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Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions
Cytotherapy, 2012; 14: 122–128
Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions
DUSKO ILIC1, EMMA STEPHENSON1, VICTORIA WOOD1, LAUREEN JACQUET1, DANIELLE STEVENSON1, ANASTASIA PETROVA2, NELI KADEVA1, STEFANO CODOGNOTTO1, HEEMA PATEL1, MAXINE SEMPLE3, GLENDA CORNWELL3, CAROLINE OGILVIE4 & PETER BRAUDE1,3 1Embryonic
Stem Cell Laboratories, Guy’s Assisted Conception Unit, Division of Women’s Health, King’s College School of Medicine, London, UK, 2St John’s Institute of Dermatology, Dermatology Research Laboratories, Guy’s Hospital, London, UK, 3Assisted Conception Unit, Guy’s and St Thomas’ Hospital Trust, London, UK, and 4Cytogenetics Department, Guy’s and St Thomas’ Hospital Trust, London, UK Abstract Background aims. Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. Methods, results and conclusions. Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington’s disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions. Key Words: clinical-grade human embryonic stem cells, extracellular matrix, Huntington’s disease, myotonic dystrophy type 1
Introduction Human embryonic stem (hES) cells are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely whilst retaining their capacity to differentiate into specialized somatic cell types. Optimism that hES cells will provide a virtually unlimited source of selected cell types for future cell therapies, as well as for drug screening and development, has resulted in considerable progress in stem cell biology over the decade since the first human cells were derived (1). The initial difficulties with regulation for obtaining embryos for stem cell research, and the lack of consensus for reporting the quality and type of suitable embryos, has largely been overcome, especially in the UK, where a regulatory route map to facilitate clinical research application has recently been produced (2,3). The clinical use of hES cells demands that all processes within production meet the requirements of regulatory bodies, whose main concern is patient safety. One of the crucial requirements is to prevent xeno-derived
infections and immunoreactions caused by animal products in cell culture; these products therefore need to be eliminated from stem cell-derivation and -culture protocols (4). For use in human subjects, hES cells need to be differentiated into specific somatic cell types. There are several methods and proprietary technologies that address this problem and, as purification methods are more or less similar, yield depends on the supply of stem cells and their differentiation protocols. Differentiation and stabilization of hES cells into various somatic cell lineages is not likely be a major obstacle technically, as long as there is an unlimited supply of undifferentiated cells (5). Therefore, it is essential to make newly derived hES cell lines suited to conditions that would support large-scale cell manufacture under xeno-free conditions in current good manufacturing practice (cGMP)-compliant facilities. The presence of feeder cells, while optimal for long-term culture of undifferentiated hES cells, limits their use, as the apparent effects of drugs,
Correspondence: Dusko Ilic, King’s College London School of Medicine, Division of Women’s Health, Women’s Health Academic Centre KHP, Assisted Conception Unit, 11th Fl., Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK. E-mail: [email protected] (Received 21 June 2011; accepted 12 September 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.623692
Xeno-free derivation and propagation of hES cells small molecules and genetic modifications could be clouded by the presence and effect of feeder cells. This requires the adaptation of stem cells to a feederfree culture environment. Early funding from the UK Medical Research Council facilitated collaboration between in vitro fertilization (IVF) units that had access to surplus human embryos, and scientists trying to derive stem cells. Since then, we have been working towards derivation of hES cell lines within a cGMP environment for use in human subjects. Step by step, the technology of hES cell line derivation has been adapted to an animal product-free environment (6,7). Extracellular matrix (ECM) constituents, such as human fibronectin, vitronectin, laminin and their combination, have reportedly supported the growth of hES cell lines with various culture media containing animal-based components (8). However, none of the conditions worked with the first commercially available xeno-free medium [KnockOut™ SR XenoFree, KO-SR XF Invitrogen (now Life Technologies, Carlsbad, CA, USA); D. Ilic, unpublished data]. The cells had either a very low rate of attachment and/or extensive spontaneous differentiation. We have resolved these initial technical obstacles and developed an optimized general protocol for entirely xeno-free derivation, propagation and adaptation of hES cells to feeder-free conditions.The protocol has been applied to normal (Supplementary Figures 1 and 2) as well as specific mutation-carrying (SMC) hES cell lines (i.e. mutations linked to Huntington’s disease and myotonic dystrophy type 1) (Supplementary Figures 3–5).
Methods Embryos The derivation of hES cells at King’s College London (London, UK) is under license from the UK Human Fertilisation and Embryology Authority (HFEA; research license number R0133) and also has local ethical approval (UK National Health Service Research Ethics Committee Reference 06/Q0702/90). No financial inducements are offered for donation. In accordance with HFEA regulations, a sample of each line that is derived is deposited in the UK Stem Cell Bank for distribution to academic and research centers internationally. Fresh embryos for the derivation were obtained from the Guy’s Assisted Conception Unit (ACU; London, UK) pre-implantation genetic diagnostics (PGD) program. Cryopreserved embryos no longer wanted for therapeutic use by patients were from both Guy’s ACU as well as external units, obtained through the human embryonic stem cell co-ordinator’s (hESCCO) network (9).
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Embryo culture Cryopreserved embryos were thawed using Quinn’s Advantage Thaw Kit (Sage, Pasadena, CA, USA) according to the manufacturer’s protocol. Fresh and thawed cleavage stage embryos were cultured in Quinn’s Advantage Protein Plus Blastocyst Medium (Sage) at 37°C, in 5% CO2 and 5% O2, up to blastocyst stage. Laser-assisted blastomere biopsy It is critically important to remove all of the cumulus and corona cells because they carry maternal genetic material and can be a source of contamination in the genetic analysis if released during the biopsy procedure. Biopsies were carried out when the embryos had reached the 6–8-cell stage. The embryo was secured in Quinn’s Advantage N2-hydroxyethylpiperazineN2-ethanesulfonic acid (HEPES)-buffered medium, Ca2⫹/Mg2⫹-free, supplemented with human serum albumin (Sage), by holding the pipette in such a way that the blastomere for the biopsy was at the 3 o’clock position. The zona was drilled with a gentle stream of acid Tyrode’s solution (Sage) by rotating the hatching air syringe clockwise until the zona began to thin and lose its structure. Once a 30–40-μm hole had been made, the blastomere was carefully aspirated, transferred into the drop away from the embryo and examined for the presence of nucleus (Figure 1a). Pre-implantation genetic haplotyping for Huntington’s disease and myotonic dystrophy type 1 Single blastomeres biopsied from embryos were washed through polyvinylpyrrolidone/phosphatebuffered saline (PBS; Sage) and collected in 2.5 μL 200 mM NaOH, 50 mM dithiothreitol using a fine polished glass capillary, and overlaid with mineral oil. The cells were lysed by incubation at 65°C for 10 min and the lysate was then neutralized with 2.5 μL 200 mM tricine before undergoing whole genome amplification by multiple displacement amplification (MDA). Cell lysates were prepared for MDA using an REPLI-g Midi kit (Qiagen, Crawley, UK) by adding 45 μL reaction master mix (15 μL nuclease-free water, 29 μL REPLI-g Midi reaction buffer, 1 μL REPLI-g Midi DNA polymerase), then incubated at 30°C for 16 h followed by inactivation at 65°C for 3 min and storage at 4°C until polymerase chain reaction (PCR) analysis was performed. Polymorphic markers flanking or intragenic to the genes of interest, myotonic dystrophy type 1 protein kinase (DMPK) and huntingtin (HTT), were identified using the University of California Santa Cruz (UCSC) Genome Browser created by the Genome Bioinformatics Group of UCSC (Santa Cruz, CA, USA; http://www.genome.ucsc.edu). Primers were then designed for 17 microsatellite markers for each
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Figure 1. The principle of normal and SMC hES cell line derivation. (a–e) Single blastomere biopsy from a cleavage-stage (day-3 post-fertilization) embryo. (a) Cleavage-stage embryo held with a holding pipette (HP) is exposed to a gentle stream of acid Tyrode’s solution coming through a Tyrode’s pipette (TP). (b) A blastomere popping through a Tyrode’s solution-drilled hole in the zona pellucida (arrow). (c, d) The blastomere is carefully aspirated using an aspiration pipette (AP). (e) The biopsied blastomere is examined for the presence of a nucleus and DNA is used for mutation diagnosis. (f–i) Laser-mediated ICM dissection. The ICM and TE of a well-developed blastocyst are separated with a series of laser pulses (double-arrow). (j, k) ICM outgrowth on mitotically inactivated HFF, 5–7 days after plating. (j) Two distinct cell populations within an outgrowth; hES cell-like colony and TE cells could be distinguished under higher magnification about 5–7 days post-plating. (k) TE is dissected out with a needle. S, needle scratches on tissues culture plastic. (l) Typical 12–15-day-old hES cell colony on mitotically inactivated HFF.
gene using Primer3 software (http://frodo.wi.mit.edu/ primer3) and checked for the presence of single nucleotide polymorphisms (SNP) using the SNPCheck bioinformatics program from the National Genetics Reference Laboratory (Manchester, UK; https:// ngrl.manchester.ac.uk/SNPCheckV2/snpcheck. htm), and for human ALU repeats using a nucleotide BLAST search (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). The primers were designed to enable testing for Huntington’s disease using a single multiplex and for myotonic dystrophy using two multiplexes, with each marker being amplified at a different PCR product size and assigned a tag (6FAM™, VIC®, NED™ or PET™ dyes; Applied Biosystems (now Life Technologies, Carlsbad, CA, USA) to ensure no overlap in marker fragments. A universal tagged primer approach was used (10,11). PCR were per-
formed in 10-μL reactions containing DNA, 0.4 μL of an optimized non-fluorescent tagged primer mix, 4 pmol fluorescently labeled universal tag primer mix, and 5 μL 2 ⫻ Qiagen Multiplex PCR Master Mix (containing HotStarTaq® DNA polymerase, Multiplex PCR buffer with 6 mM MgCl2, dNTP mix). Amplifications were performed on a G-Storm 2 thermal cycler using the following conditions: initial denaturation step of 95°C for 15 min, followed by 25 cycles of 94°C for 30 s, 59°C for 60 s, 72°C for 60 s; then 72°C for 5 min, followed by 60°C for 20 min. PCR products were run on an ABI3730 genetic analyzer using POP-6 polymer with a Genescan-600 LIZ size standard (Applied Biosystems) and analyzed using GeneMarker (SoftGenetics, State College, PA, USA). Haplotypes were constructed manually from the genotype data.
Xeno-free derivation and propagation of hES cells Laser-assisted dissection of inner cell mass Manipulation was carried out in biopsy dishes with microdrops of Ca2⫹/Mg2⫹-free HEPES-buffered medium under oil. The blastocysts were secured with a holding pipette at the region of the zona pellucida opposite the inner cell mass (ICM) and a biopsy needle prepared to aspirate the hatched area (Figure 1b). Using a Saturn Active laser (1.480 nm, 400 mW) and Cronus software (both from Research Instruments, Cornwall, UK), laser pulses were directed at the trophectoderm (TE) cell junctions adjacent to the ICM cells but outside of the safety circle. At this point, both the hatched and zona-contained cavities tended to collapse, so the biopsy needle was used to secure the hatched cells and pull them gently away from the main body of the blastocyst. The laser firing was repeated as appropriate whilst gently retracting the biopsy needle until the two sections separated. The cellular area containing the ICM was then washed and transferred to plates containing mitotically inactivated human neonatal foreskin fibroblasts (HFF). hES cell line derivation and culture ICM was plated on mitotically inactivated HFF in Quinn’s Advantage Protein Plus Blastocyst Medium supplemented with 25 ng/mL human recombinant fibroblast growth factor (hrFGF; R&D Systems, Minneapolis, MN, USA) and incubated at 37°C in 5% CO2 and 5% O2 for 3 days without disturbing. Medium was replaced, 50% each time, every 2–3 days with KO-SR XF, also supplemented with 25 ng/mL hrFGF. TE cells were removed mechanically from outgrowth (12,13). hES colonies were expanded and cryopreserved at the third passage. For initial adaptation to feeder-free conditions, the hES cells were cultured for 1–3 passages in either KO-SR XF or TeSR2 on mitotically inactivated HFF. hES cells were then transferred to native decellularized HFF ECM and continuously propagated in either KO-SR XF or TeSR2. Passage of hES cell colonies was performed manually. For differentiation assessment, hES cells were cultured for up to 3 weeks in KnockOut™ SR XenoFree (KO-SR) supplemented with 10% fetal bovine serum (FBS). Medium was exchanged every 2–3 days. Vitrification Clumps of hES cell colonies ready for cryopreservation were placed into ES-HEPES solution [20% KO-SR XF in 2 mM HEPES/Dulbecco’s modified Eagle medium (DMEM)]. Six to eight clumps were transferred to a 30–40-μL drop of 10% vitrification solution [10% ethylene glycol, 10% dimethylsulfoxide (DMSO), 80% ES-HEPES] for 1 min. The colonies were then quickly transferred into an adjacent 30–40-μL drop of 20% vitrification solution (20% ethylene glycol, 20%
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DMSO, 30% ES-HEPES, 30% 1 M sucrose solution in 80% ES-HEPES/20% KO-SR XF) for 25 s. Finally, up to eight hES cell clumps were aspirated into a vitrification straw (MTG, Bruckberg, Germany) using capillary action and snap frozen by placing the straw horizontally into liquid N2. Preparation of native decellularized HFF ECM We tested two protocols for preparation of native decellularized HFF ECM, one using sodium deoxycholate (DOC) (14) and another using Triton X-100 (15,16). In both cases, the mitotically inactivated HFF were rinsed in PBS, and incubated either at 4°C for 30 min with the DOC-based cell extraction buffer (0.5% DOC in 10 mM Tris/HCl, pH 8.0) or at 37°C for 3–5 min with the Triton X-100-based cell extraction buffer [0.5% (v/v) Triton X-100 and 20 mM NH4OH in PBS]. Complete decelluarization was confirmed with Hoechst 33342 staining (Invitrogen). Remaining decellularized HFF ECM was gently rinsed at least three times with PBS and used either immediately or stored at 4°C for up to 1 week. ECM was preserved better using Triton X-100-based cell extraction buffer, and all hES cell cultures in either KO-SR XF or TeSR™2 (StemCell Technologies, Vancouver, BC, Canada) medium were therefore carried out on native decellularized HFF ECM prepared using this protocol (see Supplementary Figure 6). Before it was released for use, each preparation was examined for the quality of the ECM with immunodetection of fibronectin, collagen IV and laminin 1 network. All antibodies are purchased from (Sigma, St. Louis, MO, USA). Pluripotency markers Pluripotency was assessed using two different techniques: enzymatic activity assay [alkaline phosphatase (AP) assay] and immunostaining. The AP assay was performed according to the manufacturer’s instructions (ATCC, Manassas, VA, USA). Undifferentiated or in vitro differentiated hES cell cultures were fixed in 3.8% paraformaldehyde for 20 min and then permeabilized in 90% acetone for 10 min or 0.2% Triton X-100 in Dulbecco’s phosphate buffer saline (DPBS) for 5 min, rinsed well and incubated overnight at 4°C with primary antibodies against tumor rejection antigen (TRA)-1-60, TRA-1-81 (Millipore, Billerica, MA, USA), nanog (R&D Systems, Minneapolis, MN, USA), octamer binding protein 4 (Oct4) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), smooth muscle actin (SMA), βIII tubulin or α-fetoprotein (αFP) (Sigma, St. Louis, MO, USA). After washing in DPBS, samples were incubated with appropriate secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at room temperature,
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rinsed again and mounted in Vectashield® mounting medium containing DNA counterstain 4′,6 diamidino2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Samples were analyzed using a Nikon 50i epifluorescence microscope equipped with appropriate filters. Images were captured with an Infinity1 CCD camera (Lumenera, Ottawa, ON, Canada) and processed using Adobe Photoshop CS5 software (Adobe, San Jose, CA, USA). Genotyping DNA was extracted from hES cell cultures using a Chemagen DNA extraction robot according to the manufacturer’s instructions. DNA was quantified using a Nanodrop spectrophotometer according to the manufacturer’s instructions, and the DNA quality was checked by agarose gel electrophoresis. Amplification of polymorphic microsatellite markers was carried out as described elsewhere (17). Briefly, DNA was amplified using two multiplexes, one of 17 PCR primer pairs for markers on chromosomes 13, 18 and 21, and one of 14 primer pairs for markers on the X and Y chromosomes. PCR products were separated on an ABI3100 capillary genetic analyzer, and results were analyzed using ABI Genotyper software. Allele sizes were recorded to give a unique fingerprint of each cell line. Array CGH DNA (1 μg) was labeled using a comparative genomic hybridization (CGH) labeling kit (Enzo Life Sciences, Farmingdale, NY, USA), following the manufacturer’s instructions. Labeled DNA was purified post-labeling using QIAquick PCR purification kit (Qiagen) following the manufacturer’s instructions. Labeling efficiency and yield were assessed by spectrophotometry (Nanodrop,Wilmington, DE, USA). An Agilent (Santa Clara, CA, USA) 4 ⫻ 44 K platform with either Wessex NGRL design 017457 or design 028469 was used; hybridization, washing and scanning of arrays were as per the manufacturer’s protocols. Image quantification, array quality control and aberration detection were performed using feature extraction and DNA analytics software packages (Agilent, Santa Clara, CA, USA). Manufacturer’s recommendations were followed. Ninety-five per cent of array data were required to pass quality control (QC). The ADM-2 algorithm at threshold 6 (with a 3-probe sliding window providing a mean detection interval of 200 kb) was used for aberration calling. HLA typing HLA-A, -B and -DRB1 typing was performed with a PCR sequence-specific oligonucleotide probe (SSOP; Luminex, Austin, TX, USA) hybridization protocol
at the certified Clinical Transplantation Laboratory, Guy’s and St Thomas’ NHS Foundation Trust and Serco Plc. (GSTS) Pathology (Guy’s Hospital, London, UK). The results were matched against the National Marrow Donor Program (NMDP) website (www.marrow.org). The MatchView® program shows potential matches on the NMDP-operated registry Be The Match Registry® at the moment of using the tool. The registry includes approximately 8 million adult donors and more than 160 000 searchable cord blood units. It is updated constantly as new donors and cord blood units are added and others removed. The registry was accessed for both KCL-19 and KCL-20 on 23 December 2010. Results and discussion PGD is used to prevent the transmission of genetic disorders to offspring. Different practices are used in various PGD centers epending on national regulations, including the method and timing of biopsy. We routinely remove single blastomeres from cleavage-stage (day 3 post-fertilization) embryos (Figure 1a–e) for the purpose of isolating DNA from the biopsied cell and subjecting it, following whole genome amplification, to haplotype analysis to identify SMC embryos (18). Derivation of hES cell lines is attempted using embryos carrying the familial mutation, and therefore unsuitable for clinical use, which couples donate for research with consent. The ICM is isolated mechanically, using a laser, from either normal or SMC well-expanded blastocysts (day 6 or 7), which have a distinct ICM (Figure 1f–i). Earlier stages or less well-expanded blastocysts are generally not used for mechanical attempts, as the risk of damage to the ICM is too great. Dissected ICM is plated in blastocyst medium (Sage) supplemented with 25 ng/mL hrFGF (R&D Systems) on a feeder layer of mitotically inactivated HFF. We start replacement of about half of the medium in the dish with KO-SR XF supplemented with 25 ng/mL hrFGF on the third day of culture. The medium is refreshed every 2–3 days. If needed, TE cells are removed mechanically from the outgrowth 5–7 days after plating (Figure 1j–k) (12,13). By 7 days, a putative hES cell colony is evident in the culture and the first subculture is performed by 12–15 days (Figure 1l). The hES cell colonies are expanded by manual passage, and vitrified when a sufficient number of cells is available. The presence of pluripotency markers and ability to differentiate into derivatives of all three germ layers is verified routinely before freezing and after thawing. Over the last 18 months, we have derived 12 normal and SMC hES cell lines under these conditions. Adapting hES cells to feeder-free conditions, although not easy, can become a standard procedure
Xeno-free derivation and propagation of hES cells that will take a skillful, experienced laboratory technician about a month to accomplish. An additional month will be needed to master the enzymatic passage steps. However, the standard protocols used for normal hES cell lines may not always work for SMC hES cell lines; defective cell–cell and cell–ECM interactions have been implicated in the phenotype of many diseases, from cancer to bleeding disorders and various neurodegenerative conditions. Indeed, the combination of CELLstart™ (Invitrogen), commercially available humanized ECM and a serum-free medium StemPro® (Invitrogen), which was successfully used routinely with more than 40 normal hES cell lines (D. Ilic, unpublished data), did not support growth of Huntington’s disease SMC hES cell lines. The most commonly used substrate Matrigel™ is a complex mixture of basement membrane ECM components prepared from mouse tumors. hES cells on Matrigel can be grown in the presence of several defined culture media, available from different suppliers but by definition not xeno-free. We hypothesized that providing normal or SMC hES cells with a native human ECM microenvironment, similar to Matrigel, might support growth of both undifferentiated normal and SMC hES cell lines (Figure 2a) (14,19,20). To define xeno- and feeder-free conditions suitable for both normal and SMC hES cell lines, we focused on two normal (KCL-19 and KCL-20) and three SMC hES cell lines (KCL-12 and KCL-13, carrying Huntington’s disease, and KCL-18, carry-
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ing myotonic dystrophy type 1 mutation; see Supplementary Figures 1–5). We tested the growth of these five hES cell lines on native decellularized HFF ECM in the presence of two commercially available xeno-free culture media, KO-SR XF and TeSR2 (Stemcell Technologies). The typical morphology of undifferentiated hES cell colonies (well-defined colony edges, a tightly packed colony and the absence of differentiated cells), presence of pluripotency markers and ability of cells to differentiate into derivatives of three germ layers, demonstrated that native decellularized HFF ECM and TeSR2 was a superb combination for maintaining the undifferentiated phenotype of all the hES lines tested (Figure 2b). KCL-12 and KCL-13 SMC hES cell lines cultured on a native decellularized HFF ECM in the presence of KO-SR XF were prone to lower cell attachment and spontaneous cell differentiation, whereas in the presence of TeSR2 we had no such difficulties. Three well-trained laboratory staff doing the evaluation independently ended up with identical results; it is, therefore, possible that this difficulty reflects the Huntington’s disease-specific phenotype, which deserves further investigation at the molecular level. Although early data from clinical trials with hES cell-derived cell therapy (21) are reassuring and the need for immunosuppression is possibly less than expected, one has to keep in mind that the therapies target immunologically privileged sites and it is likely that, for broader applications, in order to minimize the
Figure 2. Normal and SMC hES cell lines cultured on native decellularized HFF ECM. (a) Live staining of native HFF-generated ECM before processing for decellularization. Fibronectin (red) and nuclei visualized with Hoechst 33342 (blue). (b) hES cell lines propagated on native decellularized HFF ECM TeSR2 medium retain typical morphology and pluripotency marker expression. TRA-1-81 (green); fibronectin (red). (c) Fibronectin, (d) laminin 1 and (e) collagen IV and an intricate network of fibers.
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need for immunosuppression, close HLA-matching of lines to recipients will be required, in particular for a range of ethnic backgrounds (22–24). Therefore, we routinely perform HLA typing on all our normal hES cell lines. We have described a comprehensive methodology and standards for the derivation and propagation in xeno-free conditions of hES cell lines, which are therefore suitable for use in human subjects without risk of detrimental agents. The optimization of protocols is a continuous process and new commercially available media formulations and substrates, such as StemAdehereXF™ (Primorigen, Madison, WI, USA) may further simplify culture. The development of these protocols, along with methods for robust genetic characterization of the cell lines (25), represents a critically important milestone in the implementation of hES cells in the treatment of human disease. Acknowledgments This work was supported by Medical Research Council UK grants. Author contributions: DI, ES and PB designed the methods; ES, VW, JL, DS, AP, NK, SC and MS performed experiments; GC obtained patient consents; HP and CO were responsible for genomic analyzes; ES, HP and MS contributed to the writing of the methods; DI, CO and PB wrote the manuscript. Competing financial interests: The authors have declared that no competing interests exist. References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7. 2. Stephenson EL, Braude PR, Mason C. International community consensus standard for reporting derivation of human embryonic stem cell lines. Regen Med. 2007;2:349–62. 3. Interim UK Regulatory Route Map for Stem Cell Research and Manufacture, version March 12, 2009. Download available at: www.mhra.gov.uk/Howweregulate/Medicines/Medicinesregulatorynews/CON041337, accessed on October 13, 2011. 4. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med. 2005;11:228–32. 5. Salaway T, Ilic D. Logistics of stem cell isolation, preparation and delivery for heart repair: concerns of clinicians, manufacturers, investors and public health. Regen Med. 2008;3:83–91. 6. Ilic D, Giritharan G, Zdravkovic T, Caceres E, Genbacev O, Fisher SJ, et al. Derivation of human embryonic stem cell lines from biopsied blastomeres on human feeders with minimal exposure to xenomaterials. Stem Cells Dev. 2009;18: 1343–50.
Supplementary material available online Supplementary Figures 1–6
7. Stephenson EL, Braude PR. Derivation of the King’s College London human embryonic stem cell lines. In Vitro Cell Dev Biol Anim. 2010;46:178–85. 8. Skottman H, Narkilahti S, Hovatta O. Challenges and approaches to the culture of pluripotent human embryonic stem cells. Regen Med. 2007;2:265–73. 9. Franklin SB, Hunt C, Cornwell G, Peddie V, Desousa P, Stephenson EL, at al. hESCCO: development of good practice models for hES cell derivation. Regen Med. 2008;3:105–16. 10. Mann K, Donaghue C, Fox SP, Docherty Z, Ogilvie CM. Strategies for the rapid prenatal diagnosis of chromosome aneuploidy. Eur J Hum Genet. 2004;12:907–15. 11. Ahn JW, Mann K, Walsh S, Shehab M, Hoang S, Docherty Z, et al. Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance. Mol Cytogenet 2010;3:9. 12. Ilic D, Genbacev O, Krtolica A. Derivation of hESC from intact blastocysts. Curr Protoc Stem Cell Biol. 2007;Chapter 1: Unit 1A.2. 13. Ilic D, Caceres E, Lu S, Julian P, Foulk R, Krtolica A. Effect of karyotype on successful human embryonic stem cell derivation. Stem Cells Dev. 2010;19:39–46. 14. Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R. Human embryonic stem cells derived without feeder cells. Lancet. 2005;365:1636–41. 15. Beacham DA, Amantagelo MD, Cukierman E. Preparation of extracellular matrices produced by cultured and primary fibroblasts. Curr Protoc Cell Biol. 2007;Chapter 10:Unit 10.9. 16. Vlodavsky I. Preparation of extracellular matrices produced by cultured corneal endothelial and PF-HR9 endodermal cells. Curr Protoc Cell Biol. 2001;Chapter 10:Unit 10.4. 17. Heath KE, Day IN, Humphries SE. Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low-density lipoprotein receptor gene. J Med Genet. 2000;37:272–80. 18. Renwick PJ, Trussler J, Ostad-Saffari E, Fassihi H, Black C, Braude P, et al. Proof of principle and first cases using preimplantation genetic haplotyping: a paradigm shift for embryo diagnosis. Reprod Biomed Online. 2006;13:110–9. 19. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001; 294:1708–12. 20. Ilic D. Culture of human embryonic stem cells and the extracellular matrix microenvironment. Regen Med. 2006;1:95–101. 21. www.clinicaltrils.gov (ID: NCT01217008, NCT01345006 and NCT01344993), accessed on October 13, 2011. 22. Fraga AM, Sukoyan M, Rajan P, Braga DP, Iaconelli A Jr, Franco JG Jr, et al. Establishment of a Brazilian line of human embryonic stem cells in defined medium: implications for cell therapy in an ethnically diverse population. Cell Transplant. 2011;20:431–40. 23. Mosher JT, Pemberton TJ, Harter K, Wang C, Buzbas EO, Dvorak P, et al. Lack of population diversity in commonly used human embryonic stem-cell lines. N Engl J Med. 2010;362:183–185. 24. Stephenson E, Ogilvie CM, Patel H, Cornwell G, Jacquet L, Kadeva N, Braude P, et al. Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells. J R Soc Interface. 2010;7(Suppl 6):S677–88. 25. Stephenson E, Ogilvie CM, Patel H, Cornwell G, Jacquet L, Kadeva N, et al. Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells. J R Soc Interface. 2010;7(Suppl 6):S677–88.
Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions
DUSKO ILIC1, EMMA STEPHENSON1, VICTORIA WOOD1, LAUREEN JACQUET1, DANIELLE STEVENSON1, ANASTASIA PETROVA2, NELI KADEVA1, STEFANO CODOGNOTTO1, HEEMA PATEL1, MAXINE SEMPLE3, GLENDA CORNWELL3, CAROLINE OGILVIE4 & PETER BRAUDE1,3 1Embryonic
Stem Cell Laboratories, Guy’s Assisted Conception Unit, Division of Women’s Health, King’s College School of Medicine, London, UK, 2St John’s Institute of Dermatology, Dermatology Research Laboratories, Guy’s Hospital, London, UK, 3Assisted Conception Unit, Guy’s and St Thomas’ Hospital Trust, London, UK, and 4Cytogenetics Department, Guy’s and St Thomas’ Hospital Trust, London, UK Abstract Background aims. Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. Methods, results and conclusions. Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington’s disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions. Key Words: clinical-grade human embryonic stem cells, extracellular matrix, Huntington’s disease, myotonic dystrophy type 1
Introduction Human embryonic stem (hES) cells are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely whilst retaining their capacity to differentiate into specialized somatic cell types. Optimism that hES cells will provide a virtually unlimited source of selected cell types for future cell therapies, as well as for drug screening and development, has resulted in considerable progress in stem cell biology over the decade since the first human cells were derived (1). The initial difficulties with regulation for obtaining embryos for stem cell research, and the lack of consensus for reporting the quality and type of suitable embryos, has largely been overcome, especially in the UK, where a regulatory route map to facilitate clinical research application has recently been produced (2,3). The clinical use of hES cells demands that all processes within production meet the requirements of regulatory bodies, whose main concern is patient safety. One of the crucial requirements is to prevent xeno-derived
infections and immunoreactions caused by animal products in cell culture; these products therefore need to be eliminated from stem cell-derivation and -culture protocols (4). For use in human subjects, hES cells need to be differentiated into specific somatic cell types. There are several methods and proprietary technologies that address this problem and, as purification methods are more or less similar, yield depends on the supply of stem cells and their differentiation protocols. Differentiation and stabilization of hES cells into various somatic cell lineages is not likely be a major obstacle technically, as long as there is an unlimited supply of undifferentiated cells (5). Therefore, it is essential to make newly derived hES cell lines suited to conditions that would support large-scale cell manufacture under xeno-free conditions in current good manufacturing practice (cGMP)-compliant facilities. The presence of feeder cells, while optimal for long-term culture of undifferentiated hES cells, limits their use, as the apparent effects of drugs,
Correspondence: Dusko Ilic, King’s College London School of Medicine, Division of Women’s Health, Women’s Health Academic Centre KHP, Assisted Conception Unit, 11th Fl., Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK. E-mail: [email protected] (Received 21 June 2011; accepted 12 September 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.623692
Xeno-free derivation and propagation of hES cells small molecules and genetic modifications could be clouded by the presence and effect of feeder cells. This requires the adaptation of stem cells to a feederfree culture environment. Early funding from the UK Medical Research Council facilitated collaboration between in vitro fertilization (IVF) units that had access to surplus human embryos, and scientists trying to derive stem cells. Since then, we have been working towards derivation of hES cell lines within a cGMP environment for use in human subjects. Step by step, the technology of hES cell line derivation has been adapted to an animal product-free environment (6,7). Extracellular matrix (ECM) constituents, such as human fibronectin, vitronectin, laminin and their combination, have reportedly supported the growth of hES cell lines with various culture media containing animal-based components (8). However, none of the conditions worked with the first commercially available xeno-free medium [KnockOut™ SR XenoFree, KO-SR XF Invitrogen (now Life Technologies, Carlsbad, CA, USA); D. Ilic, unpublished data]. The cells had either a very low rate of attachment and/or extensive spontaneous differentiation. We have resolved these initial technical obstacles and developed an optimized general protocol for entirely xeno-free derivation, propagation and adaptation of hES cells to feeder-free conditions.The protocol has been applied to normal (Supplementary Figures 1 and 2) as well as specific mutation-carrying (SMC) hES cell lines (i.e. mutations linked to Huntington’s disease and myotonic dystrophy type 1) (Supplementary Figures 3–5).
Methods Embryos The derivation of hES cells at King’s College London (London, UK) is under license from the UK Human Fertilisation and Embryology Authority (HFEA; research license number R0133) and also has local ethical approval (UK National Health Service Research Ethics Committee Reference 06/Q0702/90). No financial inducements are offered for donation. In accordance with HFEA regulations, a sample of each line that is derived is deposited in the UK Stem Cell Bank for distribution to academic and research centers internationally. Fresh embryos for the derivation were obtained from the Guy’s Assisted Conception Unit (ACU; London, UK) pre-implantation genetic diagnostics (PGD) program. Cryopreserved embryos no longer wanted for therapeutic use by patients were from both Guy’s ACU as well as external units, obtained through the human embryonic stem cell co-ordinator’s (hESCCO) network (9).
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Embryo culture Cryopreserved embryos were thawed using Quinn’s Advantage Thaw Kit (Sage, Pasadena, CA, USA) according to the manufacturer’s protocol. Fresh and thawed cleavage stage embryos were cultured in Quinn’s Advantage Protein Plus Blastocyst Medium (Sage) at 37°C, in 5% CO2 and 5% O2, up to blastocyst stage. Laser-assisted blastomere biopsy It is critically important to remove all of the cumulus and corona cells because they carry maternal genetic material and can be a source of contamination in the genetic analysis if released during the biopsy procedure. Biopsies were carried out when the embryos had reached the 6–8-cell stage. The embryo was secured in Quinn’s Advantage N2-hydroxyethylpiperazineN2-ethanesulfonic acid (HEPES)-buffered medium, Ca2⫹/Mg2⫹-free, supplemented with human serum albumin (Sage), by holding the pipette in such a way that the blastomere for the biopsy was at the 3 o’clock position. The zona was drilled with a gentle stream of acid Tyrode’s solution (Sage) by rotating the hatching air syringe clockwise until the zona began to thin and lose its structure. Once a 30–40-μm hole had been made, the blastomere was carefully aspirated, transferred into the drop away from the embryo and examined for the presence of nucleus (Figure 1a). Pre-implantation genetic haplotyping for Huntington’s disease and myotonic dystrophy type 1 Single blastomeres biopsied from embryos were washed through polyvinylpyrrolidone/phosphatebuffered saline (PBS; Sage) and collected in 2.5 μL 200 mM NaOH, 50 mM dithiothreitol using a fine polished glass capillary, and overlaid with mineral oil. The cells were lysed by incubation at 65°C for 10 min and the lysate was then neutralized with 2.5 μL 200 mM tricine before undergoing whole genome amplification by multiple displacement amplification (MDA). Cell lysates were prepared for MDA using an REPLI-g Midi kit (Qiagen, Crawley, UK) by adding 45 μL reaction master mix (15 μL nuclease-free water, 29 μL REPLI-g Midi reaction buffer, 1 μL REPLI-g Midi DNA polymerase), then incubated at 30°C for 16 h followed by inactivation at 65°C for 3 min and storage at 4°C until polymerase chain reaction (PCR) analysis was performed. Polymorphic markers flanking or intragenic to the genes of interest, myotonic dystrophy type 1 protein kinase (DMPK) and huntingtin (HTT), were identified using the University of California Santa Cruz (UCSC) Genome Browser created by the Genome Bioinformatics Group of UCSC (Santa Cruz, CA, USA; http://www.genome.ucsc.edu). Primers were then designed for 17 microsatellite markers for each
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Figure 1. The principle of normal and SMC hES cell line derivation. (a–e) Single blastomere biopsy from a cleavage-stage (day-3 post-fertilization) embryo. (a) Cleavage-stage embryo held with a holding pipette (HP) is exposed to a gentle stream of acid Tyrode’s solution coming through a Tyrode’s pipette (TP). (b) A blastomere popping through a Tyrode’s solution-drilled hole in the zona pellucida (arrow). (c, d) The blastomere is carefully aspirated using an aspiration pipette (AP). (e) The biopsied blastomere is examined for the presence of a nucleus and DNA is used for mutation diagnosis. (f–i) Laser-mediated ICM dissection. The ICM and TE of a well-developed blastocyst are separated with a series of laser pulses (double-arrow). (j, k) ICM outgrowth on mitotically inactivated HFF, 5–7 days after plating. (j) Two distinct cell populations within an outgrowth; hES cell-like colony and TE cells could be distinguished under higher magnification about 5–7 days post-plating. (k) TE is dissected out with a needle. S, needle scratches on tissues culture plastic. (l) Typical 12–15-day-old hES cell colony on mitotically inactivated HFF.
gene using Primer3 software (http://frodo.wi.mit.edu/ primer3) and checked for the presence of single nucleotide polymorphisms (SNP) using the SNPCheck bioinformatics program from the National Genetics Reference Laboratory (Manchester, UK; https:// ngrl.manchester.ac.uk/SNPCheckV2/snpcheck. htm), and for human ALU repeats using a nucleotide BLAST search (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). The primers were designed to enable testing for Huntington’s disease using a single multiplex and for myotonic dystrophy using two multiplexes, with each marker being amplified at a different PCR product size and assigned a tag (6FAM™, VIC®, NED™ or PET™ dyes; Applied Biosystems (now Life Technologies, Carlsbad, CA, USA) to ensure no overlap in marker fragments. A universal tagged primer approach was used (10,11). PCR were per-
formed in 10-μL reactions containing DNA, 0.4 μL of an optimized non-fluorescent tagged primer mix, 4 pmol fluorescently labeled universal tag primer mix, and 5 μL 2 ⫻ Qiagen Multiplex PCR Master Mix (containing HotStarTaq® DNA polymerase, Multiplex PCR buffer with 6 mM MgCl2, dNTP mix). Amplifications were performed on a G-Storm 2 thermal cycler using the following conditions: initial denaturation step of 95°C for 15 min, followed by 25 cycles of 94°C for 30 s, 59°C for 60 s, 72°C for 60 s; then 72°C for 5 min, followed by 60°C for 20 min. PCR products were run on an ABI3730 genetic analyzer using POP-6 polymer with a Genescan-600 LIZ size standard (Applied Biosystems) and analyzed using GeneMarker (SoftGenetics, State College, PA, USA). Haplotypes were constructed manually from the genotype data.
Xeno-free derivation and propagation of hES cells Laser-assisted dissection of inner cell mass Manipulation was carried out in biopsy dishes with microdrops of Ca2⫹/Mg2⫹-free HEPES-buffered medium under oil. The blastocysts were secured with a holding pipette at the region of the zona pellucida opposite the inner cell mass (ICM) and a biopsy needle prepared to aspirate the hatched area (Figure 1b). Using a Saturn Active laser (1.480 nm, 400 mW) and Cronus software (both from Research Instruments, Cornwall, UK), laser pulses were directed at the trophectoderm (TE) cell junctions adjacent to the ICM cells but outside of the safety circle. At this point, both the hatched and zona-contained cavities tended to collapse, so the biopsy needle was used to secure the hatched cells and pull them gently away from the main body of the blastocyst. The laser firing was repeated as appropriate whilst gently retracting the biopsy needle until the two sections separated. The cellular area containing the ICM was then washed and transferred to plates containing mitotically inactivated human neonatal foreskin fibroblasts (HFF). hES cell line derivation and culture ICM was plated on mitotically inactivated HFF in Quinn’s Advantage Protein Plus Blastocyst Medium supplemented with 25 ng/mL human recombinant fibroblast growth factor (hrFGF; R&D Systems, Minneapolis, MN, USA) and incubated at 37°C in 5% CO2 and 5% O2 for 3 days without disturbing. Medium was replaced, 50% each time, every 2–3 days with KO-SR XF, also supplemented with 25 ng/mL hrFGF. TE cells were removed mechanically from outgrowth (12,13). hES colonies were expanded and cryopreserved at the third passage. For initial adaptation to feeder-free conditions, the hES cells were cultured for 1–3 passages in either KO-SR XF or TeSR2 on mitotically inactivated HFF. hES cells were then transferred to native decellularized HFF ECM and continuously propagated in either KO-SR XF or TeSR2. Passage of hES cell colonies was performed manually. For differentiation assessment, hES cells were cultured for up to 3 weeks in KnockOut™ SR XenoFree (KO-SR) supplemented with 10% fetal bovine serum (FBS). Medium was exchanged every 2–3 days. Vitrification Clumps of hES cell colonies ready for cryopreservation were placed into ES-HEPES solution [20% KO-SR XF in 2 mM HEPES/Dulbecco’s modified Eagle medium (DMEM)]. Six to eight clumps were transferred to a 30–40-μL drop of 10% vitrification solution [10% ethylene glycol, 10% dimethylsulfoxide (DMSO), 80% ES-HEPES] for 1 min. The colonies were then quickly transferred into an adjacent 30–40-μL drop of 20% vitrification solution (20% ethylene glycol, 20%
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DMSO, 30% ES-HEPES, 30% 1 M sucrose solution in 80% ES-HEPES/20% KO-SR XF) for 25 s. Finally, up to eight hES cell clumps were aspirated into a vitrification straw (MTG, Bruckberg, Germany) using capillary action and snap frozen by placing the straw horizontally into liquid N2. Preparation of native decellularized HFF ECM We tested two protocols for preparation of native decellularized HFF ECM, one using sodium deoxycholate (DOC) (14) and another using Triton X-100 (15,16). In both cases, the mitotically inactivated HFF were rinsed in PBS, and incubated either at 4°C for 30 min with the DOC-based cell extraction buffer (0.5% DOC in 10 mM Tris/HCl, pH 8.0) or at 37°C for 3–5 min with the Triton X-100-based cell extraction buffer [0.5% (v/v) Triton X-100 and 20 mM NH4OH in PBS]. Complete decelluarization was confirmed with Hoechst 33342 staining (Invitrogen). Remaining decellularized HFF ECM was gently rinsed at least three times with PBS and used either immediately or stored at 4°C for up to 1 week. ECM was preserved better using Triton X-100-based cell extraction buffer, and all hES cell cultures in either KO-SR XF or TeSR™2 (StemCell Technologies, Vancouver, BC, Canada) medium were therefore carried out on native decellularized HFF ECM prepared using this protocol (see Supplementary Figure 6). Before it was released for use, each preparation was examined for the quality of the ECM with immunodetection of fibronectin, collagen IV and laminin 1 network. All antibodies are purchased from (Sigma, St. Louis, MO, USA). Pluripotency markers Pluripotency was assessed using two different techniques: enzymatic activity assay [alkaline phosphatase (AP) assay] and immunostaining. The AP assay was performed according to the manufacturer’s instructions (ATCC, Manassas, VA, USA). Undifferentiated or in vitro differentiated hES cell cultures were fixed in 3.8% paraformaldehyde for 20 min and then permeabilized in 90% acetone for 10 min or 0.2% Triton X-100 in Dulbecco’s phosphate buffer saline (DPBS) for 5 min, rinsed well and incubated overnight at 4°C with primary antibodies against tumor rejection antigen (TRA)-1-60, TRA-1-81 (Millipore, Billerica, MA, USA), nanog (R&D Systems, Minneapolis, MN, USA), octamer binding protein 4 (Oct4) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), smooth muscle actin (SMA), βIII tubulin or α-fetoprotein (αFP) (Sigma, St. Louis, MO, USA). After washing in DPBS, samples were incubated with appropriate secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at room temperature,
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rinsed again and mounted in Vectashield® mounting medium containing DNA counterstain 4′,6 diamidino2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Samples were analyzed using a Nikon 50i epifluorescence microscope equipped with appropriate filters. Images were captured with an Infinity1 CCD camera (Lumenera, Ottawa, ON, Canada) and processed using Adobe Photoshop CS5 software (Adobe, San Jose, CA, USA). Genotyping DNA was extracted from hES cell cultures using a Chemagen DNA extraction robot according to the manufacturer’s instructions. DNA was quantified using a Nanodrop spectrophotometer according to the manufacturer’s instructions, and the DNA quality was checked by agarose gel electrophoresis. Amplification of polymorphic microsatellite markers was carried out as described elsewhere (17). Briefly, DNA was amplified using two multiplexes, one of 17 PCR primer pairs for markers on chromosomes 13, 18 and 21, and one of 14 primer pairs for markers on the X and Y chromosomes. PCR products were separated on an ABI3100 capillary genetic analyzer, and results were analyzed using ABI Genotyper software. Allele sizes were recorded to give a unique fingerprint of each cell line. Array CGH DNA (1 μg) was labeled using a comparative genomic hybridization (CGH) labeling kit (Enzo Life Sciences, Farmingdale, NY, USA), following the manufacturer’s instructions. Labeled DNA was purified post-labeling using QIAquick PCR purification kit (Qiagen) following the manufacturer’s instructions. Labeling efficiency and yield were assessed by spectrophotometry (Nanodrop,Wilmington, DE, USA). An Agilent (Santa Clara, CA, USA) 4 ⫻ 44 K platform with either Wessex NGRL design 017457 or design 028469 was used; hybridization, washing and scanning of arrays were as per the manufacturer’s protocols. Image quantification, array quality control and aberration detection were performed using feature extraction and DNA analytics software packages (Agilent, Santa Clara, CA, USA). Manufacturer’s recommendations were followed. Ninety-five per cent of array data were required to pass quality control (QC). The ADM-2 algorithm at threshold 6 (with a 3-probe sliding window providing a mean detection interval of 200 kb) was used for aberration calling. HLA typing HLA-A, -B and -DRB1 typing was performed with a PCR sequence-specific oligonucleotide probe (SSOP; Luminex, Austin, TX, USA) hybridization protocol
at the certified Clinical Transplantation Laboratory, Guy’s and St Thomas’ NHS Foundation Trust and Serco Plc. (GSTS) Pathology (Guy’s Hospital, London, UK). The results were matched against the National Marrow Donor Program (NMDP) website (www.marrow.org). The MatchView® program shows potential matches on the NMDP-operated registry Be The Match Registry® at the moment of using the tool. The registry includes approximately 8 million adult donors and more than 160 000 searchable cord blood units. It is updated constantly as new donors and cord blood units are added and others removed. The registry was accessed for both KCL-19 and KCL-20 on 23 December 2010. Results and discussion PGD is used to prevent the transmission of genetic disorders to offspring. Different practices are used in various PGD centers epending on national regulations, including the method and timing of biopsy. We routinely remove single blastomeres from cleavage-stage (day 3 post-fertilization) embryos (Figure 1a–e) for the purpose of isolating DNA from the biopsied cell and subjecting it, following whole genome amplification, to haplotype analysis to identify SMC embryos (18). Derivation of hES cell lines is attempted using embryos carrying the familial mutation, and therefore unsuitable for clinical use, which couples donate for research with consent. The ICM is isolated mechanically, using a laser, from either normal or SMC well-expanded blastocysts (day 6 or 7), which have a distinct ICM (Figure 1f–i). Earlier stages or less well-expanded blastocysts are generally not used for mechanical attempts, as the risk of damage to the ICM is too great. Dissected ICM is plated in blastocyst medium (Sage) supplemented with 25 ng/mL hrFGF (R&D Systems) on a feeder layer of mitotically inactivated HFF. We start replacement of about half of the medium in the dish with KO-SR XF supplemented with 25 ng/mL hrFGF on the third day of culture. The medium is refreshed every 2–3 days. If needed, TE cells are removed mechanically from the outgrowth 5–7 days after plating (Figure 1j–k) (12,13). By 7 days, a putative hES cell colony is evident in the culture and the first subculture is performed by 12–15 days (Figure 1l). The hES cell colonies are expanded by manual passage, and vitrified when a sufficient number of cells is available. The presence of pluripotency markers and ability to differentiate into derivatives of all three germ layers is verified routinely before freezing and after thawing. Over the last 18 months, we have derived 12 normal and SMC hES cell lines under these conditions. Adapting hES cells to feeder-free conditions, although not easy, can become a standard procedure
Xeno-free derivation and propagation of hES cells that will take a skillful, experienced laboratory technician about a month to accomplish. An additional month will be needed to master the enzymatic passage steps. However, the standard protocols used for normal hES cell lines may not always work for SMC hES cell lines; defective cell–cell and cell–ECM interactions have been implicated in the phenotype of many diseases, from cancer to bleeding disorders and various neurodegenerative conditions. Indeed, the combination of CELLstart™ (Invitrogen), commercially available humanized ECM and a serum-free medium StemPro® (Invitrogen), which was successfully used routinely with more than 40 normal hES cell lines (D. Ilic, unpublished data), did not support growth of Huntington’s disease SMC hES cell lines. The most commonly used substrate Matrigel™ is a complex mixture of basement membrane ECM components prepared from mouse tumors. hES cells on Matrigel can be grown in the presence of several defined culture media, available from different suppliers but by definition not xeno-free. We hypothesized that providing normal or SMC hES cells with a native human ECM microenvironment, similar to Matrigel, might support growth of both undifferentiated normal and SMC hES cell lines (Figure 2a) (14,19,20). To define xeno- and feeder-free conditions suitable for both normal and SMC hES cell lines, we focused on two normal (KCL-19 and KCL-20) and three SMC hES cell lines (KCL-12 and KCL-13, carrying Huntington’s disease, and KCL-18, carry-
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ing myotonic dystrophy type 1 mutation; see Supplementary Figures 1–5). We tested the growth of these five hES cell lines on native decellularized HFF ECM in the presence of two commercially available xeno-free culture media, KO-SR XF and TeSR2 (Stemcell Technologies). The typical morphology of undifferentiated hES cell colonies (well-defined colony edges, a tightly packed colony and the absence of differentiated cells), presence of pluripotency markers and ability of cells to differentiate into derivatives of three germ layers, demonstrated that native decellularized HFF ECM and TeSR2 was a superb combination for maintaining the undifferentiated phenotype of all the hES lines tested (Figure 2b). KCL-12 and KCL-13 SMC hES cell lines cultured on a native decellularized HFF ECM in the presence of KO-SR XF were prone to lower cell attachment and spontaneous cell differentiation, whereas in the presence of TeSR2 we had no such difficulties. Three well-trained laboratory staff doing the evaluation independently ended up with identical results; it is, therefore, possible that this difficulty reflects the Huntington’s disease-specific phenotype, which deserves further investigation at the molecular level. Although early data from clinical trials with hES cell-derived cell therapy (21) are reassuring and the need for immunosuppression is possibly less than expected, one has to keep in mind that the therapies target immunologically privileged sites and it is likely that, for broader applications, in order to minimize the
Figure 2. Normal and SMC hES cell lines cultured on native decellularized HFF ECM. (a) Live staining of native HFF-generated ECM before processing for decellularization. Fibronectin (red) and nuclei visualized with Hoechst 33342 (blue). (b) hES cell lines propagated on native decellularized HFF ECM TeSR2 medium retain typical morphology and pluripotency marker expression. TRA-1-81 (green); fibronectin (red). (c) Fibronectin, (d) laminin 1 and (e) collagen IV and an intricate network of fibers.
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need for immunosuppression, close HLA-matching of lines to recipients will be required, in particular for a range of ethnic backgrounds (22–24). Therefore, we routinely perform HLA typing on all our normal hES cell lines. We have described a comprehensive methodology and standards for the derivation and propagation in xeno-free conditions of hES cell lines, which are therefore suitable for use in human subjects without risk of detrimental agents. The optimization of protocols is a continuous process and new commercially available media formulations and substrates, such as StemAdehereXF™ (Primorigen, Madison, WI, USA) may further simplify culture. The development of these protocols, along with methods for robust genetic characterization of the cell lines (25), represents a critically important milestone in the implementation of hES cells in the treatment of human disease. Acknowledgments This work was supported by Medical Research Council UK grants. Author contributions: DI, ES and PB designed the methods; ES, VW, JL, DS, AP, NK, SC and MS performed experiments; GC obtained patient consents; HP and CO were responsible for genomic analyzes; ES, HP and MS contributed to the writing of the methods; DI, CO and PB wrote the manuscript. Competing financial interests: The authors have declared that no competing interests exist. References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7. 2. Stephenson EL, Braude PR, Mason C. International community consensus standard for reporting derivation of human embryonic stem cell lines. Regen Med. 2007;2:349–62. 3. Interim UK Regulatory Route Map for Stem Cell Research and Manufacture, version March 12, 2009. Download available at: www.mhra.gov.uk/Howweregulate/Medicines/Medicinesregulatorynews/CON041337, accessed on October 13, 2011. 4. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med. 2005;11:228–32. 5. Salaway T, Ilic D. Logistics of stem cell isolation, preparation and delivery for heart repair: concerns of clinicians, manufacturers, investors and public health. Regen Med. 2008;3:83–91. 6. Ilic D, Giritharan G, Zdravkovic T, Caceres E, Genbacev O, Fisher SJ, et al. Derivation of human embryonic stem cell lines from biopsied blastomeres on human feeders with minimal exposure to xenomaterials. Stem Cells Dev. 2009;18: 1343–50.
Supplementary material available online Supplementary Figures 1–6
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