Supplemental Experimental Procedures

Cell Stem Cell, Volume 1

Supplemental Data The Generation of Six Clinical-Grade Human Embryonic Stem Cell Lines Jeremy Micah Crook, Teija Tuulikki Peura, Lucy Kravets, Alexis Gina Bosman, Jeremy James Buzzard, Rachel Horne, Hannes Hentze, Norris Ray Dunn, Robert Zweigerdt, Florence Chua, Alan Upshall, and Alan Colman

Supplemental Experimental Procedures Donor Consent and Procurement of Embryos Embryos were donated following a comprehensive informed consent process (Table S1) as regulated by the Australian Commonwealth Research Involving Human Embryos Act 2002. Moreover, the research project was approved by the human ethics committees of all the relevant parties, including an IRB, namely the Sydney IVF (SIVF) Ethics Committee, as well as by the Australian NH&MRC Embryo Research Licensing Committee. The owners of embryos stored for 4 - 6 yrs were contacted for permission to declare the embryos as surplus to their reproductive needs. Once declared, the providers were offered an opportunity to meet with a counselor, after which they could allow the embryos to succumb or donate them for licensed scientific research and the clinical lines development project. In the latter instance, a thorough assessment of the donor’s medical history was performed and their personal health was screened by completion of a legally required lifestyle questionnaire for “donors of human tissue”.

All information was secured to

protect donor confidentiality. At the end of the consent process, blood samples were obtained and tested by a certified clinical pathology laboratory (Mayne

Health Laverty Pathology, Australia).

Based upon the results of the

questionnaire and blood testing (Table S2), embryos from donors with no lifestyle risk and no or low risk of infectious disease were approved for use under the program.

Embryo Production Embryos were produced following routine SIVF procedures (Henman et al., 2005), including media prepared by SIVF from components retrospectively qualified as cGMP-compliant or procured as “cGMP-manufactured” from the commercial distributor of Sydney IVF media, Cook (Australia; Table S4). Briefly, following follicular aspiration of superovulated patients, cumulus cell-oocyte complexes were immediately washed in HSA based Fertilisation Medium. Oocytes were incubated in the same medium in 4-well Nunclon dishes (Nunclon, Denmark) for 3 hr, before adding spermatozoa at a concentration of 8 - 15 x 104 per ml. The spermatozoa were prepared by 20 min centrifugation at 300g in 40% and 80% PureSperm®-gradient according to the manufacturer’s specifications (Nidacon Int., Sweden). After 1 hr co-incubation, oocytes were cleaned of loose cumulus-cells by pipetting, and transferred to clean Fertilization Medium under mineral oil (Sigma, USA). After overnight incubation putative zygotes were cleaned of cumulus cells, checked for fertilization, transferred to HSA based Cleavage Medium and cultured in groups of 5 -10 for 2 days followed by another 2 - 3 days of culture in HSA based Blastocyst Medium. Embryo culture was performed in a reduced oxygen atmosphere comprising 6% CO2, 5% O2 and 89% N2 at 37°C in a K-MINC-1000 incubator (Cook, Australia).

Embryos were preserved by control-rate freezing. Briefly, after 10 min equilibration in 5% glycerol, followed by 10 min in 9% glycerol + 0.2 M sucrose, embryos were individually loaded into 0.25 ml cryostraws and cooled to -150°C in a programmed control-rate freezer (Planer Products Ltd, England) before being plunged into liquid nitrogen (LN2). Embryos were subsequently stored in LN2 for up to 6 years.

Human Fibroblast Procurement and Culture Fibroblasts were derived from the foreskin of an approved 8 day old donor under cGMP by Ortec International (USA).

Following cell line characterization and

biosafety testing (Table S6) the FDA approved the line for clinical-use.

Written

consent was obtained from the donor’s parents for use of the cells “as helper cells or feeder cells in the development, growth and modification of hESCs” under the clinical lines project. The fibroblasts were tested and qualified for hESC derivation and culture. MCBs and WCBs were manufactured under cGMP conditions using pathogen-free fibroblast culture medium (fMedium) comprising DMEM supplemented with 10% FBS and 01% L-glutamine (Table S4). Cultures were expanded by passaging with sterile Trypsin/EDTA and cryopreserved by programmed control-rate freezing in fMedium with 10% DMSO as previously described (Table S4; Crook et al., 2007). Fibroblasts prepared for WCBs were mitotically inactivated by γ-irradiation (dose: 25 Grays) immediately prior to freezing. Frozen cell aliquots were plunged into LN2 until required for hESC derivation and culture.

BioReliance (UK) and Charles River Laboratories

(Philadelphia, USA) tested all banks negative for microbial contamination, including mycoplasma. For hESC derivation and expansion, fibroblasts were plated to gelatin-free 4 4 2 culture vessels at 6.8 x 10 and 3.8 x 10 cells/cm respectively by thawing tubes

of frozen cells in 37°C sterile water, dilution of cells with 10 ml of fMedium, centrifugation at 300 g for 10 min and removal of supernatant. After resuspension in fMedium and cell viability counting by trypan blue exclusion method, viable cells were immediately plated. fMedium was changed 1 day after plating, and plated feeders were used within 1 week.

hESC Derivation All donated embryos were transferred to the cGMP facility in LN2 dry shippers under controlled and monitored conditions and stored in a designated LN2 tank until required. The embryos were thawed by warming straws at room temperature (RT) for 30 sec and transfer to a 30ºC sterile water bath for 30 - 40 sec. Straws were swabbed with 70% EtOH, dried and opened. The contents of a straw were expelled into a culture dish and the embryo was immediately transferred to the first thaw solution. Embryos were then incubated at RT for 10 min successively in 0.5, 0.2 and 0.1 M sucrose-solutions prepared in cryopreservation buffer of a SIVF Thawing Kit (Table S4), followed by further incubation at RT for 10 min and 370C for 10 min in cryopreservation buffer alone. Embryos were subsequently transferred into SIVF Blastocyst Medium (Table S4) and cultured for 4 - 24 hrs, again in a reduced oxygen atmosphere comprising 6% CO2, 5% O2 and 89% N2 at 37°C in a K-MINC-1000 incubator. If not fully

hatched, the zona pellucida of an embryo was removed by manual bisection using a sterile Ultra-Sharp Splitting Blade (AB Technologies, USA). Depending on embryo quality, it was either bisected to separate ICM and polar trophectoderm from mural trophectoderm, re-plating only the ICM-containing part, or re-plated as a whole embryo. Culture plates comprised fibroblast coated 4 2 (high concentration: 6.8 x 10 cells/cm ) organ culture dishes with USP-grade

hESC medium containing custom made KnockOut™ DMEM (KO-DMEM) with high glucose and 20% KnockOut™ SR (KO-SR), 2 mM L-glutamine, 0.1 mM non-essential amino acids and 50 ng/ml recombinant human bFGF (Table S4). Following embryo/ICM plating, medium was changed daily. ICM-outgrowths were first passaged 4 - 7 days after plating, and every 4 14 days thereafter until either stable hESC-like growth was observed or failed to arise resulting in discontinuation of the culture. Initially manual passaging was performed by sectioning outgrowths with a splitting blade and prospective hESC colonies (ESI-014, ESI-017 and ESI-049 for 2 passages, and ESI-013, ESI-027, ESI-035, ESI-051 and ESI-053 for 3 passages) with a stem cell cutting tool (Swemed, Sweden). After establishing putative hESC lines (i.e. ESI-014: 10 days, ESI-013 and ESI-035: 11 days, ESI-049: 12 days, ESI-053: 13 days, ESI017: 14 days, ESI-051: 16 days, and ESI-027: 20 days following outgrowth formation).cultures were maintained on fibroblasts (low concentration: 3.8 x 10

4

cells/cm2) in regular incubators under standard culture conditions at 37oC with 5% CO2 and passaged enzymatically every 6 - 8 days for up to 8 passages before freezing for banking. Briefly, colonies were washed in PBS (with Ca2+ and Mg2+), incubated with 1.25 mg/ml cGMP-grade collagenase at 37oC for 5 – 10 min and

washed with hESC medium (Table S4). Cultures were scraped with a cell scraper and fluxed with a 5 ml pipette. Following centrifugation for 3 min at 250 g, the supernatant was aspirated and cells were suspended in fresh hESC medium and re-plated for further culture. Following passaging, medium was changed after 2 days and daily thereafter.

hESC Cryopreservation for Banking 6 hESC lines were banked in batches of 40 - 60 straws, with 3 x 10 cells per

straw. Briefly, cultures were harvested following 7 (ESI-049), 8 (ESI-013, ESI027, ESI-035, ESI-051 and ESI-053) or 9 (ESI-014, ESI-017) passages after embryo plating by collagenase dissociation and combined to form a single batch of cells in defined protein-free cryomedium CryoStor™ CS-5 supplemented with 5% DMSO (Table S4). Total cell number of each batch was determined following trypsinisation of a cell aliquot and Trypan blue exclusion based counting. Cells were control-rate frozen (Crook et al., 2007) in 200 µl of cryomedia in a 0.5 ml CBS™ High Security Straw (Cryo Bio System, France), hermetically sealed using a thermal sealing unit (Cryo Bio System). Sealed straws of a prospective MCB were maintained at 4oC until all straws were loaded, prior to freezing. Loaded straws were frozen to -180oC in a programmed control-rate freezer before being plunged into LN2. All MCBs were transported in LN2 under controlled and monitored conditions to a cGMP-certified storage facility (Cryosite, Australia).

hESC Culture for Line/Bank Characterization For all hESC lines, cell thawing and culture was performed as described previously (Crook et al., 2007). In addition, cell culture employed the same reagents used for cGMP cell culture described above. Briefly, thawing of straws for hESC re-culture was performed by rapid transfer from LN2 storage to sterile 37oC water. Once thawed, straws were immediately swabbed with 70% EtOH, and dried with sterile lint-free tissue. The ends of each straw were cut and the hESCs expelled by pipette to 37oC hESC medium and centrifuged at 300 g for 3 min.

o Pelleted cells were resuspended in fresh 37 C hESC medium and plated

for culture. Following post-thaw recovery, cultures were passaged every 6 - 8 days with collagenase, and expanded for subsequent assayology or pathogen testing.

Chromosome and Genotype Analyses Karyotyping was performed by Sydney Genetics (Australia). For each hESC line, a minimum of 15 metaphase spreads were examined by Giemsa-banding with a chromosome resolution ranging from 200 - 400 bands per haploid set. For DNA fingerprinting studies, samples of 1 – 2 x 103 hESCs were processed using the Chelex extraction method (Walsh et al., 1991). Cells were centrifuged at 14,000 g for 5 min and the supernatant removed. Depending on the size of the cell pellet, 100 – 200 ml of 5% Chelex (in water) solution was added together with 20 mg/ml Proteinase-K and incubated for 15 - 30 min at 56°C. After vortexing for 5 - 10 sec the sample was spun briefly, incubated in boiling water for 8 min and vortexed again for 5 - 10 sec. After spinning for 2 - 3

min the sample was subjected to PCR amplification. An Identifiler kit (Applied Biosystems), which co-amplifies 15 STR loci (CSF1P0, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, vWA) in one PCR reaction and the gender marker Amelogenin was used according to the manufacturer’s instructions. The samples were analysed using capillary electrophoresis (3100-AVANT Genetic Analyser) and the resulting electropherograms analysed for the allele profile at each locus. For ESI-013 and ESI-027 fluorescent in-situ hybridization (FISH) studies were performed essentially in accordance with Langer et al. (1981) and Lichter et al. (1988).

A probe specific for the centromere of chromosome 16 (CEP16,

Vysis, USA) was hybridized to chromosome preparations according to the manufacturers instructions.

Sterility and Pathogen Testing Sterility testing was performed under cGMP conditions by “direct inoculation method” at BioReliance (UK) in accordance with the current requirements of the 21 CFR 610 (General biological products standards, 2003), and the Q5D “Note for

guidance

characterization

on

quality of

cell

of

biotechnological

substrates

used

products,

derivation

for

production

the

and of

biotechnological/biological products CPMP/ICH/249/95”. Other in vitro and in vivo pathogen testing was performed by Charles River Laboratories in compliance with the applicable requirements of the US FDA Good Laboratory Practice regulations, Title 21 of CFR Part 58 and/or Good Manufacturing Practice regulations, Title 21 of CFR Part 211. For each hESC

bank, testing required 1 – 2 x 108 hESCs, expanded under cGMP conditions from MCB stocks.

Cell culture employed the same reagents used for cGMP cell

culture described above.

Tests included: cultivable and non-cultivable

mycoplasmas, bovine and porcine adventitious viral agents, PCR detection and quantitation of human polyomavirus (JCV), human cytomegalovirus (HCMV), PCR-based reverse transcriptase assay for retrovirus detection, in vitro safety testing on MRC-5, Vero 76 and HeLa cells, in vivo safety testing with mice and embryonated chicken eggs, and transmission electron microscopy for viral particles.

Teratoma Formation To test the differentiation potential of hESC lines in vivo, serially passaged hESCs were manually harvested.

The cell suspension was transferred to a

Falcon tube and centrifuged at 300 g for 4 min.

Following removal of

supernatant, cells were resuspended in hESC medium and injected as clumps (2 6 - 4 x 10 cells in 50 µl) into the quadricep of the right hind limb of a male SCID

mouse. Three mice were injected per hESC line. Mice were maintained under controlled conditions in accordance with the National Institutes of Health (NIH) and National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines, and with approval of the Biopolis Institutional Animal Care and Use Committee (Biopolis IACUC approval 050008, National University of Singapore IRB 05-020). Following 6 - 10 wks, teratomas typically weighing 1 - 2 g were carefully excised from the surrounding muscle tissue, separated into pieces of 0.5 g, fixed in Bouin’s solution, embedded in paraffin, sectioned and histologically

analysed following staining with hematoxylin and eosin (H&E). Teratomas were assessed by a pathologist at the Department of Pathology, National University of Singapore. Sections were also processed for standard immunocytochemistry as described below.

In Vitro Studies of Spontaneous and Directed hESC Differentiation For in vitro studies of spontaneous hESC differentiation, EBs were formed by treating hESC cultures with collagenase, scoring with a 20 µl pipette tip and scraping with an Iwaki cell scraper. Cell aggregates were transferred to a Falcon tube and centrifuged at 300 g for 5 min. Following removal of supernatant, cells were resuspended in hESC medium without bFGF, seeded to low attachment tissue culture plates (Corning Costar), and left overnight. For directed cardiomyocyte induction studies, EBs were formed by treating hESC cultures with collagenase and scoring with a 20 µl pipette tip.

Cell

aggregates were harvested with an Iwaki cell scraper, transferred to a Falcon tube and allowed to settle.

Following removal of supernatant, cells were

resuspended in serum free medium (SF; DMEM, 1x NAA, 2mM L-glutamine, 1x ITS and 0.1mM β-mercaptoethanol), seeded to low attachment tissue culture plates (Corning Costar), and left overnight. SF was then replaced by END2 cellconditioned medium generated by exposure of SF to END2 cells for 4 days (Graichen et al., 2007; Dai et al., 2007). Medium changes were performed every 3 days. Beating EBs were observed on days 9 - 21 of culture and harvested for analysis.

Pancreatic progenitor cells were derived as previously described (D’Alessandro et al., 2007; Phillips et al., 2007). Briefly, EBs were formed by resuspending freshly collagenased hESCs in ice-cold basal RPMI 1640 medium containing 20% KO-SR, 1x penicillin/streptomycin supplemented with growth factor-reduced Matrigel™ (1:6 dilution; Becton-Dickinson), 50 ng/ml Activin A (R&D) and 50 ng/ml Bmp4 (R&D). The preparation was transferred to ultra low attachment plates (2 ml per well; Corning Costar) and quickly formed a semisolid disc containing EBs of various shapes and sizes at 37° C. Medium changes and growth factor top-ups were as previously described (Phillips et al., 2007).

Growth Profiling The growth kinetics of each hESC line was determined by harvesting cells on days 2 and 4 following inoculation and daily thereafter. Cultures were collected in triplicate by collagenase dissociation. Harvested cell aggregates were treated with 0.25% Trypsin-EDTA for 5 min. Triplicate cell samples were counted by trypan blue exclusion method. Viable cell number was plotted against time, and the doubling rate (G) was calculated using the formula G = (Log10 2 / µ) x 24 or (0.301 / µ) x 24, where µ is the specific growth constant (Log10 N1 - Log10 N0 / t1 t0; where N0 and N1 are the number of cells at day 0 (t0) and day 1 (t1)) during the exponential growth phase (Choo et al., 2004).

Immunophenotyping, RT-PCR and Q-PCR hESCs were immunolabelled for standard indirect immunocytochemistry. Cells were incubated with primary antibodies for Tra 1-60 (Chemicon MAB4360; 1:100), Nanog (R&D Systems AF1997; 1:100), Oct-4 (Santa Cruz Biotechnology SC-5279; 1:100), Tra 1-81 (Chemicon MAB4381; 1:100), SSEA-4 (Chemicon MAB4304; 1:100), and Sox-2 (R&D Systems MAB2018; 1:100), followed by incubation with either Fluorescein conjugated Goat Anti Mouse (Chemicon; 1:200), R-Phycoerythrin-conjugated AffiniPure Goat Anti-Mouse IgG + IgM (Jackson ImmunoResearch Laboratories, Inc.; 1:100), or Alexa Fluor® 488 goat anti-rat IgM (Invitrogen; 1:250) secondary antibody.

For flow cytometry, hESCs

were harvested using 0.5 mM EDTA (Sigma) in PBS solution then fixed and permeabilized in Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD). The cells were then labeled with primary antibodies or isotype controls and detected with appropriate secondary antibodies. Primary antibodies included Tra 2-54 (Chemicon MAB4354; 1:50), Oct-4 (1:20), Tra 1-60 (1:40), Tra 1-81 (1:40), and SSEA-4 (1:2000). Cells were analyzed by a FACScalibur

TM

Flow Cytometer

(Becton Dickson). Ten thousand events were acquired and analyzed by CellQuest software. Paraffin sections from in vivo teratomas were stained using a standard immunoperoxidase method. After blocking in 10% goat serum/PBS, sections were incubated with primary antibodies against desmin (clone D33; Dako, Glostrup, Denmark), keratin Endo-A (clone TROMA-1, Developmental Studies Hybridoma Bank, University of Iowa, USA), GFAP (clone 6F2; AbD Serotec Oxford, England), and Ki67 (clone MM1; Novocastra, Uk), followed by a goat

anti-mouse secondary antibody (Envision + System-HRP kit; Dako, Glostrup, Denmark). Visualization of labelling was done with a DAB Substrate Kit (Dako, Glostrup, Denmark). Sections were counterstained with H&E, dehydrated, cleared and mounted in DPX and examined using brightfield microscopy. Immunocytochemistry was performed on cryosectioned EBs or cells seeded on gelatin-coated glass chamberslides (Nalgene-Nunc) using primary antibodies for Nkx2.5 (rabbit-polyclonal, Santa Cruz), myosin light chain-2a (MLC-2a; Synaptic Systems), α-myosin heavy chain (α-MHC, Hybridoma Bank, Iowa), α-actinin (Sigma) and Pdx-1 (goat-anti rabbit, kindly provided by Professor Chris Wright, Vanderbilt University) in combination with respective TRITC-labeled (goat-anti-mouse, Jackson Immuno Research; to detect MLC-2a, α-MHC and αactinin) or FITC-labeled (donkey-ani-rabbit, Chemicon; to detect Nkx2.5) secondary antibodies. For standard RT-PCR, hESCs were harvested by initially washing cultures with PBS- and incubating with 25 mM EDTA for 5 min. Following removal of EDTA, PBS+ was added and the colonies were dissociated from the feeder layer using a 1 ml pipette and filtering through a 20 µm cell strainer. hESCs were centrifuged at 300 g for 5 min and supernatant was removed. For both hESCs and EBs, RNA was isolated using an RNeasy Miniprep Kit (Qiagen). First strand cDNA was synthesised using 4 µg of RNA and reverse transcribed by M-MuLV reverse transcriptase (NEB). PCR was performed using a Biorad MyIQ iCycler

TM

thermal cycler under the following conditions: 95oC for 5 min, and then 30 cycles of 95 oC for 30 s, 60 oC for 30 s, and 72 oC for 30 s, followed by 72 oC for 10 min. The PCR was carried out with primers specific for Oct-4, Nanog, TDGF-1, UTF-1,

Sox-2, IGF-2, Hand-1, Tubulin β-3, Nestin, H19, Cerberus-1, β-Actin and GAPDH (Table S7).

PCR products were electrophoresed on a 1% agarose gel and

visualized by ethidium bromide staining. For Q-PCR total RNA was isolated using TrizolTM (Invitrogen) according to the manufacturer’s recommendations followed by cDNA synthesis using standard protocol. Approximately 50 ng of cDNA template was mixed with 2X Sybr Green PCR Master Mix (BioRad) and diluted to 1X with 100 nM primers (Pdx-1 and βActin; Table S7) and water.

PCR was performed on a BioRad iCycler.

Quantitation was performed according to ∆Ct relative to β-Actin amplification.

References Choo, A.B.H., Padmanabhan, J., Chin, A.C.P, and Oh, S.K.W. (2004). Expansion of pluripotent human embryonic stem cells on human feeders. Biotech. Bioeng. 88, 321-331.

Crook, J.M., Horne, R., and Colman, A. (2007) Standard culture of human embryonic stem cells. In Human embryonic stem cells: the practical handbook, Sullivan, S., Cowan, C.A., and Eggan K., eds (Chichester,UK: John Wiley & Sons, Ltd.), pp. 53-79.

Dai, W., Field, L.J., Rubart, M., Reuter, S., Hale SL, Zweigerdt, R., Graichen, R.E., Kay, G.L., Jyrala, A., Colman, A., Davidson, B.P., Pera, M., Kloner, R.A. (2007). Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts. JMCC. In press.

D'Alessandro, J.S., Lu, K., Fung, B.P., Colman, A., and Clarke, D.L. (2007). Rapid and efficient in vitro generation of pancreatic islet progenitor cells from nonendocrine epithelial cells in the adult human pancreas. Stem Cells Dev. 16, 75-89.

Guidance for industry derivation and characterization of cell substrates used for production of biotechnological / biological products ICH topic Q5D (2001). US Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research.

Graichen, R.E., Xu, X.Q., Braam, S.R., Balakrishnan, T., Norfiza, S., Sieh, S., Soo, S.Y., Tham, S.C., Mummery, C.L., Colman, A., Zweigerdt, R., and Davidson B.P. (2007) Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation, in press.

Henman, M., Catt, J.W., Wood, T., Bowman, M.C., de Boer, K.A., and Jansen, R.P. (2005). Elective transfer of single fresh blastocysts and later transfer of cryostored blastocysts reduces the twin pregnancy rate and can improve the in vitro fertilization live birth rate in younger women. Fertil. Steril. 84, 1620-1627.

Langer, P.R., Waldrop, A.A., and Ward, D.C. (1981). Enzymatic synthesis of biotin-lableled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acac. Sci. USA 78:4458-4460.

Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D.C. (1988). Delineationof individual human chromosomes in metaphase and interphase cells by in situ suppression hbridisation using recombinant DNA libraries. Hum. Genet. 83:358-362.

Phillips, B.W., Hentze, H., Rust, W.L., Chen, Q.P., Chipperfield, H., Tan, E.K., Abraham, S., Sadasivam, A., Soong, P.L., Wang, S.T., Lim, R., Sun, W., Colman, A., and Dunn, N.R. (2007). Directed Differentiation of Human Embryonic Stem Cells intothe Pancreatic Endocrine Lineage. Stem Cells Development. In press.

Walsh, P.S., Metzger, D.A., and Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10, 506-513.

The United States Code of Federal Regulations 21 CFR, Part 58 (1993).



Figure S1. Characterisation of cGMP hESC lines ESI-014, ESI-017, ESI-049, ESI-051 and ESI-053 indicated undifferentiated and pluripotent hESC stuatus

following

derivation,

culture

through

7

-

9

passages,

cryopreservation, thawing, and re-culture through >5 passages (A) Quantitative flow cytometry indicating robust expression by all lines of Oct-4, alkaline phosphatase (Tra 2-54), Tra 1-60, Tra 1-81 and surface-antigen SSEA4. (B) RT-PCR showing transcript expression by all lines of marker genes Oct-4, Nanog, TDGF-1, UTF-1, and Sox-2. Lane 1, 100 bp DNA ladder. (C) Histology of teratomas. Teratomas comprised ectoderm (EC; neuroepithelium), endoderm (En; gut-like epithelium) and mesoderm (Me; cartilage). (D) RT-PCR of in vitro derived EBs showing transcript for IGF-2 and Hand-1 (mesodermal markers), Tubb-3 and Nestin (ectodermal markers), and H19 and Cerberus-1 (endodermal markers). Lane 1, 100 bp DNA ladder.

Table S1. Procedure and conditions for acquiring informed embryo donor consent Excess Embryo Declaration Donors opt to allow embryos to succumb or possibly contribute to research

Stage 1 Consent

Stage 2 Consent

Final Consent

Donors informed of specific research project selected: research into hESCs Consent required for use of any cell line derived for research

Donors consent to cells derived being made available to researchers outside of SIVF Donors consent to use of cells for research cannot be withdrawn at a later date

Donors consent to clinical use of cells

Donor consent can be withdrawn at a later date

Donors agree to the implications of irrevocable consent Donors provide altruistic donation without restriction or direction of research

Donors agree to the implications of irrevocable consent No obligation to inform donors of any specific clinical use of cells

Donors provide altruistic donation

Donors consent to clinical use of cells cannot be withdrawn at a later date

Donors cannot direct the use of cells or limit their use to include or exclude any persons Donors consent to use of cells for disease and/or genetic testing

Non-consent will not affect future medical treatment SIVF may receive payments but donors will have no claim to payments Donors are assured strict confidentiality of donation

Non-consent will not affect future medical treatment Donors will not receive payment for use of cells for research

Donors consent to genetic modification of cells and testing in animal hosts Non-consent will not affect future medical treatment Donors will not receive payment for clinical use of cells

Donors are assured strict confidentiality of donation

Donors are assured strict confidentiality of donation

Traceability is restricted through SIVF only

Traceability is restricted through SIVF only

Traceability is restricted through SIVF only Donors contribute blood samples for testing and storage

Donors can contact the SIVF ethics committee to address any complaints

Donors can contact the SIVF ethics committee to address any complaints

Donors provided with counseling in the event of an unexpected result Donors can contact the SIVF ethics committee to address any complaints

Table S2. Blood and pathogen testing of donors of embryos Blood group Epstein Barr virus1:

ESI-014 F M 0+ 0+ +ve IgG +ve IgM -ve -ve -ve -ve

ESI-017 F M 0+ 0+ +ve +ve -ve -ve -ve -ve

ESI-035 F M 0+ 0+ -ve -ve -ve -ve -ve -ve

ESI-049 F M 0+ 0+ +ve +ve -ve -ve -ve -ve

ESI-051 F M 0A+ +ve +ve -ve -ve -ve -ve

ESI-053 F M 0A+ +ve +ve -ve -ve -ve -ve

Coxsackie Enterovirus2 Enteric cytopathic human -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve orphan (ECHO) Enterovirus 1 -ve -ve -ve -ve -ve -ve +ve -ve +ve -ve +ve Cytomegalovirus : IgG -ve IgM -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Syphilis -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Neisseria gonorrhoeae -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Chlamydia trachornatis: IgG -ve IgM -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Human T-Lymphotropic Virus I and II -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Hepatitus B and C -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve Human Immuno Deficiency Virus I and II -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve 1 Donors tested positive for Epstein-Barr virus IgG and Cytomegalovirus IgG were accepted into the study based on testing negative for the corresponding IgM, which indicates no active infection of the virus. Notably, Epstein-Barr virus infection and Cytomegalovirus infection are prevalent in the general population. 2 Donors were determined -ve for Coxsackie Enterovirus where a complement fixing antibody titre was ≤ 64.

Table S3. Characteristics of cGMP hESC lines and embryos hESC Line

Relationship

ESI-013

1

Karyotype

Embryo Status/Grading1 Doubling TrophectTime (h) Expansion ICM oderm

ImmunoPhenotyping

RT-PCR

Differentiation In Vivo In Vitro (3 germ layers)

XX trisomy 16

3

1

1

Not Tested

Not Tested

Not Tested

Not Tested

Not Tested

ESI-014

Sibling to ESI-017

XX

2

1

2

33

Oct-4, Tra 1-60, Tra 181,Tra 2-54, SSEA-3, SSEA-4, GCTM-2, AP

Oct-4, Sox-2, Nanog, TDGF-1, UTF-1, GAPDH, β-Actin

9

Cardiomyocytes Pdx-1

ESI-017

Sibling to ESI-014

XX

1

1

2

27

Oct-4, Tra 1-60, Tra 181, Tra 2-54, SSEA-3, SSEA-4, GCTM-2, AP


9


ESI-027

XY trisomy 16

1

2

2

Not Tested

Not Tested

Not Tested

Not Tested

Not Tested

ESI-035

XX

1

2

2

33

Oct-4, Tra 1-60, Tra 254, SSEA-3, Nanog, Sox-2, AP


9


ESI-049

XY

2

2

2

32

Oct-4, Tra 1-60, Tra 254, SSEA-3, Nanog, Sox-2, AP


9


ESI-051

Sibling to ESI-053

XX

2

1

1

29

Oct-4, Tra 1-60, Tra 254, SSEA-3, SSEA-4, Nanog, Sox-2, AP


9


ESI-053

Sibling to ESI-051

XX

2

1

2

35

Oct-4, Tra 1-60, Tra 254, SSEA-3, SSEA-4, Nanog, Sox-2, AP


9


Expansion: 1=No expansion in overall size, zona pellucida thick, 2=Some expansion in overall size, zona pellucida beginning to thin, 3=Full expansion, zona pellucida very thin. ICM: 1=Cells compacted, tightly adhered together and indistinguishable as individual cells, 2=Cells less compacted so larger in size, loosely adhered together, some visible as individual cells. Trophectoderm: 1=Many small identical cells forming a continuous trophectoderm layer, 2=Fewer, larger cells, may not form completely continuous layer. Note: As embryos were frozen and thawed at blastocyst stage and in many instances evaluated only a few hours after thawing prior to full expansion, grading was often difficult to define.

Table S4. cGMP-compliant reagents for embryo production and hESC lines derivation, culture and cryopreservation for banking Reagent Knockout DMEM

Supplier Invitrogen Corporation, USA

Catalog Number 04-0044

Knockout SerumReplacement

Invitrogen Corporation, USA

04-0095

L-Glutamine (200 mM)

Invitrogen Corporation, USA Invitrogen Corporation, USA Invitrogen Corporation, USA Invitrogen Corporation, USA Invitrogen Corporation, USA Invitrogen Corporation, USA Nordmark Arzneimittel GmbH, Germany

25030

Regulatory Comments Custom manufactured under cGMP with full USP sterility testing Custom manufactured under cGMP with full USP sterility testing Manufactured under cGMP

11140

Manufactured under cGMP

25300 14040

Manufactured under cGMP; porcine parvovirus tested Manufactured under cGMP

14190


22320 or 12320


Non-Essential Amino Acids Trypsin/EDTA PBS with Calcium and Magnesium PBS without Calcium and Magnesium DMEM Collagenase NB6

17458

Manufactured according to cGMP guidelines, sterility tested according to EP. Manufactured under cGMP

Human basic FGF

Strathmann Biotec AG, Germany

9511060 hFGFb

Fetal Bovine Serum

Fertilisation Medium

Cambrex Bio Science, USA Cambrex Bio Science, USA SIVF, Australia

14-506 F (Australian origin) 14-501 Q (USA origin) -

Cleavage Medium

SIVF, Australia

-

Blastocyst Thawing Kit

Cook, Australia

K-SIBT-5000


Blastocyst Medium

Cook, Australia

K-SIBM-20


Embryo Thawing Kit

SIVF, Australia

-

Culture Oil

Cook, Australia

K-SICO-50


Cryostor CS5 Solution

BioLife Solutions, Inc., USA Sigma-Aldrich, USA

610202


D2438

Biotechnology performance certified; USP & EP compliant

Fetal Bovine Serum

DMSO

Qualified under cGMP Qualified under cGMP cGMP-compliant cGMP-compliant

cGMP-compliant

Table S5. cGMP hESC lines sterility and pathogen testing hESC Line

Sterility

Mycoplasma1

In-Vitro Bovine Porcine In-Vivo PBRT3 Safety2 Virus Virus Test4

TEM5

hCMV6

hPolyoma hPolyoma JCV6 BKV6

ESI-014 Microbe free

-ve


-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve


-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve


-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve


-ve


-ve

-ve

-ve

-ve

-ve

1

To be tested

To be tested -ve

-ve

-ve 2

-ve

-ve

ESI-013 and ESI-027 also tested -ve for Mycoplasma; 28 day in vitro safety testing using MRC-5, Vero and Hela cell lines; RT activity detected by PCR; 4In vivo test for unapparent viruses using mice and chicken eggs; 5Transmission electron microscopy for viral particles; 6Sequences detected by quantitative-PCR 3

Table S6. Characterization and pathogen testing of fibroblast cell line Growth and Morphology Normal Karyotype XY Species Identification Human In Vivo Tumorigencicity -ve Sterility Microbe Free Mycoplasma -ve -ve In Vitro Viral Safety Test1 -ve Human T-lymphotropic Virus I and II Human Immuno Deficiency Virus I and II -ve Epstein Barr Virus -ve Human Herpes Virus 6 -ve Hepatitus A and B -ve Human Papilloma Virus -ve 1 Employed MRC-5, VERO and MDBK indicator cell lines and HSV-I/II and CMV as positive controls.

Table S7. Primer Sequences for RT-PCR Amplicon Size 105bp

Primer

Sequence (5’-3’)

Oct-4-F Oct-4-R

CAA TTT GCC AAG CTC CTG A CGT TTG GCT GAA TAC CTT CC

Nanog-F Nanog-R

TAC CTC AGC CTC CAG CAG AT TGC GTC ACA CCA TTG CTA TT

146bp

TDGF-1-F TDGF-1-R

CTT CAG AGA TGA CAG CAT TTG G CAG CAG GTT CTG TTT AGC TCC T

114bp

UTF-1-F UTF-1-R

CGC CGC TAC AAG TTC CTT A ATG AGC TTC CGG ATC TGC T

86bp

Sox-2-F Sox-2-R

TGC TGC CTC TTT AAG ACT AGG AC GCC GCC GAT GAT TGT TAT TA

117bp

Nestin-F Nestin-R

GCC CTG ACC ACT CCA GTT TA GGA GTC CTG GAT TTC CTT CC

200bp

Tubulin β-3-F Tubulin β-3-R

GCG GAT CAG CGT CTA CTA CA ATG TCC AAA GGC CCC TGA G

115bp

IGF-2-F IGF-2-R

CTG TTT CCG CAG CTG TGA C GGG GTA TCT GGG GAA GTT GT

118bp

Hand-1-F Hand-1-R

AAG CGG AAA AGG GAG CTG ACT CCA GCG CCC AGA CTT

112bp

H19-F H19-R

GCA AGA AGC GGG TCT GTT T GCT GGG TAG CAC CAT TTC TT

105bp

Cerberus-1-F Cerberus-1-R

GCC ATG AAG TAC ATT GGG AGA CAC AGC CTT CGT GGG TTA TAG

69bp

β-Actin-F β-Actin-R

CAA TGT GGC CGA GGA CTT TG CAT TCT CCT TAG AGA GAA GTG G

126bp

GAPDH-F GAPDH-R

TGC ACC ACC AAC TGC TTA GC GGC ATG GAC TGT GGT CAT GAG

87bp

Pdx-1-F Pdx-1 R

CCT TTC CCA TGG ATG AAG TC GGA ACT CCT TCT CCA GCT CTA

145bp