Derivation and Propagation of hESC Under a Therapeutic Environment

Kuldip S. Sidhu1, Sarah Walke2, Bernard E. Tuch3

1 Stem Cell Laboratory, Faculty of Medicine, School of Psychiatry, The University of New South Wales, New South Wales, Australia, 2 Diabetes Transplant Unit, The Prince of Wales Hospital and The University of New South Wales, New South Wales, Australia, 3 New South Wales Stem Cell Network, New South Wales, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1A.4
DOI:  10.1002/9780470151808.sc01a04s6
Online Posting Date:  July, 2008
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Abstract

The pluripotent nature of human embryonic stem cells (hESC) makes them very attractive as a source of various cell types that could be used therapeutically in regenerative medicine. However, eliminating all sources of contamination, animal‐derived or human cell–derived, during hESC derivation and propagation is necessary before hESC derivatives can be used clinically. Although there is continuing progress toward this goal, none of the methods to date to produce hESC lines under good manufacturing practices (GMP) has been published. The long‐term success for GMP compliance depends critically on maintaining and implementing a stringent quality control system which is also dictated by the regulatory authorities in different countries. In this unit, an approach is described based upon the experience of this author and others towards achieving clinical‐grade hESC lines systematically involving all the steps from start to finish under GMP environment. This unit provides a basic layout for GMP set up to achieve quality controls, a step‐by‐step guide to producing new hESC lines under defined conditions, and standard operating procedures used to achieve this outcome. Curr. Protoc. Stem Cell Biol. 6:1A.4.1‐1A.4.31. © 2008 by John Wiley & Sons, Inc.

Keywords: hESC; GMP; standard operating procedures; quality control

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Derivation of a New hESC Line from Human Blastocysts
  • Support Protocol 1: hESC Weekly Culture Schedule
  • Support Protocol 2: Determining Viability of hESC by Carboxyfluorescein Diacetate (CFDA) and Propidium Iodide (PI)
  • Support Protocol 3: Passaging Intact hESC Colonies by Collagenase/Dispase Treatment
  • Support Protocol 4: Subculturing hESC Colonies by TrypLE Select Treatment
  • Support Protocol 5: Slow Freezing hESC Colonies/Clumps
  • Support Protocol 6: Vitrification (Fast Freezing) and Thawing hESC Colonies/Clumps
  • Support Protocol 7: Derivation of Serum‐Free Human Fetal Fibroblasts (HFF)
  • Support Protocol 8: Preparing Feeder Plates Using Human Fetal Fibroblasts
  • Support Protocol 9: Freezing and Thawing Frozen Human Fetal Fibroblasts
  • Support Protocol 10: Reagent Supply and Batch Testing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Derivation of a New hESC Line from Human Blastocysts

  Materials
  • Frozen human embryos, preferably blastocyst stage
  • Quinn's Advantage Cleavage and Blastocyst medium (SAGE BioPharma) supplemented with 5% human serum albumin (hSA; Sage Biopharma)
  • Oil for tissue culture (SAGE BioPharma)
  • SR medium plus bFGF (see recipe)
  • Mitotically inactivated (γ‐irradiated) human fetal fibroblasts (HFF) as feeder layer (see Support Protocols protocol 87 and protocol 98)
  • 4‐ or 6‐well culture plates (Greiner bio‐one, GmbH, Germany)
  • Inverted microscope (example, Leica DM‐IRB) with CCD camera and software to manipulate images, and with laser ablation system (e.g., XYClones; Hamilton Thorne Biosciences)
  • Portable CO 2 incubator (LEC Instruments, http://www.lecinstruments.com/incubator.htm; see Fig. )
  • Dissection and biopsy pipets (e.g., Cook IVF)
  • Water‐Jacketed CO 2 incubator (e.g., Gelaire, Sydney)
  • Nalgene Cryofreezing Containers (Fisher Scientific, Nalgene cat. no. 5100‐001)
  • Microchisel, 10× (e.g., Eppendorf) or insulin syringe with 23‐G needle
  • Biological safety cabinet (BSC) with a provision to keep a microscope inside for performing hESC sub culturing
  • Pipettor with 100‐µl tip
  • Additional reagents and equipment for preparing HFF feeder cells ( protocol 9)

Support Protocol 1: hESC Weekly Culture Schedule

  Materials
  • HFF, cryopreserved ( protocol 10)
  • hESC cultures ( protocol 1)
  • SR medium for both hESCs and HFF (see recipe)
  • Collagen IV‐coated 75‐cm2 flasks (see recipe)
  • Collagen IV‐coated 6‐well plates (see recipe)
  • γ irradiator

Support Protocol 2: Determining Viability of hESC by Carboxyfluorescein Diacetate (CFDA) and Propidium Iodide (PI)

  Materials
  • Carboxyfluorescein diacetate (CFDA)
  • DMSO
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (D‐PBS; Invitrogen)
  • Propidium iodide (PI)
  • hESC removed from the culture dish ( protocol 1) and placed in a microcentrifuge tube
  • 1‐ml pipettor
  • 20‐ to 200‐µl micropipettor
  • Hemacytometer
  • Fluorescent microscope equipped with UV filters

Support Protocol 3: Passaging Intact hESC Colonies by Collagenase/Dispase Treatment

  Materials
  • hESCs cultured on HFF feeders in 6‐well plates ( protocol 1)
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (D‐PBS; Invitrogen), prewarmed
  • 1 mg/ml collagenase (Invitrogen) in PBS, sterilized with a 0.22‐µm syringe filter and prewarmed
  • 0.5 mg/ml dispase (Invitrogen, cat. no. 17105‐041) in PBS, sterilized with a 0.22‐µm syringe filter and prewarmed
  • SR medium (see recipe), prewarmed to 37°C
  • Trypsin/EDTA (Invitrogen, cat. no. 25300‐054) or TrypLE Select (Invitrogen, cat. no. 12563‐011), prewarmed
  • Microscope
  • Plastic loop (Lazy‐L‐Spreader; Cole‐Parmer Instrument)
  • 15‐ml tube
  • Biological safety cabinet (BSC) Class II hood

Support Protocol 4: Subculturing hESC Colonies by TrypLE Select Treatment

  Materials
  • hESCs cultured on HFF feeders in 6‐well plates ( protocol 1)
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (D‐PBS; Invitrogen), prewarmed at 37°C
  • TrypLE Select, prewarmed
  • SR medium (see recipe), prewarmed
  • Plastic loop (Lazy‐L‐Spreader, Cole‐Parmer Instrument)
  • 15‐ml Falcon tube

Support Protocol 5: Slow Freezing hESC Colonies/Clumps

  Materials
  • hESC colonies harvested by collagenase/dispase ( protocol 4)
  • 30% SR medium [9 ml of 20% SR medium +1 ml KOSR (Invitrogen, cat. no. 10828‐028)]
  • Cryopreservation medium II: 6 ml Knockout DMEM (Invitrogen), 2 ml of 30% SR medium, 2 ml DMSO, sterile filtered using a 0.22‐µm syringe filter
  • HFF feeder plates ( protocol 9)
  • 15‐ml tube
  • Cryovials (Greiner Bio‐One, cat. no. 122263)
  • Nalgene Cryofreezing Containers (Fisher Scientific, Nalgene 5100‐001)
  • −80°C freezer
  • Cryoboxes (Crown Scientific)
  • Liquid nitrogen tank
  • 37°C water bath

Support Protocol 6: Vitrification (Fast Freezing) and Thawing hESC Colonies/Clumps

  Materials
  • HEPES (Invitrogen, no. 15630‐080)
  • DMEM (Invitrogen, no. 11965‐092)
  • KOSR (Invitrogen, no. 10828‐028)
  • Sucrose
  • Fetal bovine serum (FBS; Invitrogen, no. 16000‐044)
  • Ethylene glycol (Sigma, no. E‐9129)
  • Dimethylsulfoxide (DMSO; Sigma, D2650)
  • hESC colonies harvested by collagenase/dispase ( protocol 4)
  • Liquid nitrogen
  • HFF feeder plates ( protocol 9)
  • SR medium (see recipe)
  • 0.22‐µm syringe filter
  • 15‐ml tube
  • Pipettor
  • Organ culture dishes (Falcon, cat. no. 353037) for vitrification, prewarmed
  • Open pulled straws (LEC Instruments)
  • 5‐ml cryovials with holes punched through the upper section, the bottom, and lid using a heated 18‐G needle, attached to a cryostraw
  • Liquid nitrogen tank
  • Forceps
NOTE: Wear safety glasses and gloves when working with liquid nitrogen.

Support Protocol 7: Derivation of Serum‐Free Human Fetal Fibroblasts (HFF)

  Materials
  • Skin from 10‐ to 12‐week fetuses
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (D‐PBS; Invitrogen)
  • Penicillin‐streptomycin (Invitrogen)
  • TrypLE Select (Invitrogen)
  • SR medium (see recipe), equilibrated
  • 35‐mm petri dish
  • Scissors
  • 15‐ml conical centrifuge tube (Fisher, cat. no. 05‐539‐2)
  • Collagen type IV–coated 75‐cm2 tissue culture flask (see recipe)
  • Additional reagents and equipment for cryopreserving using a standard slow freezing procedure ( protocol 9)

Support Protocol 8: Preparing Feeder Plates Using Human Fetal Fibroblasts

  Materials
  • HFF in 75‐cm2 flask grown for 3 to 4 days in SR medium (see protocol 8)
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (D‐PBS; Invitrogen)
  • TrypLE Select
  • SR medium (see recipe), prewarmed
  • Human collagen IV (Sigma) and/or human laminin
  • 15‐ml conical centrifuge tubes
  • Benchtop centrifuge
  • 20‐µl pipettor
  • Hemacytometer
  • 6‐well tissue culture plates (Greiner bio‐one, GmbH)
  • γ irradiator
  • Additional reagents and equipment for counting cells (Phelan, )

Support Protocol 9: Freezing and Thawing Frozen Human Fetal Fibroblasts

  Materials
  • 75‐cm2 flask with HFF ( protocol 8)
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (D‐PBS; Invitrogen), prewarmed
  • TrypLE Select, prewarmed
  • SR medium (see recipe), prewarmed
  • Cryopreservation solution: 20% (v/v) DMSO in SR medium (see recipe), sterile filtered
  • 15‐ml tubes
  • Benchtop centrifuge
  • 0.22‐µm syringe filter
  • Cryovials
  • Nalgene Cryofreezing containers (Fisher Scientific, Nalgene 5100‐001)
  • −80°C freezer
  • Cryoboxes
  • Liquid nitrogen tank 37°C water bath

Support Protocol 10: Reagent Supply and Batch Testing

  Materials
  • Human foreskin fibroblast cultures ( protocol 8)
  • SR medium prepared with test and current lots of serum or serum replacement
  • 6‐well culture plates coated with test and current (control) lots of matrix
  • Additional reagents and equipment for cell counting (Phelan, )
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Figures

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Literature Cited

Literature Cited
   Amit, M., Shariki, C., Margulets, V., and Itskovitz‐Eldor, J. 2004. Feeder layer and serum‐free culture of human embryonic stem cells. Biol. Reprod. 70:837‐845.
   Beattie, G.M., Lopez, A.D., Bucay, N., Hinton, A., Firpo, M.T., King, C.C., and Hayek, A. 2005. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23:489‐495.
   Brimble, S.N., Zeng, X., Weiler, D.A., Luo, Y, Liu, Y, Lyons, I.G., Freed, W.J., Robins, A.J., Rao, M.S., and Schulz, T.C. 2004. Karyotypic stability, genotyping, differentiation, feeder‐free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev. 13:585‐597.
   Carpenter, M.K., Rosler, E.S., Fisk, G.J., Brandenberger, R., Ares, X., Miura, T., Lucero, M., and Rao, M.S. 2004. Properties of four human embryonic stem cell lines maintained in a feeder‐free culture system. Dev. Dynamics 229:243‐253.
   Cheng, L., Hammond, H., Ye, Z., Zhan, X., and Dravid, G. 2003. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21:131‐142.
   Draper, J.S., Smith, K., Gokhale, P., Moore, H.D., Maltby, E., Johnson, J., Meisner, L., Zwaka, T.P., Thomson, J.A., and Andrews, P.W. 2004. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22:53‐54.
   Ellerstrom, C., Strehl, R., Moya, K., Andersson, K., Bergh, C., Lundin, K., Hyllner, J., and Semb, H. 2006. Derivation of a xeno‐free human embryonic stem cell line. Stem Cells 24:2170‐2176.
   Genbacev, O., Krtolica, A., Zdravkovic, T., Brunette, E., Powell, S., Nath, A., Caceres, E., McMaster, M., McDonagh, S., Li, Y., Mandalam, R., Lebkowski, J., and Fisher, S.J. 2005. Serum‐free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil. Steril. 83:1517‐1529.
   Guhr, A., Kurtz, A., Friedgen, K., and Loser, P. 2006. Current state of human embryonic stem cell research: An overview of cell lines and their use in experimental world. Stem Cells 24:2187‐2191.
   Heins, N., Englund, M.C.O., Sjoblom, C., Dahi, U., Tonning, A., Bergh, C., Lindahl, A., Hanson, C., and Semb, H. 2004. Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22:367‐376.
   Hovatta, O., Mikkola, M., Gertow, K., Stromberg, A.M., Inzunza, J., Hreinsson, J., Rozell, B., Blennow, E., Andang, M., and Ahrlund‐Richter, L. A. 2003. Culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18:1404‐1409.
   Inzunza, J., Gertow, K., Stromberg, M.A., Matilainen, E., Blennow, E., Skottman, H., Wolbank, S., Ahrlund‐Richter, L., and Hovatta, O. 2005. Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells 23:544‐549.
   Inzunza, J., Sahlen, S., Holmberg, K., Stromberg, A.M., Teerijoki, H., Blennow, E., Hovatta, O., and Malmgren, H. 2004. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long‐term cultivation. Mol. Hum. Reprod. 10:461‐466.
   James, D., Levine, A.J., Besser, D., and Hemmati‐Brivanlou, A. 2005. TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132:1273‐1282.
   Kim, H.S., Oh, S.K., Park, Y.B., Ahn, H.J., Sung, K.C., Kang, M.J., Lee, L.A., Suh, C.S., Kim, S.H., Kim, D.W., and Moon, S.Y. 2005. Methods for derivation of human embryonic stem cells. Stem Cells 23:1228‐1233.
   Klimanskaya, I., Chung, Y., Meisner, L., Johnson, J., West, M.D., and Lanza, R. 2005. Human embryonic stem cells derived without feeder cells. Lancet 365:1636‐1641.
   Klimanskaya, I., Chung, Y., Becker, S., Lu, S.J., and Lanza, R. 2006. Human embryonic stem cell lines derived from single blastomeres. Nature (letter) 444:481‐485.
   Li, Y., Powell, S., Brunette, E., Lebkowski, J., and Mandalam, R. 2005. Expansion of human embryonic stem cells in defined serum‐free medium devoid of animal‐derived products. Biotechnol. Bioeng. 91:688‐698.
   Ludwig, T.E., Levenstein, M.E., Jones, J.M., Berggren, W.T., Mitchen, E.R., Frane, J.L., Crandall, L.J., Daigh, C.A., Conard, K.R., Piekarczyk, M.S., Llanas, R.A., and Thomson, J.A. 2006. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24:185‐187.
   Mallon, B.S., Park, K.Y., Chen, K.G., Hamilton, R.S., and McKay, R.D. 2006. Toward xeno‐free culture of human embryonic stem cells. Int. J. Biochem. Cell Biol. 38:1063‐1075.
   Miyamoto, K., Hayashi, K., Suzuki, T., Ichihara, S., Yamada, T., Kano, Y., Yamabe, T., and Ito, Y. 2004. Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells. Stem Cells 22:433‐40.
   Park, S.P., Lee, Y.J., Lee, K.S., Shin, A. H., Cho, H.Y., Chung, K.S., Kim, E.Y., and Lim, J.H. 2004. Establishment of human embryonic stem cell lines from frozen‐thawed blastocysts using STO cell feeder layers. Hum. Reprod. 19:676‐84.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Rajan, P., Smotrich, D., Ross, R., Larent, L., and Loring, J.F. 2007. Derivation of embryonic stem cells from human blastocysts. In Human Stem Cell Manual a Laboratory Guide (J.F. Loring, R.L. Wesselschmidt, and P.H. Schwartz eds.). Academic Press, N.Y.
   Revazova, E.S., Turovets, N.A., Kochetkova, O.D., Kindarova, L.B., Kuzmichev, L.N., Janus, J.D., and Pryzhkova, M.V. 2007. Patient‐specific stem cell lines derived from human parthenogenetic blastocysts. Cloning and Stem Cells 9:1‐18.
   Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. 2002. Human feeders support prolonged growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20:933‐936.
   Richards, M., Tan, S., Fong, C.Y., Biswas, A., Chan, W.K., and Bongso, A. 2003 Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21:546‐556.
   Rosler, E.S., Fisk, G.J., Ares, X., Irring, J., Miura, T., Rao, M.S., and Carpenter, M.K. 2004. Long‐term culture of human embryonic stem cells in feeder‐free conditions. Dev. Dynamics 229:259‐274.
   Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A.H. 2004. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of WNT signaling by a pharmacological GSK‐3‐specific inhibitor. Nat. Med. 10:55‐63.
   Sidhu, K.S. and Tuch, B. E. 2006. Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization. Stem Cells Devel. 15:61‐69.
   Sidhu, K.S., Ryan, J.P., and Tuch, B.E. 2008. Derivation of a new hESC line, endeavour‐1 and its clonal propagation. Stem Cells Devel. 17:41‐52.
   Stacey, G.N., Cobo, F., Nieto, A., Talavera, P., Healy, L., and Concha, A. 2006. The development of ‘feeder’ cells for the preparation of clinical grade hES cell lines: Challenges and solutions. J. Biotechno l. 125:583‐588.
   Stojkovic, P., Lako, M., Przyborski, S., Stewart, R., Armstrong, L., Evans, J., Zhang, X., and Stojkovic, M. 2005a. Human‐serum matrix supports undifferentiated growth of human embryonic stem cells. Stem Cells 23:895‐902.
   Stojkovic, P., Lako, M., Stewart, R., Pryzborski, S., Armstrong, L., Evans, J., Murdoch, A., Strachan, T., and Stojkovic, M. 2005b. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23:306‐314.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshal, V.S., and Jones, J.M. 1998. Embryonic stem cell line from human blastocysts Science 282:1145‐1147.
   Vallier, L., Alexander, M., and Pederson, R. A. 2005. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J. Cell Sci. 118:4495‐4509.
   Wang, Q., Fang, Z.F., Jin, F., Lu, Y., Gai, H., and Sheng, H.Z. 2005. Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells 23:1221‐1227.
   Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D., and Carpenter, M.K.. 2001. Feeder‐free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19:971‐974.
   Xu, C., Jiang, J., Sottile, V., McWhir, J., Lebkowski, J., and Carpenter, M. K. 2004. Immortalized fibroblast‐like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22:972‐980.
   Xu, C., Rosler, E., Jiang, J., Lebkowski, J. S., Gold, J. D., O'Sullivan, C., Delevan‐Boorsma, K., Mok, M., Bronstein, A., and Carpenter, M.K. 2005a. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 23:315‐323.
   Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., and Thomson, J. A. 2005b. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat. Methods 2:185‐190.
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