cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA

Yuhui Ni1, Yuanyuan Zhao1, Luigi Warren2, Jennifer Higginbotham3, Jiwu Wang3

1 Allele Biotechnology and Pharmaceuticals, Inc, San Diego, California, 2 Cellular Programming Inc, La Jolla, California, 3 Scintillon Institute, San Diego, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.6
DOI:  10.1002/cpsc.18
Online Posting Date:  November, 2016
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Abstract

Reprogramming somatic cells to generate induced pluripotent stem cells (iPSCs) has presented the biomedical community with a powerful platform to develop new models for human disease. To fully realize the promise of this technology in cell therapy and regenerative medicine, creating iPSCs under current Good Manufacture Practice (cGMP) conditions is paramount. Some reports have described efforts in this regard, resulting in iPSC lines that are cGMP compliant. The technology developed at Allele Biotechnology for footprint‐free, feeder‐free, and xeno‐free reprogramming using only mRNA is very suitable for creating iPSC lines through an established cGMP process. This technology has resulted in a licensing agreement between Allele Biotechnology and Ocata (formerly ACT, now a wholly owned division of Astellas) for clinical applications. All reagents and vessels are certified as cGMP‐produced, all equipment and software are certifiable, and all procedures are carried out in Industry ISO 7 or Class 10,000‐grade cleanrooms. In this revised version of the unit, we describe the core improvements to implement steps toward cGMP‐compliant generation of iPSCs. Recreating a process close to cGMP production in academic research will make these findings more applicable to translational research. © 2016 by John Wiley & Sons, Inc.

Keywords: induced pluripotent stem cells; iPSC; mRNA reprogramming; feeder‐free; xeno‐free; cGMP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: cGMP Reprogramming of Human Fibroblasts to Pluripotency
  • Support Protocol 1: Live Staining of iPSC Colonies
  • Support Protocol 2: Generation of in Vitro Transcription Template Constructs
  • Support Protocol 3: PCR‐based Production of IVT Templates From Plasmid Constructs
  • Support Protocol 4: Preparation of Synthetic Messenger RNA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: cGMP Reprogramming of Human Fibroblasts to Pluripotency

  Materials
  • Synthemax II‐SC (Corning, cat. no. 3535XX1)
  • Human fibroblasts (see introduction to this protocol)
  • RiPSCell iPSC reprogramming medium (Allele Biotechnology, cat. no. ABP‐SC‐RIPSCMED)
  • Carrier‐free B18R recombinant protein (Affymetrix eBioscience, cat. no. 34‐8185)
  • 6F mRNA reprogramming premix (Allele Biotechnology, cat. no. ABP‐SC‐6FMRNA)
  • SAINT‐RED transfection reagent and HBS, pH 7.4 (Synvolux Therapeutics, cat. no. SR‐1003‐01)
  • HEPES buffered saline (HBS)
  • TeSR‐E8 culture medium (StemCell Technologies, cat. no. 05940)
  • SCLift stem cell detaching reagent (Allele Biotechnology, cat. no. ABP‐SC‐SCLIFT)
  • Dulbecco's phosphate‐buffered saline (DPBS) with calcium and magnesium (Lonza, cat. no. 17‐513F)
  • 12.5‐cm2 flask (Corning, cat. no. 353018)
  • Cell counter (e.g., Orflo Technologies, cat. no. MXC002)
  • Humidified tissue‐culture incubator set to 37°C, 5% CO 2, and (if supported) 5% O 2

Support Protocol 1: Live Staining of iPSC Colonies

  Materials
  • iPSC colonies ( protocol 1)
  • Dulbecco's phosphate‐buffered saline (DPBS) with calcium and magnesium (Lonza, cat. no. 17‐513F)
  • Directly conjugated fluorescent TRA‐1‐60 or TRA‐1‐81 antibody (e.g., StainAlive DyLight488; Stemgent, cat. no. 09‐0068/09‐0069)
  • TeSR‐E8 culture medium (StemCell Technologies, cat. no. 05940)
  • 6‐well tissue culture plates (BD Falcon, 3046)
  • Fluorescence microscope with FITC filter set and 4× or 10× objectives

Support Protocol 2: Generation of in Vitro Transcription Template Constructs

  Materials
  • Base vector for cloning (see Critical Parameters)
  • 250‐bp minigene insert sequence (Table 4.6.2)
  • HiFi HotStart ReadyMix PCR master mix (KAPA, cat. no. KK2601)
  • PCR primers (Table 4.6.2)
  • PCR purification Kit (e.g., Qiagen QIAquick, cat. no. 28104)
  • Agarose gels (e.g., 2% SYBR Safe E‐gels ThermoFisher Scientific, cat. no. G521802)
  • Low DNA mass ladder (e.g., ThermoFisher Scientific, cat. no. 10068‐013)
  • Chemically competent E. coli (e.g., ThermoFisher Scientific, One Shot TOP10, cat. no. C4040‐10)
  • 100 mm agar plates with antibiotic(s) of choice
  • LB broth supplemented with antibiotic(s) of choice
  • Plasmid Mini Kit II (e.g., E.Z.N.A. HP Omega Bio‐Tek, cat. no. D7045‐01), or equivalent
Table 4.0.2   MaterialsMinigene and Oligo Sequences


  • 8‐tube 0.2 ml PCR strips and caps (e.g., ThermoFisher Scientific, cat. no. AM12230)
  • 96‐well plate thermocycler (e.g., Bio‐Rad, cat. no. 186‐1096)
  • Agarose gel electrophoresis units (e.g., ThermoFisher Scientific, E‐gel runner and transilluminator, cat. no. G6465)
  • 37°C incubator with shaker
  • DNA sequence analysis software (e.g., CLC Sequence Viewer)

Support Protocol 3: PCR‐based Production of IVT Templates From Plasmid Constructs

  Additional Materials ( protocol 3)
  • 20 ng/μl purified plasmid containing IVT template sequence (see protocol 3)
  • 1 μM stocks of INSERT‐F and TAIL‐120 (Table 4.6.2)

Support Protocol 4: Preparation of Synthetic Messenger RNA

  Additional Materials ( protocol 3)
  • 2.5× NTP mix with cap analog (Allele Biotechnology, cat. no. ABP‐PP‐NTPMIX)
  • MEGAscript T7 IVT Kit (ThermoFisher Scientific, cat. no. AM1333)
  • PCR product templates (Allele Biotechnology, catalog customer order or user‐generated)
  • MEGAclear RNA Purification Kit (ThermoFisher Scientific, cat. no. AM1908)
  • Antarctic phosphatase with 10× buffer (New England Biolabs, cat. No. M0289S)
  • TE buffer, pH 7.0 (ThermoFisher Scientific, cat. no. AM9860)
  • Agarose gels (e.g., 2% SYBR Safe E‐gels; ThermoFisher Scientific, cat. no. G521802)
  • Nanodrop (Thermo Scientific), or equivalent UV spectrophotometer
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Figures

Videos

Literature Cited

Literature Cited
  Aasen, T., Raya, A., Barrero, M.J., Garreta, E., Consiglio, A., Gonzalez, F., Vassena, R., Bilic, J., Pekarik, V., Tiscornia, G., Edel, M., Boue, S., and Izpisua Belmonte, J.C. 2008. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26:1276‐1284. doi: 10.1038/nbt.1503.
  AbouHaidar, M.G. and Ivanov, I.G. 1999. Non‐enzymatic RNA hydrolysis promoted by the combined catalytic activity of buffers and magnesium ions. Z. Naturforsch. C 54:542‐548. doi: 10.1515/znc‐1999‐7‐813.
  Andries, O., Mc Cafferty, S., De Smedt, S.C., Weiss, R., Sanders, N.N., and Kitada, T. 2015. N(1)‐methylpseudouridine‐incorporated mRNA outperforms pseudouridine‐incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release. 217:337‐344. doi: 10.1016/j.jconrel.2015.08.051.
  Awe, J.P., Lee, P.C., Ramathal, C., Vega‐Crespo, A., Durruthy‐Durruthy, J., Cooper, A., Karumbayaram, S., Lowry, W.E., Clark, A.T., Zack, J.A., Sebastiano, V., Kohn, D.B., Pyle, A.D., Martin, M.G., Lipshutz, G.S., Phelps, P.E., Pera, R.A.R., and Byrne, J.A. 2013. Generation and characterization of transgene‐free human induced pluripotent stem cells and conversion to putative clinical‐grade status. Stem Cell Res. Ther. 4:87‐87. doi: 10.1186/scrt246.
  Baghbaderani, B.A., Tian, X., Neo, B.H., Burkall, A., Dimezzo, T., Sierra, G., Zeng, X., Warren, K., Kovarcik, D.P., Fellner, T., and Rao, M.S. 2015. cGMP‐manufactured human induced pluripotent stem cells are available for pre‐clinical and clinical applications. Stem Cell Rep. 5:647‐659. doi: 10.1016/j.stemcr.2015.08.015.
  Bhutani, K., Nazor, K.L., Williams, R., Tran, H., Dai, H., Dzakula, Z., Cho, E.H., Pang, A.W., Rao, M., Cao, H., Schork, N.J., and Loring, J.F. 2016. Whole‐genome mutational burden analysis of three pluripotency induction methods. Nat. Commun. 7:10536. doi: 10.1038/ncomms10536.
  Chan, E.M., Ratanasirintrawoot, S., Park, I.H., Manos, P.D., Loh, Y.H., Huo, H., Miller, J.D., Hartung, O., Rho, J., Ince, T.A., Daley, G.Q., and Schlaeger, T.M. 2009. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27:1033‐1037. doi: 10.1038/nbt.1580.
  Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga‐Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., Wagner, R., Lee, G.O., Antosiewicz‐Bourget, J., Teng, J.M., and Thomson, J.A. 2011. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8:424‐429. doi: 10.1038/nmeth.1593.
  Durruthy‐Durruthy, J., Briggs, S.F., Awe, J., Ramathal, C.Y., Karumbayaram, S., Lee, P.C., Heidmann, J.D., Clark, A., Karakikes, I., Loh, K.M., Wu, J.C., Hoffman, A.R., Byrne, J., Reijo Pera, R.A., and Sebastiano, V. 2014. Rapid and efficient conversion of integration‐free human induced pluripotent stem cells to GMP‐grade culture conditions. PloS One 9:e94231. doi: 10.1371/journal.pone.0094231.
  Elango, N., Elango, S., Shivshankar, P., and Katz, M.S. 2005. Optimized transfection of mRNA transcribed from a d(A/T)100 tail‐containing vector. Biochem. Biophys. Res. Commun. 330:958‐966. doi: 10.1016/j.bbrc.2005.03.067.
  Freshney, R.I. 2010. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th ed. Wiley‐Blackwell, Hoboken, N.J.
  Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., and Hasegawa, M. 2009. Efficient induction of transgene‐free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. 85:348‐362. doi: 10.2183/pjab.85.348.
  Graf, T. and Enver, T. 2009. Forcing cells to change lineages. Nature 462:587‐594. doi: 10.1038/nature08533.
  Hacein‐Bey‐Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J.I., de Saint Basile, G., Alexander, I., Wintergerst, U., Frebourg, T., Aurias, A., Stoppa‐Lyonnet, D., Romana, S., Radford‐Weiss, I., Gross, F., Valensi, F., Delabesse, E., Macintyre, E., Sigaux, F., Soulier, J., Leiva, L.E., Wissler, M., Prinz, C., Rabbitts, T.H., Le Deist, F., Fischer, A., and Cavazzana‐Calvo, M. 2003. LMO2‐associated clonal T cell proliferation in two patients after gene therapy for SCID‐X1. Science 302:415‐419. doi: 10.1126/science.1088547.
  Hanna, J., Saha, K., Pando, B., van Zon, J., Lengner, C.J., Creyghton, M.P., van Oudenaarden, A., and Jaenisch, R. 2009. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462:595‐601. doi: 10.1038/nature08592.
  Heffernan, C., Sumer, H., and Verma, P.J. 2011. Generation of clinically relevant “induced pluripotent stem” (iPS) cells. J. Stem. Cells 6:109‐127.
  Hendriks, W.T., Warren, C.R., and Cowan, C.A. 2016. Genome editing in human pluripotent stem cells: Approaches, pitfalls, and solutions. Cell Stem Cell 18:53‐65. doi: 10.1016/j.stem.2015.12.002.
  Hirai, H., Katoku‐Kikyo, N., Karian, P., Firpo, M., and Kikyo, N. 2012. Efficient iPS Cell Production with the MyoD transactivation domain in serum‐free culture. PloS One 7:e34149. doi: 10.1371/journal.pone.0034149.
  Hirai, H., Tani, T., Katoku‐Kikyo, N., Kellner, S., Karian, P., Firpo, M., and Kikyo, N. 2011. Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD. Stem Cells 29:1349‐1361.
  Holtkamp, S., Kreiter, S., Selmi, A., Simon, P., Koslowski, M., Huber, C., Tureci, O., and Sahin, U. 2006. Modification of antigen‐encoding RNA increases stability, translational efficacy, and T‐cell stimulatory capacity of dendritic cells. Blood 108:4009‐4017. doi: 10.1182/blood‐2006‐04‐015024.
  Hu, K. 2014. All roads lead to induced pluripotent stem cells: The technologies of iPSC generation. Stem Cells Dev. 23:1285‐1300. doi: 10.1089/scd.2013.0620.
  Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., and Weissman, D. 2008. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16:1833‐1840. doi: 10.1038/mt.2008.200.
  Kawai, T. and Akira, S. 2007. Antiviral signaling through pattern recognition receptors. J. Biochem. 141:137‐145. doi: 10.1093/jb/mvm032.
  Kim, H.S., Bernitz, J.M., Lee, D.F., and Lemischka, I.R. 2014. Genomic editing tools to model human diseases with isogenic pluripotent stem cells. Stem Cells Dev. 23:2673‐2686. doi: 10.1089/scd.2014.0167.
  Kimbrel, E.A. and Lanza, R. 2015. Current status of pluripotent stem cells: Moving the first therapies to the clinic. Nat. Rev. Drug Discov. 14:681‐692. doi: 10.1038/nrd4738.
  Ko, H.C. and Gelb, B.D. 2014. Concise review: Drug discovery in the age of the induced pluripotent stem cell. Stem Cells Transl. Med. 3:500‐509. doi: 10.5966/sctm.2013‐0162.
  Lancaster, M.A. and Knoblich, J.A. 2014. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science 345:1247125. doi: 10.1126/science.1247125.
  Li, M., Sancho‐Martinez, I., and Izpisua Belmonte, J.C. 2011. Cell fate conversion by mRNA. Stem Cell Res. Therapy 2:5. doi: 10.1186/scrt46.
  Li, B., Luo, X., and Dong, Y. 2016. Effects of Chemically Modified Messenger RNA on Protein Expression. Bioconjug. Chem. 27:849‐853. doi: 10.1021/acs.bioconjchem.6b00090.
  Li, M., Suzuki, K., Kim, N.Y., Liu, G.H., and Izpisua Belmonte, J.C. 2014. A cut above the rest: Targeted genome editing technologies in human pluripotent stem cells. J. Biol. Chem. 289:4594‐4599. doi: 10.1074/jbc.R113.488247.
  Mah, N., Wang, Y., Liao, M.C., Prigione, A., Jozefczuk, J., Lichtner, B., Wolfrum, K., Haltmeier, M., Flottmann, M., Schaefer, M., Hahn, A., Mrowka, R., Klipp, E., Andrade‐Navarro, M.A., and Adjaye, J. 2011. Molecular insights into reprogramming‐initiation events mediated by the OSKM gene regulatory network. PloS One 6:e24351. doi: 10.1371/journal.pone.0024351.
  Onder, T.T. and Daley, G.Q. 2012. New lessons learned from disease modeling with induced pluripotent stem cells. Curr. Opin. Genet. Dev. 22:500‐508. doi: 10.1016/j.gde.2012.05.005.
  Panopoulos, A.D., Yanes, O., Ruiz, S., Kida, Y.S., Diep, D., Tautenhahn, R., Herrerias, A., Batchelder, E.M., Plongthongkum, N., Lutz, M., Berggren, W.T., Zhang, K., Evans, R.M., Siuzdak, G., and Izpisua Belmonte, J.C. 2012. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res. 22:168‐177. doi: 10.1038/cr.2011.177.
  Raab, S., Klingenstein, M., Liebau, S., and Linta, L. 2014. A Comparative view on human somatic cell sources for iPSC generation. Stem Cells Int. 2014:768391. doi: 10.1155/2014/768391.
  Randall, R.E. and Goodbourn, S. 2008. Interferons and viruses: An interplay between induction, signalling, antiviral responses and virus countermeasures. J. Gen. Virol. 89:1‐47. doi: 10.1099/vir.0.83391‐0.
  Ross, J. 1995. mRNA stability in mammalian cells. Microbiol. Rev. 59:423‐450.
  Shui, B., Hernandez Matias, L., Guo, Y., and Peng, Y. 2016. The rise of CRISPR/Cas for genome editing in stem cells. Stem Cells Int. 2016:8140168. doi: 10.1155/2016/8140168.
  Sobol, M., Raykova, D., Cavelier, L., Khalfallah, A., Schuster, J., and Dahl, N. 2015. Methods of reprogramming to induced pluripotent stem cell associated with chromosomal integrity and delineation of a chromosome 5q candidate region for growth advantage. Stem Cells Dev. 24:2032‐2040. doi: 10.1089/scd.2015.0061.
  Strong, T.V., Hampton, T.A., Louro, I., Bilbao, G., Conry, R.M., and Curiel, D.T. 1997. Incorporation of beta‐globin untranslated regions into a Sindbis virus vector for augmentation of heterologous mRNA expression. Gene Ther. 4:624‐627. doi: 10.1038/sj.gt.3300423.
  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. doi: 10.1016/j.cell.2006.07.024.
  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. doi: 10.1016/j.cell.2007.11.019.
  Tillett, D. and Neilan, B.A. 1999. Enzyme‐free cloning: A rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res. 27:e26. doi: 10.1093/nar/27.19.e26.
  Uematsu, S. and Akira, S. 2007. Toll‐like receptors and type I interferons. J. Biol. Chem. 282:15319‐15323. doi: 10.1074/jbc.R700009200.
  Varas, F., Stadtfeld, M., de Andres‐Aguayo, L., Maherali, N., di Tullio, A., Pantano, L., Notredame, C., Hochedlinger, K., and Graf, T. 2009. Fibroblast‐derived induced pluripotent stem cells show no common retroviral vector insertions. Stem Cells 27:300‐306. doi: 10.1634/stemcells.2008‐0696.
  Waibler, Z., Anzaghe, M., Frenz, T., Schwantes, A., Pohlmann, C., Ludwig, H., Palomo‐Otero, M., Alcami, A., Sutter, G., and Kalinke, U. 2009. Vaccinia virus‐mediated inhibition of type I interferon responses is a multifactorial process involving the soluble type I interferon receptor B18 and intracellular components. J. Virol. 83:1563‐1571. doi: 10.1128/JVI.01617‐08.
  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. doi: 10.1016/j.stem.2010.08.012.
  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. doi: 10.1038/nature05944.
  Yisraeli, J.K., Melton, D.A., and James, E.D.A.J.N.A. 1989. Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol. 180:42‐50.
  Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., and Yamanaka, S. 2009. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5:237‐241. doi: 10.1016/j.stem.2009.08.001.
  Yu, J., Hu, K., Smuga‐Otto, K., Tian, S., Stewart, R., Slukvin, II, and Thomson, J.A. 2009. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324:797‐801. doi: 10.1126/science.1172482.
  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, II, and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917‐1920. doi: 10.1126/science.1151526.
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