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Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells

Abstract

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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Acknowledgements

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.

Author information

Correspondence to B. S. Mallon.

Electronic Supplementary Materials

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Supplementary Figure 1

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)

Supplementary Figure 2

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)

Supplementary Figure 3

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

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Keywords

  • Pluripotent stem cells
  • Differentiation
  • Neural precursor
  • Neurons