Clump Passaging and Expansion of Human Embryonic and Induced Pluripotent Stem Cells on Mouse Embryonic Fibroblast Feeder Cells

Odelya Hartung1, Hongguang Huo1, George Q. Daley2,1,3,4,5,6, Thorsten M. Schlaeger1,2

1 Stem Cell Program, Children's Hospital Boston, Boston, Massachusetts, 2 Harvard Stem Cell Institute, Cambridge, Massachusetts, 3 Stem Cell Transplantation Program, Division of Pediatric Hematology Oncology, Children's Hospital Boston, and Dana‐Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, 4 Manton Center for Orphan Disease Research, Boston, Massachusetts, 5 Division of Hematology, Brigham and Women's Hospital, Boston, Massachusetts, 6 Howard Hughes Medical Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.10
DOI:  10.1002/9780470151808.sc01c10s14
Online Posting Date:  August, 2010
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Abstract

The ability of human embryonic stem cells (hESCs) to differentiate into essentially all somatic cell types has made them a valuable tool for studying human development and has positioned them for broad applications in toxicology, regenerative medicine, and drug discovery. This unit describes a protocol for the large-scale expansion and maintenance of hESCs in vitro. hESC cultures must maintain a balance between the cellular states of pluripotency and differentiation; thus, researchers must use care when growing these technically demanding cells. The culture system is based largely on the use of a proprietary serum-replacement product and basic fibroblast growth factor (bFGF), with mouse embryonic fibroblasts as a feeder layer. These conditions provide the basis for relatively inexpensive maintenance and expansion of hESCs, as well as their engineered counterparts, human induced pluripotent stem cells (hiPSCs). Curr. Protoc. Stem Cell Biol. 14:1C.10.1-1C.10.15. © 2010 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; human induced pluripotent stem cells; mouse embryonic fibroblasts; bFGF

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bulk Passaging and Expansion of Human Embryonic Stem Cells (Pick to Remove)
  • Basic Protocol 2: Mechanical Passaging of Human Embryonic Stem Cells (Pick to Keep)
  • Support Protocol 1: Freezing Stocks of Human Pluripotent Stem Cells
  • Support Protocol 2: Thawing Cryogenically Preserved Human Embryonic Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Bulk Passaging and Expansion of Human Embryonic Stem Cells (Pick to Remove)

 Materials
  • 0.1% (w/v) gelatin solution (Millipore, cat. no. ES-006-B)
  • iMEF medium (see recipe)
  • 70% (v/v) isopropanol in water (VWR, cat. no. EM-PX1834-1)
  • Mitotically inactivated (gamma irradiated) CF-1 MEFs (iMEFS; Global Stem, cat. no. GSC-6001G), frozen
  • Confluent tissue culture well (9.6 cm2 or greater) of hESCs on MEFs
  • hESC medium (see recipe)
  • DMEM/F-12 (Stem Cell Technologies, cat. no. 36254)
  • 1 mg/ml collagenase IV (see recipe)
  • 6-well tissue culture plates (VWR, cat. no. 73520-906)
  • 37°C incubator
  • 15- and 50-ml conical centrifuge tubes (BD Biosciences, cat. nos. 352097 and 352070)
  • Tube rack
  • Centrifuge
  • Stereomicroscope (e.g., Discovery V8 with transmitted light darkfield base, Zeiss)
  • Vacuum aspirator
  • Cell lifter (Corning, 3008)
  • 5-ml single-use glass serological pipets (VWR, cat. no. 93000-696)
  • 2-, 5-, 10-, 25-ml plastic serological pipets (VWR, cat. no. 53283)

Basic Protocol 2: Mechanical Passaging of Human Embryonic Stem Cells (Pick to Keep)

 Materials
  • Plate or well of hESCs on iMEFs
  • hESC medium (see recipe)
  • DMEM/F-12 (Stem Cell Technologies, cat. no. 36254)
  • Stereomicroscope (e.g., Discovery V8 with transmitted light darkfield base, Zeiss)
  • 27-G needle, ½-in. long (BD, cat. no. 305109)
  • Syringe, 1-ml or 3-ml volume (BD, cat. nos. 309628 and 309585)
  • Pipet Lite LTS Pipettor, 20 to 200 µl volume (Rainin, cat. no. L-200)
  • Pipet Lite 200-µl filter tips (Rainin, cat. no. RT-L200F)
  • 15- and 50-ml conical tubes (BD Bioscience, cat. nos. 352097 and 352070)
  • 5-, 10-, or 25-ml plastic serological pipets (VWR, cat. no. 53283)
  • Additional reagents and equipment for preparing iMEF-coated tissue culture plates (Basic Protocol 1)

Support Protocol 1: Freezing Stocks of Human Pluripotent Stem Cells

 Materials
  • 2× hESC freezing medium (see recipe)
  • Confluent tissue culture well (9.6 cm2 or greater) of hESCs on iMEFs
  • hESC medium (see recipe)
  • Mr. Frosty (Nalgene, cat. no. 5100-0001)
  • Cryovials (Nalgene, cat. no. 5000-1020)
  • –80°C freezer
  • Liquid nitrogen cryostorage unit
  • Additional reagents and equipment for collecting hESCs (Basic Protocol 1)

Support Protocol 2: Thawing Cryogenically Preserved Human Embryonic Stem Cells

 Materials
  • hESC medium (see recipe)
  • 70% (v/v) isopropanol in water
  • Frozen hESCs (Support Protocol 1)
  • 6-well tissue culture plates of iMEFs (see Basic Protocol 1)
  • DMEM/F-12 (Stem Cell Technologies, cat. no. 36254)
  • 15- and 50-ml conical tubes (BD, cat. nos. 352097 and 352070)
  • Centrifuge

NOTE: The authors have found that the addition of 10 µM of ROCK inhibitor Y-27632 to the plating medium might greatly increase cell recovery after a thaw (see unit 1C.8).
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Figures

  •  FigureFigure 1C.10.1 Morphology of hESCs cultured on iMEFs. An hESC culture in good condition will display the standard hallmarks of hESC colonies, including defined borders and homogeneous cell types within the colonies. (A) hESC culture at optimal passage point. It is normal for hESC cultures to have up to 10% spontaneous differentiation. Types of spontaneous differentiation vary, but they include central differentiation (B, C) and peripheral differentiation (D). Scale bar = 250 µm.
    View Image
  •  FigureFigure 1C.10.2 Demonstration of pick-to-remove (A-C) and pick-to-keep (D-F) methods. For pick-to-remove, remove areas of spontaneous differentiation (A) using a slow vacuum aspirator attached to a filter-free 12.5-µl pipet tip (B,C). Aspirating the differentiation area slowly avoids excessive medium depletion and removal or damage to undifferentiated hESCs. For mechanical passaging, use the tip of a 27-G needle to score undifferentiated colonies into small fragments (E). Once colonies have been scored, carefully push fragments off and collect them using a 200-µl tip (F). Scale bar = 500 µm.
    View Image
  •  FigureFigure 1C.10.3 Diagram of a slow-vacuum aspirator.
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  •  FigureFigure 1C.10.4 A colony of hESCs that has been incubated in collagenase IV splitting solution for 15 min. Scale bar = 250 µm.
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  •  FigureFigure 1C.10.5 Morphologies of hESCs cultured on varying MEF densities. (A) iMEFs plated too sparsely are unable to support proper hESC pluripotency, and cause the periphery of the colony to differentiate. (B) iMEFs plated too densely do not provide hESCs the room required to grow optimally. Scale bar = 250 µm.
    View Image

Videos

Literature Cited

Literature Cited
    Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P., Harris, C.P., Waknitz, M.A., Itskovitz-Eldor, J., and Thomson, J.A. 2000. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227:271-278.
    Amit, M., Margulets, V., Segev, H., Shariki, K., Laevsky, I., Coleman, R., and Itskovitz-Eldor, J. 2003. Human feeder layers for human embryonic stem cells. Biol. Reproduct. 68:2150-2156.
    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.
    Lerou, P.H., Yabuuchi, A., Huo, H., Miller, J.D., Boyer, L.F., Schlaeger, T.M., and Daley, G.Q. 2008. Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos. Nat. Protoc. 3:923-933.
    Levenstein, M.E., Ludwig, T.E., Xu, R.H., Llanas, R.A., VanDenHeuvel-Kramer, K., Manning, D., and Thomson, J.A. 2006. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24:568-574.
    Liu, Y., Song, Z., Zhao, Y., Qin, H., Cai, J., Zhang, H., Yu, T., Jiang, S., Wang, G., Ding, M., and Deng, H. 2006. A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem. Biophys. Res. Commun. 346:131-139.
    Ludwig, T.E. and Thomson, J.A. 2009. Defined, feeder-independent medium for human embryonic stem cell culture. Curr. Protoc. Stem Cell Biol. 2:1C.2.1-1C.2.16.
    Ludwig, T.E., Bergendahl, V., Levenstein, M.E., Yu, J., Probasco, M.D., and Thomson, J.A. 2006. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3:637-646.
    Martin, M.J., Muorti, A., Gage, F., and Varki, A. 2005. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Medicine 11:228-232.
    Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147.
    Vallier, L., Alexander, M., and Pedersen, R.A. 2005. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J. Cell Sci. 118:4495-4509.
    Wang, L., Li, L., Menendez, P., Cerdan, C., and Bhatia, M. 2005. Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105:4598-4603.
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