Three independent experiments for each group were performed. Data presented as mean Data presented as mean ± SEM; Student s t-test, *p < NATURE NANOTECHNOLOGY 7

8 Supplementary Figure 4. (a) Numbers of EMF- and chemical-induced neurons on different days following EMF treatment. Data represent mean ± SEM. (b) Numbers of EMF-, Pitx3 and chemical-induced neurons on 10 days after factor transfection. Data present mean ± SEM; Student s t-test, **p < (c) qrt-pcr analysis of neuronal genes including Map2, Tuj1, Gad67, and synapsin 15 days after CFV treatment in the presence and absence of EMF + AuNPs. Data present mean ± SEM; ANOVA test, *p < 0.05, **p < (d) qrt-pcr analysis of dopaminergic (DA) neuronal genes Aadc1, Th and Dat at 15 days after chemical induced reprogramming with and without magnetized AuNP with EMF exposure. Up-regulated expression levels of dopaminergic neuronal markers, Aadc1, Th and Dat are observed in Pitx3 transfected NATURE NANOTECHNOLOGY 8

9 batch. Three independent experiments for each group were performed. Data presented as mean ± SEM; ANOVA test, *p < NATURE NANOTECHNOLOGY 9

10 Supplementary Figure 5. (a) qrt-pcr analysis of histone acetylation complex genes, Smarca2, Gcn5 and Tbp, in EMF-exposed fibroblasts growing on AuNPs. Data presented as mean ± SEM; Student s t-test, **p < Three independent experiments with replicates each were NATURE NANOTECHNOLOGY 10

11 performed. (b) Western blots of modified histones H4K12ac, H3K27ac, H3K4me3 and H3K27me3 in control and EMF treated fibroblasts. (c) Quantification of Western blots from (B). Data presented as mean ± SEM; ANOVA test, *p < (d) Immunostaining of H3K27ac and H3K4me3 on fibroblasts under EMF exposed AuNPs (left panel) and quantification of the immunostaining (right panel) (Scale bars = 100μm). Data presented as mean ± SEM; Student s t-test, **p < (e) qrt-pcr expression analysis of various histone modifiers following EMF induced direct lineage reprogramming. Data presented as mean ± SEM; ANOVA test, **p < NATURE NANOTECHNOLOGY 11

12 Supplementary Figure 6. (a) Electromagnetized AuNPs induced H3K27ac peak distribution based on peak calling in mouse genome. (b) Distance heat map of microarray data of primary DA neurons, Electromagnetized AuNPs induced ida neurons, control ida neurons and control fibroblasts. (c) Venn diagram showing the overlap between upregulated genes from microarrays and genes occupied by ChIP- Seq. (d) Boxplot illustrating the gene expression pattern distribution of the clusters NATURE NANOTECHNOLOGY 12

13 based on main Fig. 3D. The yellow dotted line shows the average of median values in each cluster. The p values were calculated by ANOVA followed by Tukey s test (***P < 0.01). (e, f, and g) Gene ontology (GO) analysis of differentially expressed genes following direct lineage reprogramming into ida neurons. Many genes related to acetylation and the neuronal phenotype were differentially expressed. (h) H3K27ac accumulation in neuronal genes NeuroD1 and NeuN on EMF induced magnetized AuNPs compared to controls. (i) Genome browser track showing H3K27ac occupancy near the transcription start site (TSS) of neuronal genes, Nf1, Med and Hpn. (j) ChIPqPCR analysis of H4K12ac at the TH promoter region in control fibroblasts and EMFexposed fibroblasts 10 days after APLN transfection. Data presented as mean ± SEM; Student s t-test, *p < 0.05, **p < NATURE NANOTECHNOLOGY 13

14 Supplementary Figure 7. (a) Number of TH+ ida neurons at 2, 4, 6, 8, 10, 12 and 14 days after Brd2 knockdown. Data presented as mean ± SEM. (b) Immunofluorescence NATURE NANOTECHNOLOGY 14

15 images of histone modification marker H3K27ac in control fibroblasts, EMF exposed fibroblasts, and ANA treated fibroblasts 10 days after APLN transfection (Scale bars = 50μm). (c) Western blot analysis of H4K12ac, H3K27ac, and H3K27me3 expression levels by EMF induced idns with and without prior Brd2 knockdown. (d) qrt-pcr analysis of the endogenous dopaminergic neuronal markers 10 days after APLN transfection in the presence and absence of shbrd2. Expression levels are normalized to Gapdh. Data presented as mean ± SEM; Student s t-test, *p < 0.05, **p < (e) ChIP-qPCR analysis of H3K27ac at the TH promoter locus in control fibroblasts and EMF-exposed fibroblasts with and without Brd2 knockdown at 10 days after APLN transfection. Data presented as mean ± SEM; Student s t-test, **p < (f) qrt- PCR analysis of the endogenous dopaminergic neuron markers Map2, Tuj1 and synapsin 10 days after APLN transfection and anacardic acid (ANA) treatment. Expression levels are normalized to Gapdh. Data presented as mean ± SEM; Student s t-test, *p < 0.05, **p < NATURE NANOTECHNOLOGY 15

16 Supplementary Figure 8. (a) Immunofluorescence images of the DA neuronal markers Vmat2 and TuJ1 in control fibroblasts and APLN-transfected ida neurons with and without Brd2 knockdown (Scale bars = 200μm). (b) Number of Vmat2+ and TuJ1+ cells in control fibroblast cultures with and without Brd2 knockdown. Data presented as mean ± SEM. NATURE NANOTECHNOLOGY 16

17 (c) qrt-pcr analysis of neuronal markers Map2, Tuj1, Gad67, and synapsin following EMF-AuNPs and chemical-induced direct lineage reprogramming. Data presented as mean ± SEM; ANOVA test, *p < 0.05 and **p < (d) Immunofluorescence of DA neuronal markers TH and TuJ1 in the anacardic acid (ANA) treatment condition (Scale bars = 100μm). (e) Number of TH+ and TuJ1+ cells following EMF- and chemicalinduced direct lineage reprogramming under the ANA treatment condition. Data presented as mean ± SEM; Student s t-test, **p < (f) Immunofluorescence images of histone modification marker H4K12ac in control fibroblasts, EMF and magnetized AuNPs treated fibroblasts, and EMF with magnetized AuNPs treated fibroblasts with Brd2 knockdown 10 days after APLN transfection(scale bars = 200μm). (g) Immunofluorescence images of histone modification marker H3K27ac in control fibroblasts, EMF with magnetized AuNPs treated fibroblasts, and EMF and magnetized AuNPs treated fibroblasts with Brd2 knockdown 10 days after APLN transfection(scale bars = 200μm). (h) Relative fluorescence intensities from (f) and (g). Data presented as mean ± SEM; Student s t-test, **p < (i) Immunofluorescence images of the histone modification marker H4K12ac in CFV-treated, EMF with magnetized AuNPs treated, and EMF with magnetized AuNPs treated / Brd2 knockdown fibroblasts (Scale bars = 250μm). (j) Immunofluorescence images of the histone modification marker H3K27ac in CFV-treated, EMF with magnetized AuNPs treated, and EMF with magnetized AuNPs treated with and without Brd2 knockdown(scale bars = 250μm). (k) Relative fluorescence intensity results from (i) and (j). Data presented as mean ± SEM; Student s t-test, **p < NATURE NANOTECHNOLOGY 17

18 Supplementary Figure 9. (a) Number of immno-positive cells in the condition of EMF only, AuNPs only and EMF + AuNPs. (b) Immunofluorescence images of TUJ1+ and MAP2+ human ida (hida) neurons reprogrammed on magnetized AuNPs under EMF exposure with and without Brd2 knockdown (Scale bars = 50μm). (c) Numbers of TUJ1+, TH+, VMAT2+, and DAT+ hida neurons with and without Brd2 knockdown. Data presented as mean ± SEM; ANOVA, *p < Three independent experiments with three replicates were performed. (d) Immunofluorescence analysis of histone modification marker H3K27AC expression in control, EMF-exposed, and EMF- NATURE NANOTECHNOLOGY 18

19 exposed BRD2-knockdown human fibroblasts 10 days after APLN transfection (Scale bars = 50μm). (e) Quantitative analysis of H3K27AC and H4K12AC immunofluorescence levels in EMF-exposed hida neurons. Three independent experiments with three replicates were performed. Data are presented as mean ± SEM; ANOVA test, *p < 0.05, **p < (f) Dopamine measurement of KCl induced dopamine release in EMF induced human ida neurons by LC-MS. Data are presented as mean ± SEM; Student s- test, **p < NATURE NANOTECHNOLOGY 19

20 Supplementary Figure 10. (a) Representative action potentials in electromagnetized AuNPs induced human ida neurons. (b) Quantification of membrane properties of human ida neurons generated under the EMF induced magnetized AuNPs condition (data represent mean ± SEM, Student-t test **p < 0.01, n = 10 for each). AP, action potential; RMP, resting membrane potential. (c) Quantification of TUJ1 positive ida neurons per 10mm 2 surface area in the Brd2 Knockdown condition. Data presented as mean ± SEM; Student s t-test, **p < (d) Western blot analysis of BRD2 and H3K27AC expression levels in hida neurons in the presence and absence of EMF+AuNPs. NATURE NANOTECHNOLOGY 20

21 Three independent experiments with three replicates were performed. (e) Western blot analysis for BRD2, H3K27AC, and H3K27me3 expression levels in EMF induced magnetized AuNPs with and without BRD2 knockdown. Protein levels were normalized to β-actin. Three independent experiments with three replicates were performed. NATURE NANOTECHNOLOGY 21

22 Supplementary Figure 11. (a) Number of EMF-AuNPs induced TH+ human ida neurons at different time points. Data represent mean ± SEM. (b) Immunofluorescence images of the DA neuronal markers VMAT2 and NEUN in human blood-derived hida neurons (Scale bars = 50μm). (c) qrt-pcr analysis of neuronal and blood markers in EMF mediated human blood-derived hida neurons. Data presented as mean ± SEM; Student s t- test, *p < 0.05, **p < (d) LC-MS analysis for DA measurement in EMF mediated human blood-derived hida neurons. (Data represent mean ± SEM, Student-t test **p < 0.01) NATURE NANOTECHNOLOGY 22

23 Supplementary Figure 12. (a) A picture of the EMF system for in vivo direct lineage reprogramming. EMF treatment is implemented every 3 hours per day for 4 weeks. (b) Number of DAB-TH+ cells in MPTP model mouse striatum with and without EMF treatment. Five brains slices with 40 μm thickness around striatum region were counted. Data presented as mean ± SEM; Student s t-test, **p < (n=7 mice for each group) (c) Movement of total distances and rearing rates from MPTP induced PD mouse model in the absence NATURE NANOTECHNOLOGY 23

24 and presence of EMF with magnetized AuNPs. (n=7 mice for each group). Two independent experiments of six sets each were performed. Data presented as mean ± SEM; Student s t-test, **p < (d) Apomorphine-induced (4 mg/kg) rotational behaviors over 60 min in 6-OHDA-lesioned mice with and without EMF exposure. Line graph represents the amount of rotation numbers. 6-OHDA-lesioned mice were used as shams and 6-OHDA lesioned mice injected with egfp lentivirus vector as mocks. 6-OHDA lesioned mice infected with APLN virus indicated as control and 6-OHDA lesioned mice with only AuNPs injection or only EMF exposure shown as AuNPs and EMF, respectively. EMF+AuNPs represents 6OHDA lesioned mice with EMF treated with magnetized AuNPs. We used 2 test groups (T1 EMF + AuNPs and T2 EMF + AuNPs) to derive this data. 20 mice were sacrificed for each test group. Data presented as mean ± SEM. (e) Immunohistochemistry of TH-positive (DAB) in vivo reprogrammed ida neurons by magnetized AuNPs in 6-OHDA-lesioned mice (Scale bars = 1mm). (f) Numbers of TH+ and DAT+ cells in striatum of 6-OHDAlesioned mice and EMF-treated 6-OHDA-lesioned mice. Five brains slices with 40 μm thickness around the lesioned site were counted. Data presented as means ± SEM; Student s t-test, **p < NATURE NANOTECHNOLOGY 24

25 Supplementary Figure 13. (a) Border distance of control MPTP induced PD model mice and EMF with magnetized AuNPs treated model mice in the border area of the open field (n=7mice for each group). Data presented as mean ± SEM; ANOVA test, *p < (b) NATURE NANOTECHNOLOGY 25

26 Immunohistochemistry of TH in MPTP (30mg/kg) treated mouse striatum without EMF treatment. WT-control (no MPTP), MPTP control (with MPTP) (Scale bars = 1mm). (c) Numbers of DA neuronal markers Map2+, TuJ1+, Th+, Vmat2+ and Pitx3+ in the MPTP treated striatum with and without EMF-AuNPs. Five brains slices with 40 μm thickness around striatum region were counted. (n=7 mice for each group). Data presented as mean ± SEM; ANOVA test, *p < 0.05 and **p < (d) qrt-pcr analysis of endogenous DA neuronal markers including Vmat2, Th, Map2 and DAT in the EMF treated MPTP PD model mouse striatum. Expression levels are normalized to Gapdh. Data present mean ± SEM; Student s t-test, *p < 0.05, **p < (e) Total travel distance and rearing rate of MPTP induced PD model mice and EMF+AuNPs treated PD model mice in the open field test at different time points (2 weeks, 3 weeks and 6 weeks). Three independent experiments of each group were performed (n=7 mice for each group). Data presented as mean ± SEM. (f) Example traces of action potentials of EMF induced in vivo reprogrammed ida neurons. (g) Quantification of apomorphine-induced rotational behavior over 60 min in 6-OHDA-lesioned mice 8 weeks after EMF exposure. Data presented as mean ± SEM; ANOVA test, *p < (n=7 mice for each group). (h) Immunofluorescence images of DA neuronal markers Dat and Th in control and EMF-AuNPs treated 6OHDA-lesioned mice (Scale bar = 100 μm). (i) Immunostaining of Pitx3 and Nurr1 in EMF induced in vivo converted ida neurons at 8-week. Transgenic mice GFAP-Cre that express Cre recombinase under the control of the GFAP promoter were injected with the flip-excision (FLEX) reporter (Flex-Synapsin:GFP) along with APNL factors and then these mice were exposed to the EMF induced AuNPs environment (Scale bar = 50 μm). NATURE NANOTECHNOLOGY 26

27 Supplementary table Supplementary table 1. GO annotation of the CellNet database. Gene set * in Gene count GO annotation (p-value) CellNet Neuron subnet_1 921 Synaptic transmission (1.49e-119) Neurotransmitter transport (7.48e-68) Regulation of transmission of nerve impulse (2.14e-47) Neuron-neuron synaptic transmission (2.25e-44) Glutamate receptor signaling pathway (2.37e-43) Neuron subnet_2 306 Synaptic transmission (1.44e-29) Neurotransmitter transport (1.1e-15) Cell morphogenesis involved in differentiation (1.86e- 12) Regulation of microtubule polymerization (2.33e-10) Axon guidance (2.43e-10) Fibroblasts 229 M phase (2.83e-240) subnet_2 Cell division (1.67e-221) Chromosome segregation (9.73e-144) DNA-dependent DNA replication (6.43e-118) DNA replication (1.31e-100) ESC subnet_1 928 M phase (1.72e-160) DNA replication (1.39e-124) Cell division (1.46e-121) Chromosome segregation (5.29e-109) DNA-dependent DNA replication (2.06e-86) NATURE NANOTECHNOLOGY 27