Skip to main content
SpringerLink
Log in

Endoderm Differentiation from Human Pluripotent Stem Cells

  • Chapter
  • First Online:
Working with Stem Cells

  • 1154 Accesses

Abstract

Human pluripotent stem cells (hPSCs) provide a virtually unlimited raw material to derive and engineer mature cell types with therapeutic value, including cell transplantation, disease modeling and drug screening. The first step to differentiate hPSCs into such cell types involves specification towards one of the three main embryonic cell populations, ecto-, endo- and mesoderm. Efficient induction into the correct lineage is critical to the success of subsequent differentiation steps and to the final yield of desired cells. Here we describe methods to generate definitive endoderm (DE), the progenitor cell population for such tissues as the thymus, liver, pancreas, stomach and intestine. In addition, we will provide methods to characterize and monitor the efficiency of DE differentiation, including expression of DE markers at the gene and protein level. Flow cytometry based methods described in this chapter can also be extended to isolate and purify cells with DE properties. Such enrichment strategies are useful to eliminate undesired cell populations, especially undifferentiated hPSCs, which harbor the potential risk for seeding tumors upon transplantation. Several of the methods for the manipulation of hPSCs and for their analysis outlined here are of general utility and are applicable to other hPSCs derivative cell populations.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Ameri J, Stahlberg A, Pedersen J, Johansson JK, Johannesson MM, Artner I, Semb H (2010) FGF2 specifies hESC-derived definitive endoderm into foregut/midgut cell lineages in a concentration-dependent manner. Stem Cells 28:45–56

    CAS  PubMed  Google Scholar 

  • Ang SL, Wierda A, Wong D, Stevens KA, Cascio S, Rossant J, Zaret KS (1993) The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development 119:1301–1315

    CAS  PubMed  Google Scholar 

  • Beddington RS, Smith JC (1993) Control of vertebrate gastrulation: inducing signals and responding genes. Curr Opin Genet Dev 3:655–661

    Article  CAS  PubMed  Google Scholar 

  • Bone HK, Nelson AS, Goldring CE, Tosh D, Welham MJ (2011) A novel chemically directed route for the generation of definitive endoderm from human embryonic stem cells based on inhibition of GSK-3. J Cell Sci 124:1992–2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Borowiak M, Maeh R, Chen S, Chen AE, Tang W, Fox JL, Scheiber SL, Melton DA (2009) Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell 4:348–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brafman DA, Moya N, Allen-Soltero S, Fellner T, Robinson M, McMillen ZL, Gaasterland T, Willert K (2013a) Analysis of SOX2-expressing cell populations derived from human pluripotent stem cells. Stem Cell Rep 1:464–478

    Article  CAS  Google Scholar 

  • Brafman DA, Phung C, Kumar N, Willert K (2013b) Regulation of endodermal differentiation of human embryonic stem cells through integrin-ECM interactions. Cell Death Differ 20:369–381

    Article  CAS  PubMed  Google Scholar 

  • Cai J, Zhao Y, Liu Y, Ye F, Song Z, Qin H, Meng S, Chen Y, Zhou R, Song X, Guo Y, Ding M, Deng H (2007) Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 45:1229–1239

    Article  CAS  PubMed  Google Scholar 

  • Chen S, Borowiak M, Fox JL, Maeh R, Osafune K, Davidow L, Lam K, Peng LF, Scheiber SL, Rubin LL, Melton D (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5:258–265

    Article  CAS  PubMed  Google Scholar 

  • Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng X, Ying L, Lu L, Galvao AM, Mills JA, Lin HC, Kotton DN, Shen SS, Nostro MC, Choi JK, Weiss MJ, French DL, Gadue P (2012) Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell Stem Cell 10:371–384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Conlon FL, Lyons KM, Takaesu N, Barth KS, Kispert A, Herrmann B, Robertson EJ (1994) A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120:1919–1928

    CAS  PubMed  Google Scholar 

  • D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23:1534–1541

    Article  PubMed  Google Scholar 

  • D’Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24:1392–1401

    Article  PubMed  Google Scholar 

  • Faust C, Magnuson T (1993) Genetic control of gastrulation in the mouse. Curr Opin Genet Dev 3:491–498

    Article  CAS  PubMed  Google Scholar 

  • Haegel H, Larue L, Ohsugi M, Fedorov L, Herrenknecht K, Kemler R (1995) Lack of beta-catenin affects mouse development at gastrulation. Development 121:3529–3537

    CAS  PubMed  Google Scholar 

  • Hoveizi E, Khodadadi S, Tavakol S, Karima O, Nasiri-Khalili MA (2014) Small molecules differentiate definitive endoderm from human induced pluripotent stem cells on PCL scaffold. Appl Biochem Biotechnol 173:1727–1736

    Article  CAS  PubMed  Google Scholar 

  • Kadzik RS, Morrisey EE (2012) Directing lung endoderm differentiation in pluripotent stem cells. Cell Stem Cell 10:355–361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kelly OG, Pinson KI, Skarnes WC (2004) The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice. Development 131:2803–2815

    Article  CAS  PubMed  Google Scholar 

  • Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, Young H, Richardson M, Smart NG, Cunningham J, Agulnick AD, D’Amour KA, Carpenter MK, Baetge EE (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26:443–452

    Article  CAS  PubMed  Google Scholar 

  • Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, Fehling HJ, Keller G (2004) Development of definitive endoderm from embryonic stem cells in culture. Development 131:1651–1662

    Article  CAS  PubMed  Google Scholar 

  • Liu P, Wakamiya M, Shea MJ, Albrecht U, Behinger RR, Bradley A (1999) Requirement for Wnt3 in vertebrate axis formation. Nat Genet 22:361–365

    Article  CAS  PubMed  Google Scholar 

  • Longmire TA, Ikonomou L, Hawkins F, Chistodoulou C, Cao Y, Jean JC, Kwok LW, Mou H, Rajagopal J, Shen SS, Dowton AA, Serra M, Weiss DJ, Green MD, Snoeck HW, Ramirez MI, Kotton DN (2012) Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10:398–411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Naujok O, Diekmann U, Lenzen S (2014) The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling. Stem Cell Rev 10:480–493

    Article  PubMed  Google Scholar 

  • Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA (2014) Generation of functional human pancreatic beta cells in vitro. Cell 159:428–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Parent AV, Russ HA, Khan IS, LaFlam TN, Metzger TC, Anderson MS, Hebrok M (2013) Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development. Cell Stem Cell 13:219–229

    Article  CAS  PubMed  Google Scholar 

  • Sasaki H, Hogan BL (1993) Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118:47–59

    CAS  PubMed  Google Scholar 

  • Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (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

    Article  CAS  PubMed  Google Scholar 

  • Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, Shoyer NF, Wells JM (2011) Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470:105–109

    Article  PubMed  Google Scholar 

  • Su M, Hu R, Jin J, Yan Y, Song Y, Sullivan R, Lai L (2015) Efficient in vitro generation of functional thymic epithelial progenitors from human embryonic stem cells. Sci Rep 5:9882

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takeuchi H, Nakatsuji N, Suemori H (2014) Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 4:4488

    PubMed  PubMed Central  Google Scholar 

  • Tam PP, Behinger RR (1997) Mouse gastrulation: the formation of a mammalian body plan. Mech Dev 68:3–25

    Article  CAS  PubMed  Google Scholar 

  • Touboul T, Hannan NR, Corbineau S, Martinez A, Martinet C, Branchereau S, Mainot S, Strick-Marchand H, Pedersen R, Di Santo J, Weber A, Vallier L (2010) Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology 51:1754–1765

    Article  CAS  PubMed  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034

    Google Scholar 

  • VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626

    Article  CAS  PubMed  Google Scholar 

  • Vincent SD, Dunn NR, Hayashi S, Norris DP, Robertson EJ (2003) Cell fate decisions within the mouse organizer are governed by graded Nodal signals. Genes Dev 17:1646–1662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang P, Rodriguez RT, Wang J, Ghodasara A, Kim SK (2011) Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm. Cell Stem Cell 8:335–346

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang P, McKnight KD, Wong DJ, Rodriguez RT, Sugiyama T, Gu X, Ghodasara A, Qu K, Chang HY, Kim SK (2012) A molecular signature for purified definitive endoderm guides differentiation and isolation of endoderm from mouse and human embryonic stem cells. Stem Cells Dev 21:2273–2287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Willert KH (2008) Isolation and application of bioactive Wnt proteins. Methods Mol Biol 468:17–29

    Article  CAS  PubMed  Google Scholar 

  • Willert K, Nusse R (2012) Wnt proteins. Cold Spring Harb Perspect Biol 4:a007864

    Article  PubMed  PubMed Central  Google Scholar 

  • Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR 3rd, Nusse R (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423:448–452

    Article  CAS  PubMed  Google Scholar 

  • Yasunaga M, Tada S, Torikai-Nishikawa S, Nakano Y, Okada M, Jakt LM, Nishikawa S, Chiba T, Era T, Nishikawa S (2005) Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotechnol 23:1542–1550

    Article  CAS  PubMed  Google Scholar 

  • Zhu S, Wurdak H, Wang J, Lyssiotis CA, Peters EC, Cho CY, Wu X, Schultz PG (2009) A small molecule primes embryonic stem cells for differentiation. Cell Stem Cell 4:416–426

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Bioengineering, UCSD, La Jolla, CA, 92093, USA

    Nathan Kumar

  2. School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, 85287, USA

    David Brafman

  3. Department of Cellular and Molecular Medicine, UCSD, La Jolla, CA, 92093, USA

    Karl Willert

  4. Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Dr. – Room 3009, La Jolla, CA, 92093, USA

    Karl Willert

Authors
  1. Nathan Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Brafman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karl Willert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karl Willert .

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Kumar, N., Brafman, D., Willert, K. (2016). Endoderm Differentiation from Human Pluripotent Stem Cells. In: Working with Stem Cells. Springer, Cham. https://doi.org/10.1007/978-3-319-30582-0_14

Download citation

Publish with us

Policies and ethics

Search

Navigation