Regulation and compliance
This study was approved by the Local Research Ethics Committee (Sunderland Research Ethics Committee) and was licensed by the UK Human Fertilisation and Embryology Authority. Blastocysts were obtained after informed donor consent. Human foreskins were obtained after parental consent. The premises for the production of the clinical-grade fibroblast line has been licensed by the UK Human Tissue Authority (HTA) for testing, processing, storage, distribution, and import/export of human tissue (HTA license number 22111). All processes associated with the derivation, expansion, and cryopreservation of the master cell bank (MCB) of NclFed1A were carried out in accordance with the Newcastle University Biomanufacturing Facility Quality Management System (QMS), which operates in accordance with appropriate legislation, guidance, and regulation published by the Medicines and Healthcare products Regulatory Agency (MHRA) and the HTA. All documentation related to the QMS and to the production process was created and managed by using Q-Pulse software (Gael Ltd, UK.
Derivation and expansion of human fibroblasts
We derived human foreskin fibroblasts from tissue obtained from donors deemed to be of low risk based on their medical history. This included healthy children of ~6 months of age undergoing circumcision for religious reasons with no known infection or disease. Because no vertical transmission of prions has been documented in humans, the use of tissue from a young child minimizes the risk of prion contamination . All human tissue was transferred to the processing laboratory in PBS. The tissue was dissociated with a scalpel (VWR, UK) and incubated with Collagenase Type IV (Invitrogen, USA Cat. No. 17104-019) at 37°C for 40 minutes. Samples were washed by centrifugation and plated in a T25 or T75 flask (TPP; Switzerland) with either FBS growth medium (DMEM (Invitrogen, Cat. No. 11995-065), 10% FBS (Invitrogen, Cat. No. 10099-141) and 1 × glutamine (Invitrogen, Cat. No. 25030) supplemented with 1× Pen/Strep (Invitrogen)) or with xeno-free hESC medium; KOSR-XF (KO DMEM (Invitrogen), 15% KnockOut Serum Replacement-XenoFree (Invitrogen), 0.1 mM NEAA (Invitrogen); 0.1 mM β-mercaptoethanol; 2 mM Glutamax; 8 ng/ml FGF2 (Invitrogen)). Cells were incubated at 37°C and 5% CO2, medium was changed every 48 to 72 hours, and cells were passaged when cells were confluent. Cells were deemed confluent when the growth surface of the flask was covered by cells (Additional file 1, Figure S1).
For passaging, the culture medium was removed from flasks and replaced with Tryple Select (Invitrogen); the fibroblasts were incubated for 5 minutes at 37°C. The cells were washed by centrifugation and passaged. Cells cultured from the pre-seed bank (PSB) to the master cell bank (MCB) were cultured in FBS growth medium without penicillin and streptomycin. Cells were passaged at a ratio of 1:6 in T75, T150, or T300 flasks with an estimated plating density of 3.5 × 104 cells/cm2). When confluent at P5, the MCB was cryopreserved; the choice of flask was based on the maximum number of flasks an operator could handle in one session.
Cryopreservation of fibroblasts
Fibroblasts were dissociated with Tryple Select, washed by centrifugation, and resuspended in freeze medium (10% DMSO (Sigma, UK) and 90% FBS (Invitrogen)). The resuspended cells were aliquoted into 1-ml aliquots in 2-ml cryovials (TPP) that were cooled by using controlled-rate freezing (Mr Frosty; Nalgene, USA) at 1°C/min. After cryopreservation, cell counts and viability were carried out by using a Vi-Cell (Beckman Coulter, USA).
Inactivation of fibroblasts
Fibroblasts were inactivated by using either mitomycin C or X-ray irradiation. For mitomyocin C inactivation, fibroblasts were incubated with 10 μg/ml of mitomyocin C for 2.5 hours at 37°C and 5% CO2. The mitomyocin C was washed out by using FBS growth medium, and washing was repeated 7 times. Samples were incubated overnight in the FBS growth medium and cryopreserved, as described previously. For X-ray inactivation, fibroblasts were exposed to 50 Gy (Faxitron, USA). Samples were incubated overnight and cryopreserved.
Source of human blastocysts
Embryos used to determine the efficiency of hESC derivation were donated by couples undergoing assisted-conception treatment. Embryos were produced in vitro by conventional oocyte insemination or by intracytoplasmic sperm injection (ICSI) and cultured in G1 medium (Vitrolife, Sweden) for 2 to 3 days until the best-quality embryos were selected for transfer to the uterus or for cryopreservation. The embryos used in this study were cryopreserved but were no longer required for treatment. Cryopreservation and thawing was performed by using a Vitrolife Freeze medium (Vitrolife) and Thaw medium (Vitrolife). Thawed embryos were cultured in G2 medium (Vitrolife) for 3 to 4 days until they developed to the blastocyst stage. All blastocysts, regardless of quality, were included in the study and were randomly allocated to explantation on three different feeder cell lines.
hESC stem cell derivation and culture
hESC derivations were carried out in a fully enclosed isolator cabinet (Vitrosafe). Human blastocysts were dissociated by using two insulin needles (Becton Dickinson, USA); the inner cell mass (ICM) was removed and plated on Cellstart (Invitrogen) with either inactivated human foreskin fibroblasts (NclFed1A) or MEFs. Samples were incubated in hESC medium; KOSR (KO DMEM (Invitrogen), 20% KnockOut Serum Replacement (Invitrogen), 0.1 mM nonessential amino acids NEAA (Invitrogen); 0.1 mM β-mercaptoethanol; 2 mM Glutamax; 8 ng/ml FGF2 (Invitrogen)) supplemented with 5% Quinns Advantage Protein Supplement (Rochford Medical, Ltd, UK). The plated ICMs were incubated for 3 days at 37°C, 5%CO2, and 5%O2, and were checked daily for the presence of outgrowths. Initial hESC colonies were dissected by using insulin needles and passaged on to fresh feeder cells in medium that was changed every 2 to 3 days. For enzymic passaging, once the cells became 65% to 85% confluent, they were washed once in PBS medium and then incubated for 5 to 15 minutes in Tryple Select (Invitrogen). They were washed once with centrifugation in hESC medium and passaged at a ratio of 1:3 or 1:6.
RNA extraction was carried out by using Dynabeads mRNA Direct (Invitrogen) as described in the user manual, and cDNA was synthesized by using Superscript III (Invitrogen). The PCR primers are described in Additional file 2, Table S1. PCR reactions were carried out by adding Biomix red (Bioline, UK), as described in the user manual. Conditions for the PCR were 94°C for 2 minutes, 30 × (94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds), and 72°C for 15 minutes. PCR products were run on a 1% agarose gel.
Population doubling time
NclFed1A was thawed and seeded in T25 flasks. After incubation for 48 hours, cell counts were determined by using a Vi-Cell. This was repeated at 24-hour intervals for 4 days. Counts were repeated 3 times for each sample.
Estimation of the number of cells in a confluent flask
NclFed1A was passaged into 8 × T150 flasks; when deemed confluent (Additional file 2, Figure S1), the cells were dissociated by using Tryple Express (Invitrogen), resuspened, and three cell counts were measured for each flask by using a Vi-Cell. The number of cells in a confluent flask and per square centimeter was then determined (Additional file 2, Figure S1).
hESC colonies or fibroblasts were grown on coverslips. The cells were fixed in 4% PFA, blocked with a 10% wt/vol milk-powder solution for 1 hour at room temperature and incubated overnight at 4°C with an anti-NANOG human polyclonal antibody raised in goat (R and D Systems, USA), an anti-OCT4 polyclonal antibody raised in rabbit (Abcam, UK), HFF-Cellect (Cellartis, Sweden), or 5B5 (Abcam). The cells were washed and incubated with either Alexa Fluor 488 donkey anti-goat immunoglobulin (Molecular Probes, USA), Alexa Fluor 555 donkey anti-rabbit immunoglobulin (Molecular Probes), or Alexa Fluor 488 donkey anti-mouse immunoglobulin (Molecular Probes). Nuclear staining was carried out by using Draq5 (Biostatus, UK). Samples were washed 3 times before imaging. Samples were imaged by using an inverted confocal microscope (Zeiss, Germany).
Flow-cytometry analysis for cell-surface markers
FACS analysis was carried out as described . In brief, cells were dissociated with Tryple and incubated for 1 hour with the fibroblast-specific marker HFF-Cellect (Cellartis), and an antibody raised against either Tra-1-60, Tra-1-81, Tra-2-54, SSEA3, or SSEA4. Cells were washed and incubated for 30 minutes with the appropriate secondary antibody. Analysis was carried out by using a flow cytometer (Becton Dickinson).
Determination of Neu5GC concentrations
Cells were treated with trifluoracetic acid at 80°C for 1 hour and derivatized by using DMB solution (7 mM 1,2-diamino-4,5-methylene dioxybenzene (Sigma); 1.4 M acetic acid (Sigma), 0.75 M β-mercaptoethanol (Sigma), and 18 mM sodium hydrosulfite (Sigma)) for 2 hours 30 minutes at 50°C and run on an HPLC (Dionex) by using a C8 column (Agilent AD-LC-139, USA). Samples were run at 0.90 ml/min in an isocratic solution of 7% methanol, 9% acetic acid, and 84% water. Neu5Gc detection was carried out by using a fluorescence detector (Dionex, UK) with an excitation wavelength of 373 nm and emission wavelength of 448 nm.
Karyotyping and genotyping
Karyotyping and genotyping were contracted to The Doctors Laboratory, which is accredited by the National External Quality Assessment Service (NEQAS; UK).
Analysis of variance was used to investigate the effect of the fibroblast cell line or the passage number of fibroblasts on the expression of pluripotency markers in hESCs. Χ2 analysis was used to compare fibroblast cell lines for hESC derivation efficiency. All data are expressed as mean ± SD. As it is envisaged that NclFed1A will be used only as feeder cells derived from the MCB, therefore all population-doubling data (PD) are expressed from the MCB.