Induced Pluripotent Stem Cells from Ovarian Tissue

Sophia Salas1, Nicholas Ng1, Behzad Gerami‐Naini2, Raymond M. Anchan3

1 Department of Obstetrics, Gynecology and Reproductive Biology, Division of Reproductive Endocrinology and Infertility, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, 2 School of Dental Medicine, Tufts University, Boston, Massachusetts, 3 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.10
DOI:  10.1002/cphg.47
Online Posting Date:  October, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Yamanaka and colleagues revolutionized stem cell biology and regenerative medicine by observing that somatic cells can be reprogrammed into pluripotent stem cells. Evidence indicates that induced pluripotent stem (iPS) cells retain epigenetic memories that bias their spontaneous differentiation into the originating somatic cell type, therefore epigenetic memory may be exploited to improve tissue specific regeneration. We recently showed that iPS cells reprogrammed from ovarian granulosa cells using mouse and human tissue overwhelmingly differentiate homotypically into ovarian steroidogenic and primordial germ cells. Herein we detail a protocol for the culture of human ovarian granulosa cells. We review approaches for reprogramming human ovarian granulosa cells into iPS cells. Standard methods to induce pluripotency are outlined, concentrating on integrative retroviruses. Additionally, alternative protocols for lentivirus and Sendai virus are provided. Each approach has inherent limitations, such as reprogramming efficiency, insertional mutagenesis, and partial reprogramming. Major advances continue to be made in somatic cell reprogramming to identify an optimal approach and utilization in cell‐based therapies. © 2017 by John Wiley & Sons, Inc.

Keywords: induced pluripotent stem cells; human ovarian tissue; retrovirus; homotypic; granulosa cells

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Human Granulosa Cell (hGC) Harvest and Culture
  • Basic Protocol 2: Retrovirus Generation and Infection
  • Alternate Protocol 1: Lentivirus Generation and Infection
  • Alternate Protocol 2: Single Construct Virus Generation and Infection
  • Alternate Protocol 3: Sendai Virus Infection
  • Basic Protocol 3: Stem Cell Confirmation
  • Support Protocol 1: Teratoma Formation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Human Granulosa Cell (hGC) Harvest and Culture

  Materials
  • Aspirated follicular fluid
  • Granulosa cell (GC) medium (see recipe)
  • FICOLL Plus (Amersham Pharmacia Biotech, cat. no. 17‐1440‐03)
  • Gelatin (Sigma‐Aldrich, cat. no. G1393)
  • PBS (without calcium and magnesium)
  • PBS with calcium and magnesium (PBS+/+; Cell Grow, cat. no. 21‐02‐CM)
  • 16‐Gauge needle
  • 37°C incubator
  • Thermo Scientific Sorvall Legend XTR centrifuge

Basic Protocol 2: Retrovirus Generation and Infection

  Materials
  • 293T medium (see recipe)
  • Granulosa cell (GC) medium (see recipe)
  • Viral constructs
    • p‐CMV OCT3/4 (Addgene, mouse, cat. no. 13366; human, cat. no. 17217)
    • p‐CMV SOX2 (Addgene, mouse, cat. no. 13367; human, cat. no. 17218)
    • p‐CMV KLF4 (Addgene, mouse, cat. no. 13370; human, cat. no. 17219)
    • pMX‐CMYC (Addgene, mouse, cat. no. 13375; human, cat. no. 17220)
    • p‐CMV EcoEnv (Addgene, mouse, cat. no. 15802)
    • p‐CMV AmpEnv (Addgene, human, cat. no. 15799)
    • p‐CAG VSVG (Addgene, mammalian, cat. no. 11914)
  • Escherichia coli dh5α (Thermo Fischer Scientific, cat. no. 18288019)
  • Ampicillin containing LB agar (Sigma‐Aldrich, cat. no. L3147)
  • LB broth (Sigma‐Aldrich, cat. no. L3522)
  • Maxiprep kit (Qiagen, cat. no. 12165)
  • 70% ethyl alcohol
  • Isopropyl alcohol
  • TE buffer (see recipe)
  • 293T cells (Takara, cat. no. 632180)
  • Gelatin (Sigma‐Aldrich, cat. no. G1393)
  • DMEM (Thermo Fisher Scientific, Invitrogen brand, cat. no. 11960)
  • Fugene 6 (Thermo Fisher Scientific, cat. no. PRE2692)
  • Polybrene transfection reagent (Millipore Sigma, cat. no. TR‐1003‐G)
  • EsgroLIF (Millipore Sigma, cat. no. ESG1106)
  • 0.45‐µm filter (Thermo Fisher Scientific, cat. no. 09‐740‐106)
  • Beckman TL100 ultracentrifuge
  • Benchtop swinging bucket centrifuge (Heraeus Megafuge 8, 230V; Thermo Fisher Scientific, cat. no. 75007211)
  • Thermo Fisher Scientific NanoDrop 2000C
  • 37°C incubator
  • −80°C freezer
  • 4°C refrigerator
NOTE: To generate viruses, plasmids are first prepared by transforming constructs into E. coli. This is performed for retrovirus, lentivirus, and single construct preparations. Once the constructs are transformed into E. coli, the protocol does not differ in virus generation between viral types. All constructs for retrovirus, lentivirus, and single strand lentivirus were purchased from Addgene.All mouse vectors were employed for mouse granulosa cell (mGC) transformation and human vectors for hGC transformation.

Alternate Protocol 1: Lentivirus Generation and Infection

  Additional Materials (also see protocol 2)
  • 293T medium (see recipe)
  • Granulosa cell (GC) medium (see recipe)
  • Viral constructs
    • pLenti OCT 3/4 (mammalian; Addgene, cat. no. 35391)
    • pLenti SOX2 (mammalian; Addgene, cat. no. 35388)
    • pLenti KLF4 (mammalian; Addgene, cat. no. 35389)
    • pLenti CMYC (mammalian; Addgene, cat. no. 46970)
    • pCMV‐dR8.2 dvpr
    • p‐CAG VSVG
  • E. coli dh5α (Thermo Fisher Scientific, cat. no. 18288019)
  • Ampicillin containing LB agar (Sigma‐Aldrich, cat. no. L3147)
  • LB broth (Sigma‐Aldrich, cat. no. L3522)
  • Maxiprep kit (Qiagen, cat. no. 12165)
  • 70% ethyl alcohol
  • Isopropyl alcohol
  • 293T cells (Takara, cat. no. 632180)
  • Gelatin (Sigma‐Aldrich, cat. no. G1393)
  • DMEM (Thermo Fisher Scientific, Invitrogen brand, cat. no. 11960)
  • Fugene 6 (Thermo Fisher Scientific, cat. no. PRE2692)
  • Polybrene transfection reagent (Millipore, cat. no. TR‐1003‐G)
  • EsgroLIF (Millipore, cat. no. ESG1106)
  • 0.45‐µm filter (Thermo Fisher Scientific, cat. no. 09‐740‐106)
  • Beckman TL100 ultracentrifuge
  • Benchtop swinging bucket centrifuge (Heraeus Megafuge 8, 230V; Thermo Fisher Scientific, cat. no. 75007211)
  • Thermo Fisher Scientific NanoDrop 2000C
  • 37°C incubator
  • −80°C freezer

Alternate Protocol 2: Single Construct Virus Generation and Infection

  Materials
  • 293T medium (see recipe)
  • Granulosa cell (GC) medium (see recipe)
  • Viral construct (Addgene, cat. no. 20325)
  • E. coli dh5α (Thermo Fisher Scientific, cat. no. 18288019)
  • Ampicillin containing LB agar (Sigma‐Aldrich, cat. no. L3147)
  • LB broth (Sigma‐Aldrich, cat. no. L3522)
  • Maxiprep kit (Qiagen, cat. no. 12165)
  • 70% ethyl alcohol
  • 293T cells (Takara, cat. no. 632180)
  • Gelatin (Sigma‐Aldrich, cat. no. G1393)
  • DMEM (Thermo Fisher Scientific, Invitrogen brand, cat. no. 11960)
  • Fugene 6 (Thermo Fisher Scientific, cat. no. PRE2692)
  • TE buffer (see recipe)
  • Polybrene transfection reagent (Millipore, cat. no. TR‐1003‐G)
  • EsgroLIF (Millipore, cat. no. ESG1106)
  • 0.45‐µm filter (Thermo Fisher Scientific, cat. no. 09‐740‐106)
  • Beckman TL100 ultracentrifuge
  • Benchtop swinging bucket centrifuge (Heraeus Megafuge 8, 230V; Thermo Fisher Scientific, cat. no. 75007211)
  • Thermo Fisher Scientific NanoDrop 2000C
  • 37°C incubator
  • −80°C freezer

Alternate Protocol 3: Sendai Virus Infection

  Additional Materials (also see protocol 2)
  • Sendai virus (Thermo Fisher Scientific, Invitrogen brand, cat. no. A168518)

Basic Protocol 3: Stem Cell Confirmation

  Materials
  • Human embryonic stem (hES) cell medium (see recipe)
  • Mouse embryonic stem (mES) cell medium (see recipe)
  • Freezing medium (see recipe)
  • Mouse embryonic fibroblasts (MEFs; Global Stem, cat. no. GSC‐6001)
  • Mitomycin C (Sigma‐Aldrich, cat. no. M4287)
  • Human embryonic stem (hES) cell characterization kit (Millipore, cat. no. SCR001)
  • PBS with calcium and magnesium (PBS+/+; Cell Grow, cat. no. 21‐02‐CM)
  • Trypsin (Thermo Fisher Scientific, Invitrogen brand, cat. no. 25300‐054)
  • Collagenase type IV (Gibco, cat. no. 17104019)
  • FBS‐containing medium
  • Stem cell antibodies
    • OCT4 (Abcam, cat. no. AB18976)
    • SSEA4, human (Millipore kit, cat. no. SCR001)
    • SSEA1, mouse (Millipore kit, cat. no. SCR001)
    • TRA1‐80‐1, human; TRA 1‐60, human (Millipore kit, cat. no. SCR001)
    • Nanog (Abcam, cat. no. AB80892)
    • GDF3 (Abcam, cat. no. AB93892)
    • CKIT (Santa Cruz, cat. no. SC5535)
  • Alkaline phosphatase leukocyte staining kit (Sigma‐Aldrich, cat. no. 86R)
  • RNeasy Plus Mini kit (Qiagen, cat. no. 74136)
  • qScript cDNA Synthesis kit (Quanta Biosciences, cat. no. 95047)
  • GoTaq PCR Core System I (Promega, cat. no. M7660)
  • Agarose powder (Thermo Fisher Scientific, Invitrogen brand, cat. no. 16500‐500)
  • Ethidium bromide (Fischer BioReagents, cat. no. 1302‐10)
  • Paraformaldehyde (Sigma‐Aldrich, cat. no. 441244‐1KG)
  • Sucrose (Sigma‐Aldrich, cat. no. S7903‐1KG)
  • Optimal cutting temperature (OCT) compound (Fischer Healthcare, cat. no. 4585)
  • MEF coated plates
  • 0.22‐µm syringe filter (Millipore, cat. no. SLGV033RS)
  • Ultra‐low attachment spheroid formation 24‐well plates (Corning Costar, cat. no. 07‐200‐602)
  • Thermo Fisher Scientific Sorvall Legend XTR centrifuge
  • BioRad thermal cycler C1000
  • Gel Logic 112 High Performance UV transiluminator
  • Mr. Frosty isopropyl alcohol freezing container (Thermo Fisher Scientific, cat. no. 5100‐0001)
  • 37°C incubator
  • −80°C freezer
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Anchan, R., Gerami‐Naini, B., Lindsey, J. S., Ho, J. W. K., Kiezun, A., Lipskind, S., … Williams, Z. (2015). Efficient differentiation of steroidogenic and germ‐like cells from epigenetically‐related iPSCs derived from ovarian granulosa cells. PLoS One, 10, e0119275. doi: 10.1371/journal.pone.0119275.
  Anchan, R. M., Quaas, P., Gerami‐Naini, B., Bartake, H., Griffin, A., Zhou, Y., … Maas, R. L. (2011). Amniocytes can serve a dual function as a source of iPS cells and feeder layers. Human Molecular Genetics, 20, 962–974. doi: 10.1093/hmg/ddq542.
  Bar‐Nur, O., Russ, H. A., Efrat, S., & Benvenisty, N. (2011). Epigenetic memory and preferential lineage‐specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells. Cell Stem Cell, 9, 17–23. doi: 10.1016/j.stem.2011.06.007.
  Carey, B. W., Markoulaki, S., Hanna, J., Saha, K., Gao, Q., Mitalipova, M., & Jaenisch, R. (2008). Reprogramming of murine and human somatic cells using a single polycistronic vector. Proceedings of the National Academy of Sciences of the United States of America, 106, 157–162. doi: 10.1073/pnas.0811426106.
  Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion. Division of Reproductive Health (2014). Assisted Reproductive Technology (ART) Data.
  Connolly, J. B. (2002). Lentiviruses in gene therapy clinical research. Gene Therapy, 9, 1730–1734. doi: 10.1038/sj.gt.3301893.
  Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., & 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. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, 85, 348–362. doi: 10.2183/pjab.85.348.
  Gonzales, K. A., & Ng, H. H. (2011). Choreographing pluripotency and cell fate with transcription factors. Biochimica et Biophysica Acta, 1809, 337–349. doi: 10.1016/j.bbagrm.2011.06.009.
  Hanna, J., Wernig, M., Markoulaki, S., Sun, C. W., Meissner, A., Cassady, J. P., … Jaenisch, R. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 318, 1920–1923. doi: 10.1126/science.1152092.
  Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A. E., & Melton, D. A. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small‐molecule compounds. Nature Biotechnology, 26, 795–797. doi: 10.1038/nbt1418.
  Kim, K., Doi, A., Wen, B., Ng, K., Zhao, R., Cahan, P., … Daley, G. Q. (2010). Epigenetic memory in induced pluripotent stem cells. Nature, 467, 285–290. doi: 10.1038/nature09342.
  Li, M., Cascino, P., Ummarino, S., & Di Ruscio, A. (2017). Application of induced pluripotent stem cell technology to the study of hematological diseases. Cells, 6, 7. doi: 10.3390/cells6010007.
  Lin, S. L., Chang, D. C., Lin, C. H., Ying, S. Y., Leu, D., & Wu, D. T. (2011). Regulation of somatic cell reprogramming through inducible mir‐302 expression. Nucleic Acids Research, 39, 1054–1065. doi: 10.1093/nar/gkq850.
  Liu, T., Zou, G., Gao, Y., Zhao, X., Wang, H., Huang, Q., … Cheng, W. (2012). High efficiency of reprogramming CD34+ cells derived from human amniotic fluid into induced pluripotent stem cells with Oct4. Stem Cells and Development, 21, 2322–2332. doi: 10.1089/scd.2011.0715.
  Lois, C., Hong, E. J., Pease, S., Brown, E. J., & Baltimore, D. (2002). Germline transmission and tissue‐specific expression of transgenes delivered by lentiviral vectors. Science, 295, 868–872. doi: 10.1126/science.1067081.
  Mao, J., Zhang, Q., Ye, X., Liu, K., & Liu, L. (2014). Efficient induction of pluripotent stem cells from granulosa cells by Oct4 and Sox2. Stem Cells and Development, 23, 779–789. doi: 10.1089/scd.2013.0325.
  Miller, D. G., Adam, M. A., & Miller, A. D. (1990). Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Molecular and Cellular Biology, 10, 4239–4242. doi: 10.1128/MCB.10.8.4239.
  Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., … Yamanaka, S. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology, 26, 101–106. doi: 10.1038/nbt1374.
  Naldini, L., Blome, R. U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., … Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 272, 263–267. doi: 10.1126/science.272.5259.263.
  Narsinh, K. H., Jia, F., Robbins, R. C., Kay, M. A., Longaker, M. T., & Wu, J. C. (2011). Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA. Nature Protocols, 6, 78–88. doi: 10.1038/nprot.2010.173.
  Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline‐competent induced pluripotent stem cells. Nature, 448, 313–317. doi: 10.1038/nature05934.
  Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A., Okamoto, S., … Yamanaka, S. (2011). A more efficient method to generate integration‐free human iPS cells. Nature Methods, 8, 409–412. doi: 10.1038/nmeth.1591.
  Polo, J. M., Liu, S., Figueroa, M. E., Kulalert, W., Eminli, S., Tan, K. Y., … Hochedlinger, K. (2010). Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nature Biotechnology, 28, 848–855. doi: 10.1038/nbt.1667.
  Schlaeger, T. M., Daheron, L., Brickler, T. R., Entwisle, S., Chan, K., Cianci, A., … Daley, G. Q. (2015). A comparison of non‐integrating reprogramming methods. Nature Biotechnology, 33, 58–63. doi: 10.1038/nbt.3070.
  Sommer, C. A., Stadtfeld, M., Murphy, G. J., Hochedlinger, K., Kotton, D. N., & Mostoslavsky, G. (2009). Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cell, 27, 543–549. doi: 10.1634/stemcells.2008‐1075.
  Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., & Hochedlinger, K. (2008). Induced pluripotent stem cells generated without viral integration. Science, 322, 945–949. doi: 10.1126/science.1162494.
  Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & 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.
  Takahashi, K., & 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., & Yamanaka, S. (2013). Induced pluripotent stem cells in medicine and biology. Development (Cambridge, England), 140, 2457–2461. doi: 10.1242/dev.092551.
  Tashiro, M., McQueen, N. L., & Seto, J. T. (1999). Determinants of organ tropism of Sendai virus. Frontiers in Bioscience, 4, D642–645. doi: 10.2741/A460.
  Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., … 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., Lengner, C. J., Hanna, J., Lodato, M. A., Steine, E., Foreman, R., … Jaenisch, R. (2008). A drug‐inducible transgenic system for direct reprogramming of multiple somatic cell types. Nature Biotechnology, 26, 916–924. doi: 10.1038/nbt1483.
  Wernig, M., Zhao, J. P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., … Jaenisch, R. (2008). Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proceedings of the National Academy of Sciences of the United States of America, 105, 5856–5861. doi: 10.1073/pnas.0801677105.
  Yu, J., Hu, K., Smuga‐Otto, K., Tian, S., Stewart, R., Slukvin, I. I., & Thomson, J. A. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science, 324(5928), 797–801. doi: 10.1126/science.1172482.
  Yu, H., & Kwon, Y. J. (2008). Preparation and quantification of pseudotyped retroviral vectors. Methods in Molecular Biology, 433, 1–16.
  Zhen, W., Wang, Y., Chang, T., Huang, H., & Yee, J. K. (2013). Significant difference in genotoxicity by retrovirus integration in human T cells and induced pluripotent stem cells. Gene, 519, 142–149. doi: 10.1016/j.gene.2013.01.009.
  Zhu, S., Li, W., Zhou, H., Wei, W., Ambasudhan, R., Lin, T., … Ding, S. (2010). Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell, 7, 651–655. doi: 10.1016/j.stem.2010.11.015.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library