Regimen of follicular stimulation
Female rhesus monkeys exhibiting regular menstrual cycles were induced with exogenous gonadotropins [29
]. The expression of monkey endogenous gonadotropins was down regulated at the beginning of mensis (day one to day two) by daily subcutaneous injections of Gonadotropin-releasing hormone (GnRH) antagonist (Antide; Ares Serono, 0.5 mg/kg body weight) for six days and by twice daily injection of recombinant human follicle-stimulating hormone [r-FSH: Organon Inc. 30 IU, intramuscular injection (i.m.)] concurrently. This was followed by the injection of r-FSH + recombinant human luteinizing hormone (r-hLH; Ares Serono; 30 IU each, i.m., twice daily) on the last three days. Ultrasonography was performed on day seven of the stimulation to confirm follicular responses. An i.m. injection of 1,000 IU recombinant human chorionic gonadotropin (r-hCG; Ares Serono,) was administered for ovulation induction when there were follicles at 3-4 mm in diameter. In general, r-hCG was administered at approximately 37 hours prior to oocyte retrieval for optimal maturation of metaphase II arrested oocytes.
In vitro Maturation (IVM)
Oocytes were matured in modified CMRL-1066 containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT) supplemented with 40 μg/mL Sodium pyruvate, 150 μg/mL Glutamine, 550 μg/mL Calcium lactate, 100 ng/ml estradiol and 3 ug/ml of Progesterone for up to 36 hours in 35-μl drops of medium under mineral oil at 37°C with 5% CO2, 5% O2 and 90% N2.
Generation of transgenic HD monkeys
High titer lentiviruses carryiing (1) exon 1 of human htt
gene with 84 CAG repeats and (2) green fluorescent protein (GFP
) gene under the regulation of human polyubiquitin C promoter, were injected into the PVS of metaphase II (MII) arrested monkey oocytes followed by intracytoplasmic sperm injection (ICSI) [10
]. The resultant embryos were transferred into surrogate females for the generation of transgenic monkeys. Transgenic status was confirmed by PCR.
Characterization and preparation of donor skin cells
Donor skin cells were primary cultures of skin tissue derived from miscarried transgenic HD monkey (rHD) at four months of gestation. The transgenic status of the skin cells was confirmed by PCR, immunostaining and Western analysis [10
Production of transgenic HD monkey tetraploid embryos
MII arrested oocytes were placed in TL-HEPES [31
] with 5 μg/ml of cytochalasin B (Sigma) for 15 minutes. The 1st
polar body (PB) was gently squeezed out through a small slit at the zona pellucida (ZP). After thorough washes of the oocytes, skin cell was placed under the ZP. The couplet was fused by electrofusion using fusion electrodes in 0.3 M Manitol fusion medium (two direct currents, 30 volts 30 μsec; Electro cell fusion system LF-101, Nepa Gene Company). The reconstructed embryos were cultured in medium supplemented with 50 nM trichostatin A (TSA; Sigma) for 10-12 hours. Two hours after fusion, the reconstructed embryos were activated by 5 μM Ionomycin for five minutes and then incubated in 2 mM 6-Dimethylaminopurine (6-DMAP; Sigma) for five hours at 37°C with 5% CO2
, 5% O2
, 90% N2
. The reconstructed embryos were further cultured in HECM 9 medium for eight days with 5% FBS added on Day two of culture. Fresh medium was replaced every two days.
Establishment and maintenance of Huntington's monkey ES cells from tetraploid blastocyst
Tetraploid blastocysts were cultured for ten to 14 days until attached onto MFFs to form an outgrowth. The outgrowths, the exhibited prominent stem cell morphology, were mechanically removed, transferred onto freshly prepared MFFs and continued to culture for the derivation of monkey ES cells. Monkey ES cells were cultured in medium composed of knockout-Dulbecco's modified Eagle's medium (KO-DMEM) supplemented with 20% Knock-out Serum Replacement (KSR; Invitrogen), 1 mM glutamine, 1% non-essential amino acids and supplemented with 4 ng/ml of human basic fibroblast growth factor (bFGF; Chemicon). The HD monkey ES cells derived from tetraploid HD monkey embryos were named, TrES1.
Transgenic status of the HD monkey ES cells
For detecting the htt-84Q gene, ubiquitin forward primer (5'-GAGGCGTCAGTTTCTTTGGTC-3') and htt-84Q-R reverse primer (5'-GCTGGGTCACTCTGTCTCTG-3') were used to yield an 818-bp product after amplification of genomic DNA from the HD monkey tissues. Genomic DNA (100 ng) from different tissues were subjected to PCR for 35 cycles at 96°C for 5 min, 96°C for 45 sec, 62°C for 45 sec, and 72°C for 150 sec, followed by 72°C for 7 min. To determinate the number of CAG repeats in HD monkeys, the PCR products were sequenced using HD exon 1-F primer (5'-GGCGACCCTGGAAAAGCTGA-3'). For GFP gene, ubiquitin forward primer (5'-GAGGCGTCAGTTTCTTTGGTC-3') and GFP-R reverse primer (5'-TAGTGGTTGTCGGGCAGCAG-3') were used for amplification for 35 cycles at 94°C for 5 min, at 94°C for 30 s, 64°C for 30 s, and 72°C for 20 s, followed by 72°C for 5 min, which yielded a product of 869 bp. DNA from WT-monkeys was used as the negative control, and plasmid htt-84Q and GFP were used as the positive controls.
Genotyping was executed using a panel of 13 microsatellites, known to be highly polymorphic and possessing high levels of heterozygosity in other rhesus macaque populations [32
]. Primers for each microsatellite were obtained with one of the standard Applied Biosytems (AB) five-dye labels. Amplification reactions were performed on AB 9700 thermal cyclers using MgCl2
concentrations of either 1.5 mM or 2.0 mM. Electrophoresis was carried out using an AB 3730 genetic analyzer, with all subsequent genotyping analysis using Genemapper 4.0. All genotyping was performed blind, with a positive and negative control included for each reaction.
Immunostaining of mutant htt
For cell samples, differentiated TrES1 were fixed using 4% paraformaldehyde (PFA) for 15 mins. Then they were permeabilized and blocked. The sample was next incubated with primary antibody mEM48 (1:50) followed by incubation with secondary antibody conjugated with Alexa Red (Molecular Probe). DNA was counterstained with Hoechst 33342 (5 μg/ml), mounted in Vectashield antifade solution (Vector Labs), and sealed with nail polish. The specimen was examined with an epifluorescent microscope. For mouse brains, the mice were anesthetized and perfused using 4% PFA. Brain tissues were post-fixed in 4% PFA overnight at 4°C, transferred to 30% sucrose, stored at 4°C, embedded in Optimal Cutting Temperature (OCT) medium (Sakura) and cut at 50 μm, followed by DAB staining. For DAB staining, sections were incubated with 0.3% H2O2 for 15 mins, blocked for one hour, and incubated with mEM48 (1:50) at 4°C overnight. After washing with DPBS, the brain sections were processed with avidin-biotin using the Vectastain Elite ABC kit (Vector Laboratories), and immediately stained with DAB (Vector Laboratories) for 30-40 secs. Brain sections were mounted on the slides with mounting media (Sigma), and images were examined and captured by MetaMorph software (Universal Imaging).
Immunostaining of stem cell markers
TrES1 were placed onto MFF in a four-well plate followed by two to three days culture, and was then fixed in 4% PFA, permeabilized by 1% Triton-X (excluded for cell surface markers), blocked with 2% BSA and 130 mM glycine in phosphate buffer saline (PBS). After overnight incubation with primary antibodies [Oct4 (Santa Cruz Biotechnology), SSEA-4 (Chemicon), TRA-1-60 (Chemicon)] followed by thorough washes, a secondary antibody conjugated with Alexa Red (Molecular Probe) was used for detection of the primary antibodies. DNA was counterstained with Hoechst 33342 (5 μg/ml). The specimen was examined with an epifluorescent microscope. Alkaline phosphatase assay was performed following manufacturer's instruction (Vector Lab).
Quantitative RT-PCR (Q-PCR) of stemness factors
The total RNA of cell samples was extracted using RNeasy Mini Kit (Qiagen). RNA quality was determined by BioPhotometer (Eppendorf). Reverse transcription was performed by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and the resulted cDNA was used for Q-PCR. 2× Power SYBR® Green PCR Master Mix (Applied Biosystems) was mixed with specific primers and cDNA, and subjected to the iQ5 real-time PCR detection system (Bio-Rad). for one cycle at 96°C for 12 mins; then at 96°C for 15 secs and 60°C for 30 secs for 50 cycles. The specific primers for mutant htt specific primers were: HD Exon 1-F: ATGGCGACCCTGGAAAAGCT and HD Exon 1-R: TGCTGCTGGAAGGACTTGAG. The specific primer for 18S: 18S F: CGGCTACCACATCCAAGGAA and 18S R: CCTGTATTGTTATTTTTCGTCACTACCT. Specific-qPCR primer sets targeting stem cell markers were: Oct 4 (Oct4-F: 5'-GCA ACC TGG AGA ATT TGT TCC T-3' and Oct4-R: 5'-GGG CGA TGT GGC TGA TCT-3'), Sox2 (Sox2-F: 5' GCA GGT TGA CAT CGT TGG TAA T-3' and Sox2-R: 5'CCC CCC GAA GTT TGC TGC G 3'), Nanog (Nanog-F: 5'-TGA AGC ATC CGA CTG TAA AGA ATC-3' and Nanog-R: 5'-CAT CTC AGC AGA AGA CAT TTG CA-3').
Mitochondria Inheritance Analysis
Sequencing primers were designed in primer 3 http://frodo.wi.mit.edu/
in order to amplify two regions of rhesus mitochondrial DNA (Macaca mulatta
NCBI reference sequence NC_005943). PCRs were performed using standard amplification reactions on AB 9700 thermal cyclers using 2.0 mM MgCl2
concentration. PCR products were checked for expected size by electrophoresis on agarose gels. Shrimp alkaline phosphatase and Exonuclease I were added to remove single strand DNA. Sequencing reactions were done using AB Big Dye terminator on a 9700 thermal cycler. The reaction was purified and sequencing reactions were performed on an AB 3730 genetic analyzer. Subsequent analysis was done using SeqScape genetic software. Positive and negative controls were sequenced along with experimental samples for each region.
Cytogenetic analysis/G-banding analysis
TrES1 at passage 25 was treated with KaryoMax® colcemid (Invitrogen) for 20 mins, dislodged with 0.05% Trypsin-EDTA, centrifuged and resuspended in hypotonic 0.075 M KCl solution for 20 mins. Following centrifugation, the cells were fixed three times in a 3:1 ratio of methanol to glacial acetic acid. The cell pellet was resuspended in 1 ml of fixative and stored at 4°C. For GTL-Banding, the fixed cell suspension was dropped on wet slides, air dried, and baked at 90°C for one hour. Slides were immersed in 0.5× Trypsin-EDTA (Invitrogen) with two drops of 67 mM Na2HPO4 for 20 to 30 secs, rinsed in distilled water and stained with Leishman Stain (Sigma) for 90 secs. Twenty metaphases were analyzed for numerical and structural chromosome abnormalities using an Olympus BX-40 microscope. Images were captured, and at least two cells were karyotyped using the CytoVysion® digital imaging system (Applied Imaging).
In vitro differentiation to neuronal lineage
TrES1 cell clumps were cultured in suspension for seven days for the formation of embryoid bodies (EBs). EBs were then allowed to attach onto a gelatin coated plate and cultured in N1 medium for seven days, N2 medium for 14 days and N3 medium for seven days to allow for differentiation into mature neuronal cell types. A four weeks extended culture at N3 stage was added to enhance maturation of neurons and mimic post-mitotic condition in the brain. The N1 medium was composed of KO-DMEM (Invitrogen) supplemented with minimum essential amino acid (Invitrogen), 200 mM of L-glutamine (Invitrogen) and N2 supplement (Invitrogen). The N2 medium was composed of N1 medium supplemented with 20 ng/ml bFGF. The N3 medium was composed of KO-DMEM supplemented with 1% FBS (Hyclone) and B27 supplement (Invitrogen). NPCs were immunostained with nestin, whereas successful differentiation of neuronal cell types was confirmed by the expression of neuron specific βIII tubulin and MAP2 [13
Counting of TrES1 with nuclear aggregate and NIs
After immunostaining with mEM48, each sample was examined and images were taken at different regions of the culture. A total of three 35 mm dishes of differentiated TrES1 at different differentiation stages (N2, N3-1w and N3-4w) were used in this study. All images were taken at the same magnification, and the total number of cells in each image were counted and categorized as those with nuclear staining with mEm48, and those with nuclear staining that contained one to five, six to 10 and more than 10 NIs.
Western Blot Analysis
Total proteins were extracted from TrES1 cells and equal amounts (20-30 μg) of protein extract were loaded into a 9% polyacrylamide gel (Bio-Rad). Following electrophoresis, proteins were transferred onto a PVDF membrane followed by blocking in 5% skim milk for two hours. The membrane was then incubated with primary antibodies, mouse mEM48 (1:50), and γ-tubulin (1:2000; Sigma), followed by secondary antibody conjugated with peroxidase (Jackson Immuno Research Laboratories) for detecting proteins with a Western Lightning Chemiluminescence Reagent Plus. (PerkinElmer).
In vivo differentiation of TrES1 and formation of teratoma in SCID mice
Undifferentiated TrES1 cell clumps were collected mechanically. TrES1 derived NPCs at N2 stage were collected by brief treatment with 0.05% trypsin/EDTA (Invitrogen) to produce single cell suspension. An estimate of 1 × 105 undifferentiated TrES cells and NPCs were resuspended in 5 ul of DPBS and implanted into the striatum of SCID mice. At four to 10 weeks after implantation, animals were euthanized, and the brain was recovered for further analysis. All surgical and animal procedures were approved by YNPRC/Emory Animal Care and Biosafety Committees. For staining of neuronal markers, the sections were incubated with primary antibodies (nestin, βIII tubulin, MAP2; Chemicon) at 4°C over night followed by thorough washes. A secondary antibody conjugated with Alexa Red (Molecular Probe) was used for detection of the primary antibodies. DNA was counterstained with Hoechst 33342 (5 μg/ml). The specimen was examined with an epifluorescent microscope.
Immunhistochemical staining of mutant htt
Mice were anesthetized and perfused using 4% paraformaldehyde (PFA). Brain tissues were post-fixed in 4% PFA overnight at 4°C, transferred to 30% sucrose at 4°C, embedded in Optimal Cutting Temperature (OCT) medium (Sakura), cut at 50 μm, followed by DAB staining. Sections were incubated with 0.3% H2O2 for 15 mins, blocked for 1 hr, and incubated with mEM48 (1:50) at 4°C overnight. After washing with DPBS, the brain sections were processed with avidin-biotin using the Vectastain Elite ABC kit (Vector Laboratories), and immediately stained with DAB (Vector Laboratories) for 30-40 secs. Brain sections were mounted on the slides with mounting media (Sigma), and images were examined and captured by MetaMorph software (Universal Imaging).
Student t-test was used for statistical analysis. Differences of P < 0.05 were considered statistically significant.