Reprogramming of Mouse, Rat, Pig, and Human Fibroblasts into iPS Cells

Kuppusamy Rajarajan1, Marc C. Engels1, Sean M. Wu2

1 Cardiovascular Research Center, Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, 2 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.15
DOI:  10.1002/0471142727.mb2315s97
Online Posting Date:  January, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The induction of pluripotency in somatic cells by transcription‐factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient‐specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically modified large animal models or potentially transplantable donor tissues or organs. This unit describes the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts. Curr. Protoc. Mol. Biol. 97:23.15.1‐23.15.32. © 2012 by John Wiley & Sons, Inc.

Keywords: induced pluripotent stem cells; reprogramming; fibroblasts; mouse; rat; human; pig; lentivirus; retrovirus

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Culture of Fibroblasts
  • Support Protocol 1: Production of Lentivirus
  • Basic Protocol 2: Infection of Fibroblasts Using Pluripotency Factors
  • Basic Protocol 3: Establishment and Maintenance of Mouse iPS Cells
  • Alternate Protocol 1: Establishment and Maintenance of Rat iPS Cells
  • Alternate Protocol 2: Establishment and Maintenance of Pig iPS Cells
  • Alternate Protocol 3: Establishment and Maintenance of Human iPS Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation and Culture of Fibroblasts

  Materials
  • Pregnant female mice
  • Sterile Dulbecco's PBS (DPBS; with CaCl 2 and MgCl 2; Invitrogen, cat. no. 14040141)
  • Penicillin and streptomycin (Invitrogen, cat. no. 15140‐155)
  • 0.25% trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25200‐056)
  • MEF medium (see recipe)
  • Freezing/cryopreservation medium (see recipe)
  • Liquid N 2
  • Dissecting equipment including sterile forceps and sterile scalpel blades
  • 100‐mm petri dishes (BD Falcon 351029)
  • 15‐ and 50‐ml conical tubes
  • Centrifuge
  • Pipettors with 1000‐µl (P1000) pipet tips
  • 100‐mm cell culture dishes (Corning 430167), pre‐coated with 0.1% gelatin (see recipe)
  • Cryovials
  • Additional reagents and equipment for counting cells ( appendix 3F)

Support Protocol 1: Production of Lentivirus

  Materials
  • 293FT cells (Invitrogen, cat. no. R700‐07)
  • 293FT medium (see recipe)
  • FuGENE HD Transfection Reagent (Roche, cat. no. 04 709 705 001)
  • DMEM/high glucose (Invitrogen, cat. no. 11965092)
  • VSV‐G plasmid (Addgene, cat. no. 8454)
  • D8.9/psPAX2 plasmid (Addgene, cat. no. 12260)
  • STEMCCA (OKSM) Lentivirus Reprogramming Kit (Millipore, cat. no. SCR511)
  • 10% bleach disinfectant
  • Serum‐free DMEM medium (e.g., Invitrogen)
  • 100‐mm cell culture dishes (Corning, cat. no. 430167) precoated with 0.1% gelatin
  • 0.45‐µm disposable filters
  • Ultracentrifugation tubes (Beckman, cat. no. 344058)
  • Beckman Coulter Optima L‐90K ultracentrifuge with SW‐32 rotor

Basic Protocol 2: Infection of Fibroblasts Using Pluripotency Factors

  Materials
  • Embryonic fibroblasts or dermal fibroblasts (passage ≤3; see protocol 1)
  • MEF medium (see recipe)
  • Sterile Dulbecco's PBS (DPBS; with CaCl 2 and MgCl 2; Invitrogen, cat. no. 14040141)
  • 0.25% trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25200‐056)
  • Polybrene (Millipore TR‐1003‐G)
  • Pre‐aliquotted virus (see protocol 2)
  • 6‐well plate precoated with 0.1% gelatin
  • 15‐ml conical centrifuge tubes
  • Additional reagents and equipment for counting cells ( appendix 3F)
NOTE: Using fresh virus gives dramatically better infection efficiency. Virus should not be stored for longer than 1 week at 4°C before use. Virus should never undergo more than 1 freeze/thaw cycle, as multiple freeze/thaw cycles considerably reduce infection efficiency.NOTE: For mouse and rat fibroblasts, mouse four factor plasmids are most widely used for making iPSCs. For pig and human fibroblasts it is preferable to use human four‐in‐one factor constructs, but individual human factors can also be used. A STEMCCA Cre‐Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit is commercially available (Millipore).NOTE: For increased viral infection rate, multiple infections can be performed by aspirating and replenishing medium with fresh virus‐containing medium every 12 hr up until 36 hr after initial infection.NOTE: As a feeder layer for iPS cells, MEFs can be used for all four species. The MEFs should be growth‐inhibited by irradiation or mitomycin C treatment. For best results, a single monolayer of feeder cells should be plated onto gelatinized culture plates (for mouse and rat) or Matrigel‐coated culture plates (for pig and human).

Basic Protocol 3: Establishment and Maintenance of Mouse iPS Cells

  Materials
  • Mouse fibroblasts infected with pluripotency factors ( protocol 3); for timing, see Figure
  • MEF medium (see recipe)
  • 0.25% trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25200‐056)
  • Mouse iPS cell medium (see recipe) with double concentration of LIF and with normal concentration of mouse LIF as specified in Reagents and Solutions
  • Doxycyline (Sigma, cat. no. D9891); optional
  • 24‐ and 6‐well plates of inactivated mouse fibroblast feeder cells (unit 23.2)
  • Phosphate‐buffered saline (PBS) without CaCl 2 and MgCl 2 (Invitrogen 14190‐250)
  • Freezing/cryopreservation medium (see recipe)
  • Isopropanol
  • Liquid N 2
  • 70% ethanol
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • Light microscope with camera
  • Marker pen
  • 20‐µl (P20) and 1000‐µl (P1000) pipet tips
  • 2.0‐ml cryovials
  • Freezing chamber (e.g., Mr. Frosty; Thermo Scientific, cat, no. 5100‐0001)
  • Liquid nitrogen storage tank
  • Cryogenic handling gloves and eye protectors
  • Forceps
  • Additional reagents and equipment for preparing inactivated mouse embryonic fibroblast feeder cells (unit 23.2) and counting cells using a hemacytometer ( appendix 3F)

Alternate Protocol 1: Establishment and Maintenance of Rat iPS Cells

  Materials
  • Rat fibroblasts infected with pluripotency factors ( protocol 3); for timing, see Figure
  • MEF medium (see recipe)
  • 0.25% trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25200‐056)
  • Rat iPS cell medium (see recipe for serum‐free rat iPS medium or KOSR‐containing rat iPS medium) with double concentration of LIF and with normal concentration of mouse LIF as specified in Reagents and Solutions
  • Doxycyline (Sigma, cat. no. D9891); optional
  • 24‐ and 6‐well plates of inactivated mouse fibroblast feeder cells (unit 23.2)
  • Phosphate‐buffered saline (PBS) without CaCl 2 and MgCl 2 (Invitrogen 14190‐250)
  • Freezing/cryopreservation medium (see recipe)
  • Isopropanol
  • Liquid N 2
  • 70% ethanol
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • Light microscope with camera
  • Marker pen
  • 20‐µl (P20) and 1000‐µl (P1000) pipet tips
  • 2.0‐ml cryovials
  • Freezing chamber (e.g., Mr. Frosty; Thermo Scientific, cat. no. 5100‐0001)
  • Liquid nitrogen storage tank
  • Cryogenic handling gloves and eye protectors
  • Additional reagents and equipment for preparing inactivated mouse embryonic fibroblast feeder cells (unit 23.2) and counting cells using a hemacytometer ( appendix 3F)
NOTE: There are two recipes for rat iPS medium (see Reagents and Solutions): one for serum‐free rat iPS medium and one for KOSR‐containing rat iPS medium. Either of these media can be used for culturing rat iPSCs.

Alternate Protocol 2: Establishment and Maintenance of Pig iPS Cells

  Materials
  • Pig fibroblasts infected with pluripotency factors ( protocol 3); for timing, see Figure
  • MEF medium (see recipe)
  • 0.25% trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25200‐056)
  • 24‐ and 6‐well plates of inactivated mouse fibroblast feeder cells (unit 23.2)
  • Matrigel‐coated plates (see recipe; optional)
  • Pig iPS cell culture medium (see recipe)
  • mTeSR1 medium (optional)
  • Doxycyline (Sigma, cat. no. D9891); optional
  • Phosphate‐buffered saline (PBS) without CaCl 2 and MgCl 2 (Invitrogen, cat. no. 14190‐250)
  • Collagenase IV
  • Accutase (optional)
  • Dispase (optional)
  • ROCK inhibitor Y27632 (Calbiochem, cat. no. 688000)
  • TeSR2 (STEMCELL Technologies, cat. no. 05860; optional)
  • mFreSR (STEMCELL Technologies, cat. no. 05855; optional)
  • Freezing/cryopreservation medium (see recipe)
  • Isopropanol
  • Liquid N 2
  • 70% ethanol
  • 15‐ml conical tubes
  • Marker pen
  • Light microscope with camera
  • 20‐µl (P20) and 1000‐µl (P1000) pipet tips
  • Cell lifter (Corning, cat. no. 3008)
  • Eppendorf Model 5810R benchtop centrifuge (ore equivalent)
  • 2.0‐ml cryovials
  • Freezing chamber (e.g., Mr. Frosty; Thermo Scientific)
  • Liquid nitrogen storage tank
  • Cryogenic handling gloves and eye protectors
  • Forceps
  • Additional reagents and equipment for preparing inactivated mouse embryonic fibroblast feeder cells (unit 23.2) and counting cells using a hemacytometer ( appendix 3F)

Alternate Protocol 3: Establishment and Maintenance of Human iPS Cells

  Materials
  • Human fibroblasts infected with pluripotency factors ( protocol 3); for timing, see Figure
  • MEF medium (see recipe)
  • 0.25% trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25200‐056)
  • 24‐ and 6‐well plates of inactivated mouse fibroblast feeder cells (unit 23.2)
  • Human iPS cell culture medium (see recipe)
  • Doxycyline (Sigma, cat. no. D9891); optional
  • Matrigel‐coated plates (see recipe; optional)
  • mTeSR1 medium (optional)
  • Phosphate‐buffered saline (PBS) without CaCl 2 and MgCl 2 (Invitrogen 14190‐250)
  • Collagenase IV
  • Accutase (optional)
  • Dispase (optional)
  • ROCK inhibitor Y27632 (Calbiochem, cat. no. 688000)
  • TeSR2 (STEMCELL Technologies, cat. no. 05860; optional)
  • mFreSR (STEMCELL Technologies, cat. no. 05855; optional)
  • Freezing/cryopreservation medium (see recipe)
  • Isopropanol
  • Liquid N 2
  • 70% ethanol
  • 15‐ml conical tubes
  • Marker pen
  • Light microscope with camera
  • 20‐µl (P20) and 1000‐µl (P1000) pipet tips
  • Cell lifter (Corning, cat. no. 3008)
  • Eppendorf Model 5810R benchtop centrifuge (or equivalent)
  • 2.0‐ml cryovials
  • Freezing chamber (e.g., Mr. Frosty; Thermo Scientific)
  • Liquid nitrogen storage tank
  • Cryogenic handling gloves and eye protectors
  • Forceps
  • Additional reagents and equipment for preparing inactivated mouse embryonic fibroblast feeder cells (unit 23.2) and counting cells using a hemacytometer ( appendix 3F)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Boland, M.J., Hazen, J.L., Nazor, K.L., Rodriguez, A.R., Gifford, W., Martin, G., Kupriyanov, S. and Baldwin, K.K. 2009. Adult mice generated from induced pluripotent stem cells. Nature 461:91‐94.
   Byrne, J.A., Pedersen, D.A., Clepper, L.L., Nelson, M., Sanger, W.G., Gokhale, S., Wolf, D.P., and Mitalipov, S.M. 2007. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 22:450:497‐502.
   Campbell, K.H., McWhir, J., Ritchie, W.A., and Wilmut, I. 1996. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380:64‐66.
   Carey, B.W., Markoulaki, S., Hanna, J., Saha, K., Gao, Q., Mitalipova, M., and Jaenisch, R. 2009. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc. Natl. Acad. Sci. U.S.A. 106:157‐162.
   Chan, A.W., Dominko, T., Luetjens, C.M., Neuber, E., Martinovich, C., Hewitson, L., Simerly, C.R., and Schatten, G.P. 2000. Clonal propagation of primate offspring by embryo splitting. Science 287:317‐319.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Eggan, K., Akutsu, H., Loring, J., Jackson‐Grusby, L., Klemm, M., Rideout, W.M. 3rd, Yanagimachi, R., and Jaenisch, R. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. U.S.A. 98:6209‐6214.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., and Roberts, R.M. 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proc. Natl. Acad. Sci. U.S.A. 106:10993‐10998.
   Friedrich Ben‐Nun, I., Montague, S.C., Houck, M.L., Tran, H.T., Garitaonandia, I., Leonardo, T.R., Wang, Y.C., Charter, S.J., Laurent, L.C., Ryder, O.A., and Loring, J.F. 2011. Induced pluripotent stem cells from highly endangered species. Nat. Methods. 8:829‐831.
   Gurdon, J.B. 1962. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol. 10:622‐640.
   Hogan, B.L. 1976. Changes in the behavior of teratocarcinoma cells cultivated in vitro. Nature 263:136‐137.
   Jia, F., Wilson, K.D., Sun, N., Gupta, D.M., Huang, M., Li, Z., Panetta, N.J., Chen, Z.Y., Robbins, R.C., Kay, M.A., Longaker, M.T., and Wu, J.C. 2010. A nonviral minicircle vector for deriving human iPS cells. Nat. Methods. 7:197‐199.
   Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P., and Woltjen, K. 2009. Virus‐free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458:771‐775.
   Kang, L., Wang, J., Zhang, Y., Kou, Z., and Gao, S. 2009. iPS cells can support full‐term development of tetraploid blastocyst‐complemented embryos. Cell Stem Cell. 5:135‐138.
   Kobayashi, T., Yamaguchi, T., Hamanaka, S., Kato‐Itoh, M., Yamazaki, Y., Ibata, M., Sato, H., Lee, Y.S., Usui, J., Knisely, A.S., Hirabayashi, M., and Nakauchi, H. 2010. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142:787‐799.
   Li, W., Wei, W., Zhu, S., Zhu, J., Shi, Y., Lin, T., Hao, E., Hayek, A., Deng, H., and Ding, S. 2009. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell. 4:16‐19.
   Li, X., Meng, G., Krawetz, R., Liu, S., and Rancourt, D.E. 2008. The ROCK inhibitor Y‐27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev. 17:1079‐1085.
   Li, Y., Cang, M., Lee, A.S., Zhang, K., and Liu, D. 2011. Reprogramming of sheep fibroblasts into pluripotency under a drug‐inducible expression of mouse‐derived defined factors. PLoS One 6:e15947.
   Liu, H., Zhu, F., Yong, J., Zhang, P., Hou, P., Li, H., Jiang, W., Cai, J., Liu, M., Cui, K., Qu, X., Xiang, T., Lu, D., Chi, X., Gao, G., Ji, W., Ding, M., and Deng, H. 2008. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3:587‐590.
   Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R., Plath, K., and Hochedlinger, K. 2007. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1:55‐70.
   Nagy, A., Gócza, E., Diaz, E.M., Prideaux, V.R., Iványi, E., Markkula, M., and Rossant, J. 1990. Embryonic stem cells alone are able to support fetal development in the mouse. Development 110:815‐821.
   Okita, K., Ichisaka, T., and Yamanaka, S. 2007. Generation of germline‐competent induced pluripotent stem cells. Nature 448:313‐317.
   Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., and Yamanaka, S. 2008. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322:949‐953.
   Park, I.H., Lerou, P.H., Zhao, R., Huo, H., and Daley, G.Q. 2008a. Generation of human‐induced pluripotent stem cells. Nat. Protoc. 3:1180‐1186.
   Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., and Daley, G.Q. 2008b. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141‐146.
   Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., Isacson, O., and Jaenisch, R. 2009. Parkinson's disease patient‐derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136:964‐977.
   Sommer, C.A., Stadtfeld, M., Murphy, G.J., Hochedlinger, K., Kotton, D.N., and Mostoslavsky, G. 2009. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27:543‐549.
   Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., and Hochedlinger, K. 2008. Induced pluripotent stem cells generated without viral integration. Science 322:945‐949.
   Stadtfeld, M., Apostolou, E., Akutsu, H., Fukuda, A., Follett, P., Natesan, S., Kono, T., Shioda, T., and Hochedlinger, K. 2010. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465:175‐181.
   Takahashi, K. and Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663‐676.
   Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861‐872.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1827.
   Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.H., Li, H., Lau, F., Ebina, W., Mandal, P.K., Smith, Z.D., Meissner, A., Daley, G.Q., Brack, A.S., Collins, J.J., Cowan, C., Schlaeger, T.M., and Rossi, D.J. 2010. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7:618‐630.
   Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. 2007. In vitro reprogramming of fibroblasts into a pluripotent ES‐cell‐like state. Nature 448:318‐324.
   Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hämäläinen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., Kaji, K., Sung, H.K., and Nagy, A. 2009. PiggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458:766‐770.
   Wu, S.M. and Hochedlinger, K. 2011. Harnessing the potential of induced pluripotent stem cells for regenerative medicine. Nat. Cell Biol. 13:497‐505.
   Yu, J., Vodyanik, M.A., Smuga‐Otto, K., Antosiewicz‐Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917‐1920.
   Yusa, K., Rad, R., Takeda, J., and Bradley, A. 2009. Generation of transgene‐free induced pluripotent mouse stem cells by the piggyBac transposon. Nat. Methods 6:363‐369.
   Zhao, X.Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C.L., Ma, Q.W., Wang, L., Zeng, F., and Zhou, Q. 2009. iPS cells produce viable mice through tetraploid complementation. Nature 461:86‐90.
   Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Schöler, H.R., Duan, L., and Ding, S. 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4:381‐384.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library