Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAMpos and Tra-1-60neg by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments.
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Schwartz, P. H., Brick, D. J., Stover, A. E., Loring, J. F., & Muller, F. J. (2008). Differentiation of neural lineage cells from human pluripotent stem cells. Methods, 45(2), 142–158.
Falk, A., Koch, P., Kesavan, J., et al. (2012). Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PloS One, 7(1), e29597.
Nemati, S., Hatami, M., Kiani, S., et al. (2011). Long-term self-renewable feeder-free human induced pluripotent stem cell-derived neural progenitors. Stem Cells and Development, 20(3), 503–514.
Nistor, G., Siegenthaler, M. M., Poirier, S. N., et al. (2011). Derivation of high purity neuronal progenitors from human embryonic stem cells. PloS One, 6(6), e20692.
Cohen, M. A., Itsykson, P., & Reubinoff, B. E. (2007). Neural differentiation of human ES cells. Curr Protoc Cell Biol. Chapter 23, Unit 23 27.
Iacovitti, L., Donaldson, A. E., Marshall, C. E., Suon, S., & Yang, M. (2007). A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo. Brain Research, 1127(1), 19–25.
Koch, P., Opitz, T., Steinbeck, J. A., Ladewig, J., & Brustle, O. (2009). A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proceedings of the National Academy of Sciences, USA, 106(9), 3225–3230.
Kumar, M., Bagchi, B., Gupta, S. K., Meena, A. S., Gressens, P., & Mani, S. (2007). Neurospheres derived from human embryoid bodies treated with retinoic Acid show an increase in nestin and ngn2 expression that correlates with the proportion of tyrosine hydroxylase-positive cells. Stem Cells and Development, 16(4), 667–681.
Lim, U. M., Sidhu, K. S., & Tuch, B. E. (2006). Derivation of motor neurons from three clonal human embryonic stem cell lines. Current Neurovascular Research, 3(4), 281–288.
Ma, W., Tavakoli, T., Derby, E., Serebryakova, Y., Rao, M. S., & Mattson, M. P. (2008). Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Developmental Biology, 8, 90.
Schuldiner, M., Eiges, R., Eden, A., et al. (2001). Induced neuronal differentiation of human embryonic stem cells. Brain Research, 913(2), 201–205.
Schulz, T. C., Palmarini, G. M., Noggle, S. A., Weiler, D. A., Mitalipova, M. M., & Condie, B. G. (2003). Directed neuronal differentiation of human embryonic stem cells. BMC Neuroscience, 4, 27.
Swistowski, A., Peng, J., Liu, Q., et al. (2010). Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions. Stem Cells, 28(10), 1893–1904.
Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., & Thomson, J. A. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotechnology, 19(12), 1129–1133.
Bauwens, C. L., Peerani, R., Niebruegge, S., et al. (2008). Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells, 26(9), 2300–2310.
Sachlos, E., & Auguste, D. T. (2008). Embryoid body morphology influences diffusive transport of inductive biochemicals: a strategy for stem cell differentiation. Biomaterials, 29(34), 4471–4480.
Axell, M. Z., Zlateva, S., & Curtis, M. (2009). A method for rapid derivation and propagation of neural progenitors from human embryonic stem cells. Journal of Neuroscience Methods, 184(2), 275–284.
Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 27(3), 275–280.
Erceg, S., Lainez, S., Ronaghi, M., et al. (2008). Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions. PloS One, 3(5), e2122.
Gerrard, L., Rodgers, L., & Cui, W. (2005). Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells, 23(9), 1234–1241.
Joannides, A. J., Fiore-Heriche, C., Battersby, A. A., et al. (2007). A scaleable and defined system for generating neural stem cells from human embryonic stem cells. Stem Cells, 25(3), 731–737.
Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., et al. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell, 3(5), 519–532.
Lee, S. H., Lumelsky, N., Studer, L., Auerbach, J. M., & McKay, R. D. (2000). Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nature Biotechnology, 18(6), 675–679.
Watanabe, K., Ueno, M., Kamiya, D., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25(6), 681–686.
Okabe, S., Forsberg-Nilsson, K., Spiro, A. C., Segal, M., & McKay, R. D. (1996). Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mechanisms of Development, 59(1), 89–102.
Itsykson, P., Ilouz, N., Turetsky, T., et al. (2005). Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Molecular and Cellular Neuroscience, 30(1), 24–36.
Bianco, P., Kuznetsov, S. A., Riminucci, M., & Gehron Robey, P. (2006). Postnatal skeletal stem cells. Methods in Enzymology, 419, 117–148.
Takahashi, K., Tanabe, K., Ohnuki, M., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861–872.
Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663–676.
He, J. Q., Ma, Y., Lee, Y., Thomson, J. A., & Kamp, T. J. (2003). Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circulation Research, 93(1), 32–39.
Si-Tayeb, K., Noto, F. K., Nagaoka, M., et al. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology, 51(1), 297–305.
Mallon, B. S., Park, K. Y., Chen, K. G., Hamilton, R. S., & McKay, R. D. (2006). Toward xeno-free culture of human embryonic stem cells. The International Journal of Biochemistry & Cell Biology, 38(7), 1063–1075.
We gratefully acknowledge Dr. Sergei Kuznetsov and Dr. Pamela Robey of the National Institute for Dental and Craniofacial Research for providing the bone marrow stromal cells from which the iPSC line was derived. We would also like to thank Dr. Ron McKay and Dr. Josh Chenoweth of the Lieber Institute for Brain Development for helpful discussions. This research was supported by the Intramural Research Program of the NIH, NINDS.
Conflicts of Interest
The authors declare no potential conflicts of interest.
Electronic Supplementary Materials
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Thawed neural precursor cells. A&B) H1-derived NPCs p10, 0.5 well frozen and thawed in the presence of 10 μM Y27632 - A) 4 h post thaw; B) 24 h post thaw; C&D) SCU-i10-derived NPCs p5, 1 well frozen and thawed in the presence of 10 μM Y27632 - C) 4 h post thaw; D) 24 h post thaw. Scale bars = 100 μm. (PPT 3105 kb)
Immunostaining of SCU-i10 NPCs differentiated for 11 days with antibodies to MAP2 (top row; red) and TuJ1 (second row; green). Merged images of both immunostains with Hoechst nuclear stain (third row; blue) are shown in the bottom row. Scale bars = 100 μm. Three differentiation media were compared - standard Neurobasal medium containing BDNF and GDNF (left column), mTeSR1 (center column) and NDM (right column). (PPT 3859 kb)
Characterization of the SCU-i10 human iPSC line. A) FACS analysis shows SCU-i10s are positive for SSEA-4, Tra-1-60 and Tra-1-81 and negative for SSEA-1; B) Karyotype is normal at p40 (performed by Cell Line Genetics, Madison, WI); C) Immunostaining of cells differentiated to endodermal lineage with antibodies to hepatocyte markers HNF4A (red) and albumin (green) with Hoechst nuclear stain (blue). Scale bar = 100 μm; D) Image captured from Supplementary Movie 1 which shows spontaneously beating area of differentiated cells. (PPT 1022 kb)
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Kozhich, O.A., Hamilton, R.S. & Mallon, B.S. Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells. Stem Cell Rev and Rep 9, 531–536 (2013) doi:10.1007/s12015-012-9357-8
- Pluripotent stem cells
- Neural precursor