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.

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% CO₂ 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^TM medium, and Naïve-like hES cells were cultured in 20% O₂, 5% CO₂ 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 10⁶ 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^+/- 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^+/- 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 et al., 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 et al., 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 MgCl₂ (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 H₂O 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 et al., 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; MgSO₄, 1; NaH₂PO₄, 1.25; NaHCO₃, 26; CaCl₂, 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% O₂ and 5% CO₂, at 34°C for 30 min, and then placed in ACSF without FFN102 and continuously bubbled with 95% O₂ and 5% CO₂, 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, CaCl₂ 2, MgCl₂ 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, Na₃GTP 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 (>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⁺ and K⁺ 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; MgSO₄, 1; NaH₂PO₄, 1.25; NaHCO₃, 26; CaCl₂, 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% O₂ and 5% CO₂, 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; Na₃-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; Na₃GTP, 0.5; Alexa Fluor 555, 0.05 (pH 7.2, 290-300 mOsmol/kg).

Action potentials and Na⁺/K⁺ 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

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 p values reported in the figure legends. Significance was assessed as p < 0.05.