DOI: 10.5061/dryad.1ns1rn91d
title: Interspecies chimerism with human embryonic stem cells generates functional human dopamine neurons at low efficiency

description: Interspecies chimeras offer great potential for regenerative medicine and
 creation of human disease models. Whether human pluripotent stem cell
 (hPSC) derived neurons in an interspecies chimera can differentiate into
 functional neurons and integrate into host neural circuity is not known.
 Here we show, using Engrailed 1 (En1) as a development niche that human
 naïve-like ES cells can incorporate into embryonic and adult mouse brains.
 Human-derived neurons including tyrosine hydroxylase (TH) positive neurons
 integrate into the mouse brain at low efficiency.  These
 TH-positive neurons have electrophysiologic properties consistent with
 their human origin. Additionally, these human-derived neurons in the mouse
 brain accumulate pathologic phosphorylated α-synuclein in response to
 α-synuclein preformed fibrils.  Optimization of human/mouse
 chimeras could be utilized to study human neuronal differentiation and
 human brain disorders.
description: Culture of Human ES Cells
 Human ES cell line RUES1 and RUES2
 were obtained from the WiCell Research Institute. The formulation of hESC
 medium is: DMEM/F12 supplemented with 20% knockout serum replacement
 (KSR), 1% GlutaMAX, 1% nonessential amino acids, 0.1 mM β-Mercaptoethanol,
 and 10 ng/ml bFGF. The primed hES cells were cultured on a feeder layer of
 irradiated mouse embryonic fibroblast (MEF) cells in hESC medium under 5%
 CO<sub>2</sub> at 37 °C. To maintain primed hESCs in an
 undifferentiated state, the irradiated feeder cells were freshly prepared
 one day before the passage. Primed hESCs were routinely passaged every 5-7
 days at a split ratio of 1:3 by a PBS wash followed by Collagenase
 IV.  hES cells labelling
 For chimeras, RUES1 or RUES2 were made
 to express a CAG-GFP or CAG-dsRed expression cassette by piggyBac
 transposon transposition. The CAG-GFP or CAG-dsRed plasmid (2.5 µg) and
 Transposase (2.5 µg) were transfected into ES cells using a nucleofector
 device (Lonza). Four days after transfection, cells were treated with
 Puromycin (350 µg/ml) during the first and second week.
 Puromycin-resistant hES cell colonies were manually picked using a pipette
 and transferred to a 12-well culture plate pre-coated with irradiated MEFs
 and cultured with hES cell medium. hES cells Naïve-like
 conversion RUES1 or RUES2 naïve-like conversion
 was usually conducted on day 3 after single cell passage of primed hESC to
 be added to the medium to promote hES cell proliferation. Naïve-like RUES1
 or RUES2 were cultured in RSeT<sup>TM</sup> medium, and
 Naïve-like hES cells were cultured in 20% O<sub>2</sub>, 5%
 CO<sub>2</sub> at 37 °C. Naïve-like hES cells were digested
 into single cells by Accutase for passage. Karyotype Analysis
 For karyotype analysis, on the day of
 sampling cultured hES cells reached 70% confluence. Colcemid solution
 (0.25 µg /ml) was added to the cultured medium and incubated for 4 hr.
 Cells were washed in PBS, dissociated into single cells, and spun down for
 5 min at 1200 rpm. The pellet was re-suspended in prewarmed 0.075 M KCl
 for 7 min at room temperature. After spinning and removing the hypotonic
 solution, freshly prepared 5 mL of ice-cold methanol: acetic acid (3:1,
 v/v) was added by gently pipetting and left at room temperature for 5 min.
 Fixation was repeated for an additional three times after centrifugation
 for 5 min at 1200 rpm. Finally, the pellet was resuspended and then
 dropped onto slides and stained with Giemsa. 
 Teratoma Assay
 Naïve-like human ES cells were
 collected by Accutase before injection. Approximately
 10<sup>6</sup> cells were resuspended in PBS supplemented with
 30% Matrigel and subcutaneously injected into immunodeficient NOD/SCID
 mice. After 4–5 weeks, teratomas developed and were removed before the
 tumor size exceeded 1.5 cm in diameter. The teratomas were then fixed by
 4% PFA and processed for hematoxylin and eosin
 staining. Blastocyst
 injection To produce embryos,
 En1<sup>+/-</sup> female mice (6-8 weeks of age) were
 super-ovulated by intraperitoneal injection of pregnant mare serum
 gonadotropin (PMSG, 7.5 IU each), followed 48 hr later by injection of
 human chorionic gonadotropin (hCG, 7.5 IU each), and then mated with a
 En1<sup>+/-</sup> male mouse. Vaginal plugs were checked the
 following morning. Blastocysts were collected at E3.5 by flushing the
 oviduct and the uterus and then cultured in KSOM
 medium.  Naïve-like hES cells (RUES1 or RURS2)
 were harvested for injection and placed on ice. The single cell suspension
 on ice was used for injection within 1 hr. Blastocyst injection was
 carried out by the Johns Hopkins Transgenic Mouse Core Facility. Forty to
 fifty blastocysts were injected with single naïve-like cells (8-12 per
 blastocyst), and then cultured in KSOM for at least 1 hr until the embryo
 transfer. To induce pseudo-pregnant females, 8-week-old CD1 female mice
 were mated with vasectomized male mice. Around 12 blastocysts were
 transferred into both uterine horns of female recipients at
 E2.5.  Tissue clearing and whole mount
 immunostaining Conceptuses were dissected at the
 different developmental stages, perfused using PBS and perfused with 4%
 PFA in PBS, then post-fixed in 4% PFA overnight at 4 °C.  For
 immunostaining, the following solutions were used, blocking solution: 5%
 donkey serum, 1% bovine serum albumin (BSA), 0.2% Triton X-100, 0.02%
 sodium azide in PBS; primary antibody solution containing primary
 antibodies in blocking solution; secondary antibody solution containing
 secondary antibodies also in blocking solution. Post-fixed embryos were
 washed in PBS 3 times, permeabilized in PBS with 0.5% Triton X-100 for 2
 hrs at room temperature, blocked in block solution for 2 hrs, and then
 incubated with primary antibodies at 4 °C overnight. Embryos were then
 washed with PBS containing 0.2% Tween-20 three times for 30 min each,
 incubated with Alexa Fluor-conjugated secondary antibodies solution with
 DAPI for 1–2 h at 37 °C. After 3 washes with PBS containing 0.2% Tween-20
 for 30 min each, samples were immersed into the tissue-clearing reagent
 for 1–2 days. A confocal or light sheet microscope was used to capture the
 images. Antibodies used are listed in Table S1.
 The entire mouse embryo or brain was
 subjected to tissue clearing using the CUBIC tissue-clearing protocol
 previously described (Susaki<em> et
 al.</em>, 2015). Optimized CUBIC-clearing and
 immunostaining protocols were used before confocal imaging. The clearing
 reagent was composed of 125 g of urea (25% by wt), 125 g of N, N, N´,
 N´-tetrakis (2-hydroxy-propyl) ethylenediamine (25% by wt), 75 g of Triton
 X-100 (15% by wt) and 175 g of distilled water (35% by wt).
 Three-dimensional (3D) reconstruction of confocal z-stacks was performed
 using IMARIS software (x64, v.9.0.2 Bitplane AG, Zürich, Switzerland).
 Background noise of fluorescent channel was minimized via the display
 adjustments panel.  Primary neuronal
 culture Primary cortical neurons or midbrain
 neurons were prepared from E15.5-18.5 human-mouse chimera embryos, and
 cultured in Neurobasal media supplemented with B27, 0.5 mM L-glutamine on
 24-well plate coated with poly-L-lysine. The neurons were maintained by
 exchanging half of the medium with fresh medium every 3–4 days later.
 Neurons cultured for 10–20 days were harvested for indirect
 immunofluorescence, DNA or mRNA extraction or were used for integration
 examination or electrophysiological recordings.
 Quantitative PCR
 analysis Total mRNA was isolated using an RNasy
 Mini Kit (Qiagen) or PicoPure RNA Extraction Kit for cultured hES cells,
 neuron or microdissected cells. RNA was subsequently quantified and
 genomic DNA removed. Complementary DNA was generated using TransScript
 First-Strand cDNA Synthesis Kit, PCR was conducted using SYBR Green qPCR
 Master Mix, and performed on a Vii A7 Quantitative PCR System (Applied
 Biosystem). The data were analyzed using ΔΔCT method and normalized with
 GAPDH or other endogenous control genes. Primers are listed in Table
 S2. Genomic PCR
 Genomic PCR was used to analyze the
 integration of human cells in mouse brain. Total genomic DNA of cells and
 embryos was extracted using DNeasy Blood & Tissue Kit. For
 detecting the integration using PCR, 100 ng of total DNA/sample was used,
 quantitative PCR for human cells corporation was performed using SYBR
 Green PCR Master Mix, genomic PCRs were conducted using DNA polymerase.
 The PCR product was purified and sequenced for confirmation. Primers are
 listed in Table S2. A standard curve was established by adding serial
 dilutions of human ES cells to approximate integrated cells (Preston
 Campbell<em> et
 al.</em>, 2015). To determine the genotypes of En1
 mutant mice, the tail tip was used for genomic DNA extraction. Reagents
 and protocol for the PCR were the same as described above. The primer
 sequences are listed in Table S2. PFF preparation and Stereotactic
 injection procedure Recombinant α-Syn protein and PFF
 preparation was performed according to published protocols (Kam et al.,
 2018; Mao et al., 2016). Before the injection, PFF was diluted in sterile
 cold PBS and placed on ice. All surgical procedures were performed
 using aseptic techniques. 3–5-month-old human-mouse chimeric mice were
 anesthetized with sodium pentobarbital. PFF (5 µg/side) was
 stereotactically delivered into the striatum using a 2 µl syringe. After
 making a midline incision of the scalp, a burr hole was drilled in the
 appropriate location for the striatum (+0.2 mm Medial-lateral; +2.0 mm
 antero-posterior and +3.0 mm dorso-ventral from the bregma). Bilateral
 Injections were performed at a rate of 0.1 µl/min, the needle was left in
 place for an additional 5 minutes after each injection, and then slowly
 withdrawn. Mice were monitored daily after surgery, and post-surgical care
 was provided. Histology
 Mice were euthanized with sodium
 pentobarbital, followed by intracardial perfusion with PBS and 4% PFA
 overnight. After cryoprotections with 30% sucrose solution, the brain was
 embedded in Tissue-Tek OCT solution and then sectioned with a Leica
 Cryostat to 10–40 μm thickness.  For double or triple immunofluorescent
 staining, sections were rinsed three times with PBS and mounted on the
 slide, incubated with PBS-0.5% Triton -100 (BST) for 15 mins, then
 incubated with PBS-0.1% Triton X-100 plus 0.1% sodium azide, 5% donkey
 serum plus 1% BSA and antibodies listed in Table S1 overnight in the dark.
 After rinsing 3 times with PBST, the sections were incubated with
 fluorescent-conjugated secondary antibodies for 2 h in the dark at 37 °C.
 After the sections were washed with PBST 3 times, the sections were
 covered with a coverslip using Prolong antifade mounting media containing
 DAPI. Images were acquired by using a ZEISS LSM 710 confocal
 microscope. Thioflavin S
 staining Before being processed for
 Thioflavin-S staining, The GFP-positive cells in coronal brain sections
 were stained using two-step antibody staining with a rabbit anti-GFP
 primary and anti-rabbit-Alexa Flour 594 conjugated
 secondary antibody. Sections were then incubated with 0.05% filtered ThS
 at room temperature in the dark for 15 seconds, followed by
 differentiation in 80% ethanol for 10 seconds and then washed with large
 volumes of distilled water. The nuclei were stained with DAPI, washed with
 PBS, and then mounted with a coverslip using Prolong antifade mounting
 media, Thioflavin-S staining of brain sections were scanned with a 488 nm
 laser. Laser capture microdissection, RNA
 extraction and preamplification Thin coronal sections (15 μm) were
 prepared in a cryostat at -20°C. Free-floating brain sections were stained
 with primary and secondary antibody, placed onto the membrane dish (Lumox
 Dish 35, hydrophilic), and placed into the dish holder of the microscope
 and cells were captured using the ZEISS PALM Microbeam Ultra-violet laser
 microdissection system. Cells were cut at 20 × magnification while keeping
 laser power to a minimum. Single neurons were dissected after double
 staining and directly collected in the cap of the collection tube
 (AdhesiveCap 500 opaque) containing a small volume of lysis buffer.
 Samples in collection tubes were stored at -80°C until further
 processing.  RNA from LCM dissected single neurons
 was extracted using the Arcturus PicoPure RNA Isolation Kit. Another 40 μl
 of extraction buffer was added into the tube after the frozen sample was
 thawed on ice, spun them down for 1 min and incubated for 30 min at 42 °C.
 The subsequent extraction steps followed the manufacturer’s instructions.
 Throughout the extraction period, all equipment as well as workbenches
 were cleaned with RNaseZAP.  All steps were conducted on ice unless
 otherwise specified. 6 μl RNA sample was placed into a
 0.2-ml thin-walled PCR tube followed by adding 1 μl of 10 mM dNTP mix and
 1 μl of 10 μM oligo dT. Samples were incubated at 72 °C for 3 min after
 quickly vortexing and spinning down the solution. Immediately afterward,
 samples were placed back on ice. 5.7 μl of RT mix containing: 0.25 μl
 RNAse inhibitor, 2 μl Superscript II first-strand buffer (5 ×), 0.5 μl
 100 mM DTT, 0.5 μl 200 U/μl SuperScript II reverse transcriptase, 2 μl 5 M
 betaine (Sigma-Aldrich), 0.06 μl 1 M MgCl<sub>2</sub>
 (Sigma-Aldrich), 0.1 μl 100 μM TSO and 0.29 μl nuclease-free water were
 added to the samples. The samples were spun down and incubated in a
 thermal cycler. The RT reaction protocol was as follows: 90 min at 42 °C,
 then 10 cycles of (2 min at 50 °C and 2 min at 42°), and finally 15 s at
 70 °C. After 1st-strand reverse transcription, cDNA pre-amplification was
 performed by combining and mixing with 12.5 μl of KAPA HiFi HotStart Ready
 mix (2 X), 0.2 μl of 10 μM ISPCR primers and 2.3 μl
 H<sub>2</sub>O and then cycling as follow: 3 min at 98 °C,
 then 20 cycles of (20 s at 98 °C, 15 s at 67 °C, 6 min at 72 °C), and
 finally 5 min at 72 °C. Pre-amplified cDNA (1 μl) was used to examine the
 marker gene expression by PCR. Primers are listed in Table
 S1. Fluorescent dye
 loading Fluorescent dopamine tracer FFN102 was
 used to trace human-derived dopamine neurons. The labeling protocol was
 followed as previously described with minor modifications
 (Rodriguez<em>
 et al.</em>, 2013). Briefly, the chimeric mice (12-16
 months old) were anesthetized deeply with isoflurane and decapitated. The
 brains were placed in ice-cold artificial cerebrospinal fluid (ACSF)
 containing (in mM): NaCl, 125; KCl, 2.5; MgSO<sub>4</sub>, 1;
 NaH<sub>2</sub>PO<sub>4</sub>, 1.25;
 NaHCO<sub>3</sub>, 26; CaCl<sub>2</sub>, 2; and
 D-glucose, 10. Acute transverse brain slices (100 μm) containing
 substantia nigra (SNc) were prepared using a vibratome (Leica VT1200S).
 Sections were incubated in ACSF containing FFN102 (10 μM) and continuously
 bubbled with 95% O<sub>2</sub> and 5%
 CO<sub>2</sub>, at 34°C for 30 min, and then placed in ACSF
 without FFN102 and continuously bubbled with 95% O<sub>2</sub>
 and 5% CO<sub>2</sub>, at 34°C for 30 min. Brain slices loaded
 with FFN102 were confirmed by immunostaining. 
 Electrophysiological
 recordings Neuronal culture
 recording Whole-cell patch-clamp recordings were
 performed using HEKA EPC10 amplifier (HEKA Elektronik, Lambrech, Germany).
 Cultured dopaminergic (DA) or cortical neurons from chimeric embryos were
 visualized under a 40 X water immersion objective by fluorescence and DIC
 optics (Carl Zeiss, Germany), and the chamber was constantly perfused at a
 rate of 1–2 ml/min at 32 °C with a bath solution containing (in mM): NaCl
 137, KCl 5, CaCl<sub>2</sub> 2, MgCl<sub>2</sub>
 1, HEPES 10, and glucose 10. The pH of bath solution was adjusted to 7.4
 with NaOH, and osmolarity was at 300–310 mOsmol/kg. Patch pipettes (4–10
 MΩ) were pulled from borosilicate glass (BF-150, Sutter Instruments,
 Novato, CA, USA) using a Flaming-Brown micropipette puller (P-1000, Sutter
 Instruments, Novato, CA, USA) and filled with a pipette solution
 containing (in mM): K-gluconate 126, KCl 8, HEPES 20, EGTA 0.2, NaCl 2,
 MgATP 3, Na<sub>3</sub>GTP 0.5, Alexa Fluor 647 (cortical
 neuron cultures) or 555 (DA neuron cultures) 0.05 (adjusted to pH 7.3 with
 KOH; adjusted to 290-300 mOsmol/kg with sucrose). Resting membrane
 potential (RMP) was recorded in current clamp mode at 0 pA immediately
 after establishing whole-cell configuration. Series resistance (Rseries)
 and input resistance (Rin) were calculated from a 5-mV pulse and monitored
 throughout the experiment. Unstable recordings (&gt;10% fluctuation of
 Rseries value) during experiments were rejected from further
 analysis. For voltage-clamp experiments, the
 membrane potential was typically held at –70 mV. A series of
 hyperpolarizing and depolarizing step currents were injected to measure
 intrinsic properties and to elicit action potentials. For
 voltage-dependent sodium and potassium currents, voltage steps ranging
 from –100 mV to +60 mV were delivered at 20-mV increments. Drugs were
 applied through a gravity-driven drug delivery system (VC-6, Warner
 Hamden, CT, USA). Data were acquired by PatchMaster software (HEKA
 Elektronik, Lambrech, Germany), sampled at 10 kHz, and filtered at 2.9
 kHz. Na<sup>+</sup> and K<sup>+</sup> currents and
 action potentials were analyzed using Clampfit 10.5 software (Molecular
 devices, Palo Alto, CA, USA).  After recording, immunocytochemistry
 was performed to label neurons injected with the fluorescent dye Alexa
 Fluor 647 or 555 to enable confirmation of the cell type and definition of
 the cell origination. Images were acquired using a LSM880 confocal
 laser-scanning microscope (Carl Zeiss). Brain slices
 recordings The experiments were performed in
 acute transverse brain slices containing substantia nigra (SNc) or
 hippocampus prepared from chimeric mice (12-16 weeks old). The mice were
 anesthetized deeply with isoflurane and decapitated. The brains were
 placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in
 mM): NaCl, 125; KCl, 2.5; MgSO<sub>4</sub>, 1;
 NaH<sub>2</sub>PO<sub>4</sub>, 1.25;
 NaHCO<sub>3</sub>, 26; CaCl<sub>2</sub>, 2; and
 D-glucose, 10. Slices (350 μm) were prepared using a vibratome (Leica
 VT1200S). Sections were incubated in ACSF and continuously bubbled with
 95% O<sub>2</sub> and 5% CO<sub>2</sub>, at 34°C
 for 60 min, and then at room temperature (22-24 °C) until use. A single
 slice was transferred into a submerged recording chamber and perfused
 constantly with carbogen-equilibrated ACSF at a rate of 2 ml/min.
 Recordings were performed at room temperature. 
 The patch electrodes had a resistance
 of 3–5 MΩ. For hippocampal slice recording, electrodes were filled with
 solution containing (in mM): Cs gluconate, 122.5; CsCl, 17.5; HEPES, 10;
 EGTA,0.2; NaCl, 8; Mg-ATP, 2; Na<sub>3</sub>-GTP, 0.3, Alexa
 Fluor 555, 0.05. The pH was adjusted to 7.2, and the osmolarity was
 adjusted to 290–300 mOsmol/kg. NMDA/AMPA EPSCs were recorded from CA1
 pyramidal cells upon stimulation of the Schaffer collateral fibers (0.1 ms
 rectangular pulses delivered each 10 s), in the presence of picrotoxin
 (100 μM). Stimulus intensity was set to evoke AMPA responses (at –90 mV)
 that were half the maximum. NMDA receptor-mediated EPSCs were isolated by
 blocking AMPA currents with
 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide
 (NBQX, 10 μM), raising the holding potential to +40 mV, and finally
 verified by elimination with D-2-amino-5-phosphonovaleric acid (D-APV, 50
 μM) superfusion. Ratiometric measurements of NMDA/AMPA currents were
 calculated from peak amplitudes at +40 and –90 mV. mEPSCs were recorded in
 gap-free mode for 5-10 min and analyzed by using the Mini Analysis Program
 (Synaptosoft) (amplitude threshold (15), area threshold (20), period to
 search local maximum (15,000 μs), time before peak for baseline (10,000
 μs), period to average baseline (10,000 μs), period to search decay time
 (5,000 μs), fraction to find decay (0.001)). For substantia nigra slice
 recording, electrodes were filled with solution containing (in mM):
 K-gluconate, 126; KCl, 8; HEPES, 20; EGTA, 0.2; NaCl, 2; MgATP, 3;
 Na<sub>3</sub>GTP, 0.5; Alexa Fluor 555, 0.05 (pH 7.2, 290-300
 mOsmol/kg). Action potentials and
 Na<sup>+</sup>/K<sup>+</sup> currents were
 analyzed using Clampfit 10.5 software (Molecular devices). During
 recording, holding current and series resistance were continuously
 monitored throughout the experiment, and if any varied significantly, the
 experiment was discarded. Neurons labelled with Alexa Fluor 555 were
 confirmed by immunohistochemistry after recording. Before imaging, brain
 slices were cleared using clarity method shown
 above. Quantification and statistical
 analysis<br> Data are represented as mean ± SEM.
 Statistical analysis was performed using GraphPad Prism 7 software
 (GraphPad Software, Inc.). We used the unpaired t test to calculate
 <em>p</em> values reported in the figure legends. Significance
 was assessed as <em>p</em> &lt;
 0.05.
description: # Interspecies chimerism with human embryonic stems cells generates
 functional human dopamine neurons at low efficiency
 **In this study, we used
 Engrailed 1 (En1) as a development niche that human naïve-like ES cells
 can incorporate into the embryonic and adult mouse brain. Human-derived
 neurons including tyrosine hydroxylase (TH) positive neurons integrate
 into the mouse brain at low efficiency.  These TH positive neurons have
 electrophysiologic properties consistent with their human origin.
 Additionally, these human-derived neurons in the mouse brain accumulate
 pathologic phosphorylated α-synuclein in response to α-synuclein preformed
 fibrils.**    **Description of the data and file structure** **Figure
 Files** 1.      Figure 1: Chimeric contribution of human ES RUES1-derived
 cells in En1 knockout mouse brain   a.      Figure 1a: Summary of
 blastocyst injections collected chimeric embryos and offspring b.     
 Figure 1b: Representative bright field image of mouse embryo at E10.5
 derived from blastocyst with RUES1 injection c.      Figure 1c:
 Representative integration of RUES1-derived cells into E10.5 embryonic
 midbrain (En1-/-) region d.      Figure 1d: Representative confocal
 immunofluorescence images showing the integration of RUES1-derived cells
 into an E10.5 embryo e.      Figure 1e: Quantification of GFP positive
 fluorescence intensity in the midbrain in En1+/+, En1+/- , En1-/-
 genotypes f.       Figure 1f: Representative images showing the
 integration of RUES1-derived cells in chimeric mouse forepaw at E10.5.
 Files include: hetero  injected paw-1 g.      Figure 1g: Flow cytometry
 analysis of DA neurons dissociated from chimeric embryos at E18 h.     
 Figure 1h: Representative images (bright view and GFP channel) of neuronal
 culture from E16 embryos 2.      Figure 2               : Chimeric
 contribution of human ES RUES1-derived cells in 23-day-old En1 knockout
 mouse brain a.      Figure 2a: Representative dorsal and ventral view of
 brain from chimeric mice. Hetero, KO and WT b.      Figure 2b:
 Representative horizontal and sagittal light sheet confocal images of
 chimeric mouse brain. WT and KO c.      Figure 2c: Representative
 immunofluorescence images showing the integration of RUES1-derived cells
 in 23-day-old chimeric brain 3.      Figure 3: Characterization of human
 ES RUES1-derived neurons in En1+/- mouse brain a.      Figure 3a:
 Representative images showing the GFP positive neuron in midbrain (E18
 mouse embryos derived from blastocyst injection of naïve-like RUES1-GFP)
 culture b.      Figure 3b: Quantification of GFP+ MAP2 or TH positive
 cells in neuronal culture c.      Figure 3c: Representative images showing
 the integration of RUES1-derived neurons in the SNc, SNR, RMC region of
 4-month-old chimeric mouse (En1+/-) d.      Figure 3d: Colocalization of
 GFP in TH positive neuron from chimeric mouse midbrain e.      Figure 3e:
 Summary of RUES1-GFP derived neurons from 3-month-old and 6-month-old
 EN1+/+ and EN1+/- chimeric mice f.       Figure 3f: Quantification of GFP
 and TH positive neurons in e 4.      Figure 4: Characterization of human
 ES RUES1-derived DA neurons in En1+/- mouse brain a.      Figure 4a: Laser
 capture microdissection (LCM) of GFP and TH positive and negative neurons
 in four-month-old chimeric mouse brain stained with anti-GFP and anti-TH
 antibodies b.      Figure 4b: PCR analysis of dopamine neuron related
 marker gene expression in neurons captured by LCM c.      Figure 4c:
 Representative images of RUES1-derived GFP+/TH+ neurons expressing
 dopamine transporter (DAT) d.      Figure 4d: Schematic experimental
 procedure of FFN102-based dopamine tracing e.      Figure 4e: Dopamine
 tracing in hES derived EN1+/- GFP+/TH+ cells using FFN102 f.       Figure
 4f: Quantification of FFN102 traced GFP+/TH+ cells 5.      Figure 5:
 Electrophysiological analysis of RUES1-derived neurons in neuronal culture
 and brain slice a.      Figure 5a: Post-immunostaining showing recorded
 neurons in neuronal culture. b.      Figure 5b: Electrophysiological
 properties of human DA neurons and human non-DA neurons c.      Figure 5c:
 Post-immunostaining showing recorded neurons in midbrain slice d.     
 Figure 5d: Electrophysiological properties of RUES1-derived DA neurons and
 RUES1-derived non-DA neurons in midbrain slice e.      Figure 5d: Summary
 of electrophysiological data from neuronal cultures and brain slices
 6.      Figure 6: RUES1-derived neurons can respond to α-syn PFFs a.     
 Figure 6a: Schematic diagram of mPFF or hPFF injection in RUES1-GFP
 En1+/-  chimeric mice b.      Figure 6b: Representative images of
 Thioflavin S (ThS) staining in SNc of En1+/-  chimeric mice c.      Figure
 6c: Representative images of pSyn positive immunostaining in SNc region of
 En1+/-  chimeric mice injected with mPFF or hPFF d.      Figure 6d:
 Quantification of α-Syn pathology in the midbrain of mPFF and hPFF
 injected En1+/-  chimeric mice e.      Figure 6e: Representative images of
 pSyn and Ubiquitin co-immunostaining in the SNc region of En1+/-  chimeric
 mice injected with mPFF or hPFF  **Supplementary Figure Files** S1.   S
 Figure 1: Human ES cell naïve-like state conversion and characterization
 a.      S Figure 1a: GFP-labelled human ES cell line RUES1 and RUES2
 naïve-like conversion b.      S Figure 1b: Naïve conversion of human ES
 cell lines H1 and RUES1-GFP in different conversion conditions c.      S
 Figure 1c: Quantification of colony thickness of RUES1-GFP cells cultured
 in RSeT medium and published cocktail medium d.      S Figure 1d:
 Morphology of RUES and RUES2 cultured in conventional HESM (primed) and
 RSeT medium (naïve-like) e.      S Figure 1e: In vivo teratoma formation
 of naïve-like RUES1 and RUES2 cells examined by H&E staining S2.   S
 Figure 2: Chimeric contribution of RUES1-derived cells in chimeric mouse
 brain a.      S Figure 2a: En1 embryo and pup generation summary from
 En1+/- matings b.      S Figure 2b: Representative images of 4% PFA fixed
 mouse embryo E12.5 before and after CUBIC clearing c.      S Figure 2c:
 Representative images of GFP and MAP2 staining in neuronal culture from
 E16 chimeric mouse embryonic brain (En1-/-). d.      S Figure 2d:
 Representative images showing the integration of RUES1-derived cells into
 E9.5 embryo (En1+/-). e.      S Figure 2e: Representative images showing
 the integration of RUES1-derived cells into E11.5 embryos f.       S
 Figure 2f: Representative images showing the integration of GFP positive
 RUES1-derived cells into E15.5 embryo (En-1+/-) forepaw g.      S Figure
 2g: Quantification of chimerism of RUES1 contribution to the chimeric
 embryonic brain examined by qPCR h.      S Figure 2h: PCR analysis of
 En1+/-chimeric embryonic neuronal culture i.       S Figure 2i:
 Representative confocal immunofluorescence images showing RUES1 and RUES2
 derived TH neurons in E15.5 En1+/- human-mouse chimera j.       S Figure
 2j: Representative images of neuronal marker genes expression in
 RUES1-derived neuron in the adult En1+/-  chimeric midbrain k.      S
 Figure 2k: Representative images of RUES1-derived glial cell in the adult
 En1+/-  chimeric brain l.       S Figure 2l: Representative images of
 RUES1-derived cells in the cerebellum of adult En1+/-  chimeric brain
 m.    S Figure 2m: Representative images of RUES1-derived cells in the
 brain stem of adult En1+/-  chimeric brain. LRt: lateral reticular nucleus
 n.      S Figure 2n: Representative images of RUES1-derived cells in the
 hippocampus region of adult En1+/-  chimeric brain S3.   S Figure 3:
 Characterization of neuronal cell types in chimeric brain a.      S Figure
 3a: Representative images showing the integration of GFP positive
 RUES1-derived cells in the region of raphe nuclei and tectum b.      S
 Figure 3b: Representative images showing other types of neuronal marker
 c.      S Figure 3c: Representative images showing dopaminergic neuron
 specific transcript factor d.      S Figure 3d: Representative images
 showing En1 expression in En1+/- mice with or without injection e.      S
 Figure 3e: Quantification of En1 expression by fluorescence intensity and
 positive number in total per section S4.   S Figure 4:
 Electrophysiological analysis in neuronal culture and brain slice a.     
 S Figure 4a: Representative images of recorded primary cortex neuronal
 cultures b.      S Figure 4b: Electrophysiological properties of hES
 derived cortical neurons from En1+/- human-mouse chimera c.      S Figure
 4c: Representative images of recorded hippocampal region d.      S Figure
 4d: Electrophysiological properties of RUES1-derived neurons in
 hippocampal slice recordings e.      S Figure 4e: Electrophysiological
 parameters from RUES1-derived neurons and mouse neurons S5.   S Figure 5:
 RUES1-derived neurons generate pathologic α-synuclein in response to
 administration of α-synuclein PFF a.      S Figure 5a: Representative
 images of bilateral injection in the striatum of 4-month-old En1+/-
 human-mouse chimeric mice b.      S Figure 5b: Representative images of
 anti-pSyn staining in the VTA and SNc region of the midbrain from En1+/-
 chimeric mice c.      S Figure 5c: Representative images of anti-pSyn
 co-stained with different neuronal marker in the midbrain region d.      S
 Figure 5d: Representative images of anti-pSyn co-stained with TH in the
 VTA and SNc from mPFF and hPFF injected chimeric mice  **Supplementary
 Movie Files** SM1.     Supplementary Movie 1. Light sheet imaging of GFP
 staining (horizontal view) of E9.5 En1-/- chimeric brain, related to Fig
 1C SM2.     Supplementary Movie 2. Light sheet imaging of GFP staining
 (sagittal view) of E9.5 En1-/- chimeric brain, related to Fig 1C