Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
361–364
Year published:
DOI:
doi:10.1038/nbt.1616
Received
Accepted
Published online

Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies1, 2, 3, 4, 5, 6, 7. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies8. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.

At a glance

Figures

  1. Human ESCs remain pluripotent after 10 weeks propagation in suspension.
    Figure 1: Human ESCs remain pluripotent after 10 weeks propagation in suspension.

    (a) FACS analysis of HES1 cells after cultivation in suspension and on feeders showing that in both culture conditions >90% of the cells express markers of pluripotent stem cells, whereas <2% express PSA-NCAM, which is a marker of early neural differentiation (n = 3). Data are presented as mean ± s.d. (b) RT-PCR analysis of free-floating clusters of hESCs confirming the expression of transcripts of markers of pluripotency, whereas the expression of the primitive ectoderm marker FGF5 is not detected. (c) Immunostaining of cells dissociated from the clusters and plated for 24 h, demonstrating that the majority of cells express OCT-4. (d) Darkfield micrograph of the clusters of hESCs in suspension. (e) Alkaline phosphatase activity within the hESC aggregates is demonstrated (fluorescence image). (f,g) After plating of the clusters on feeders, they give rise to colonies with morphological characteristics of colonies of undifferentiated hESCs (f, phase contrast image), which are comprised of cells harboring alkaline phosphatase activity (g, fluorescence image). (h) Proliferation curve showing the fold increase in the number of cells cultivated in suspension or as colonies on feeders. Cell number was monitored each week and the cumulative fold increase in cell number from the starting population was calculated in three independent experiments. (il) Histological analysis of H&E-stained sections of teratoma tumors that developed after inoculation of the hESC-clusters under the testes capsule of NOD/SCID mice showing differentiated tissues representing the three germ layers (low magnitude image (i), cartilage (j), neural rosette (k) and glandular structure (l)). (mo) Immunostaining of in vitro–differentiated progeny, representing the three embryonic germ layers, within the outgrowth of plated embryoid bodies (human muscle actin (m), and SOX-17, (o)) and neural spheres (β-III Tubulin, (n)). (p) G-banding analysis showing a normal karyotype. Nuclei are counterstained by DAPI in (c) and (mo). Scale bars, 20 μm (c,k,mo); 50 μm (d,e,j,l); 100 μm (f,g); 500 μm (i).

  2. Derivation of hESCs in suspension.
    Figure 2: Derivation of hESCs in suspension.

    (a,b) Darkfield micrograph of an inner cell mass after transfer to suspension culture conditions (a), and of the clusters of cells that were derived from the inner cell mass after 10 weeks of cultivation (b). (c) Fluorescence image showing alkaline phosphatase activity within a cluster. (dh) After plating on feeders, the clusters gave rise to colonies with morphological characteristics of colonies of undifferentiated hESCs (d, phase contrast image), which were comprised of cells immunoreactive with anti-SSEA-4 (e), SSEA-3 (f), TRA-1-60 (g) and TRA-1-81 (h) (fluorescence images). (ik) Immunostaining of in vitro–differentiated progeny, representing the three embryonic germ layers, within the outgrowth of plated embryoid bodies (β-III tubulin, (i); SOX-17, (j); human muscle actin, (k)). (l) G-banding analysis showing a normal karyotype after 10 weeks of cultivation in suspension. Nuclei are counterstained by DAPI in ik. Scale bars, 20 μm (a, ek); 50 μm (c); 100 μm (b,d). HAD17 hESC line.

  3. Controlled conversion of the hESC clusters in suspension into neural precursor spheres.
    Figure 3: Controlled conversion of the hESC clusters in suspension into neural precursor spheres.

    Clusters of H7 cells, cultivated in suspension for 7 weeks, were transferred and further cultured 4 weeks in a chemically defined medium supplemented with noggin and FGF2. (a) FACS analysis of one representative experiment showing that 91% of the cells expressed PSA-NCAM, whereas 1.2% expressed TRA-1-81. (bh) After plating and culturing for 1 week on laminin, indirect immunofluorescence staining showed cells within rosettes expressing markers of neural precursors such as nestin and Pax6 (b), the neural stem/radial glial cell marker 3CB2 (c), subtypes of neurons expressing β-III tubulin and tyrosine hydroxylase (TH) (d), GABA (e) and glutamate (f), as well as cells expressing the astrocyte marker GFAP (g) and the marker of oligodendroglial progenitors NG2 (h). Nuclei are counterstained by DAPI. Scale bars, 20 μm.

References

  1. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399404 (2000).
  2. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 11451147 (1998).
  3. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185187 (2006).
  4. Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C. & Bongso, A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933936 (2002).
  5. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971974 (2001).
  6. Braam, S.R. et al. Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification. Nat. Protoc. 3, 14351443 (2008).
  7. Wang, L. et al. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110, 41114119 (2007).
  8. Kurosawa, H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J. Biosci. Bioeng. 103, 389398 (2007).
  9. McDevitt, T.C. & Palecek, S.P. Innovation in the culture and derivation of pluripotent human stem cells. Curr. Opin. Biotechnol. 19, 527533 (2008).
  10. Lock, L.T. & Tzanakakis, E.S. Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture. Tissue Eng. Part A 15, 20512063 (2009).
  11. Nie, Y., Bergendahl, V., Hei, D.J., Jones, J.M. & Palecek, S.P. Scalable culture and cryopreservation of human embryonic stem cells on microcarriers. Biotechnol. Prog. 25, 2031 (2009).
  12. Oh, S.K. et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. (Amst.) 4, 4 (2009).
  13. Krawetz, R. et al. Large-scale expansion of pluripotent human embryonic stem cells in stirred suspension bioreactors. Tissue Eng. Part C Methods 8, 8 (2009).
  14. Itsykson, P. et al. Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Mol. Cell. Neurosci. 30, 2436 (2005).
  15. Hoover, C.S. & Martin, R.L. Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements. Biotechniques 8, 7682 (1990).
  16. Stockinger, H. Serum-free medium for mammalian cells. US patent 5,063,157 (1991).
  17. Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837845 (2004).
  18. Xu, C. et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 23, 315323 (2005).
  19. Pyle, A.D., Lock, L.F. & Donovan, P.J. Neurotrophins mediate human embryonic stem cell survival. Nat. Biotechnol. 24, 344350 (2006).
  20. Beattie, G.M. et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489495 (2005).
  21. Furue, M.K. et al. Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc. Natl. Acad. Sci. USA 105, 1340913414 (2008).
  22. Turetsky, T. et al. Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum. Reprod. 23, 4653 (2008).
  23. Herszfeld, D. et al. CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat. Biotechnol. 24, 351357 (2006).
  24. Baker, D.E. et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo . Nat. Biotechnol. 25, 207215 (2007).
  25. Mitalipova, M.M. et al. Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23, 1920 (2005).
  26. Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131137 (1994).
  27. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681686 (2007).
  28. Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M. & McKay, R.D. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675679 (2000).
  29. Yan, Y. et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23, 781790 (2005).
  30. Gropp, M. & Reubinoff, B. Lentiviral vector-mediated gene delivery into human embryonic stem cells. Methods Enzymol. 420, 6481 (2006).

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Author information

Affiliations

  1. The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy.

    • Debora Steiner,
    • Hanita Khaner,
    • Malkiel Cohen,
    • Sharona Even-Ram,
    • Yaniv Gil,
    • Pavel Itsykson,
    • Tikva Turetsky,
    • Maria Idelson,
    • Yael Berman-Zaken &
    • Benjamin Reubinoff
  2. Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

    • Einat Aizenman
  3. Department of Genetics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

    • Rita Ram

Contributions

D.S. designed and performed the experiments, analyzed the data and wrote the manuscript; H.K. and M.C. performed the neural differentiation study; S.E.-R. conducted immunostainings and confocal analysis; Y.G. performed the teratoma studies; P.I. contributed to developing the concept of suspension culture; T.T. contributed to the experiments; M.I. performed PCR analysis; E.A. contributed to embryo recruitment, culture and isolation of inner cell masses; R.R. and Y.B.-Z. conducted karyotype analysis. B.R. conceived the study and wrote the paper.

Competing financial interests

B.R. is the CSO and holds shares in CellCure Neurosciences Ltd. However, the project was not funded by CellCure Neurosciences Ltd. and the company has no rights in its results.

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