Integrated Platform for Production and Purification of Human Pluripotent Stem Cell-Derived Neural Precursors

Abstract

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications, such as transplantation of clinically engineered tissues and organs, and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study, the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80 % efficiency, by means of the dual-SMAD inhibition protocol, based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted, and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these “contaminating” cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5 % and less than 1.5 % of Tra-1-60 positive cells were present in the purified populations. After re-plating, the purified neural precursors maintained their phenotype, and the success of the preparative purification with MACS was further confirmed with a decrease of 94.3 % in the number of Oct4-positive proliferating hPSC colonies. Thus, the integration of the MACS depletion step with the neural commitment protocol paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC-derived neural cells for different applications.

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Acknowledgments

G.M.C. Rodrigues, T.G. Fernandes and C.A.V. Rodrigues acknowledge support from Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BD/89374/2012, SFRH/BPD/86316/2012 and SFRH/BPD/82056/2011, respectively). This work was financially supported by Fundação para a Ciência e a Tecnologia (grants PTDC/EQU-ERQ/105277/2008 and PTDC/EBB-BIO/122504/2010), and the European Union’s Seventh Framework Program (NeuroStemcell project, grant 222943). LT-NES® is a registered trademark of LIFE&BRAIN GmbH.

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Correspondence to Maria Margarida Diogo.

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Gonçalo M. C. Rodrigues and Andreia F.S. Matos have contributed equally

All co-authors of the manuscript have agreed to the submission of the manuscript. Oliver Brüstle is co-founder of and has stock in LIFE & BRAIN GmbH. All other authors declare no conflict of interest.

Electronic supplementary material

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Video showing the first 24 h of the neural commitment of hiPSCs. (M4V 8324 kb)

Supplementary Figure S1
figure6

Immunocytochemistry of hESC-derived cells after the neural commitment protocol for Sox2 (A), Pax6 (B) and Nestin (C). Nuclei were stained with DAPI. Scale bars: in (A) and (C) - 50 μm; (B) - 100 μm. (JPEG 5062 kb)

Supplementary Figure S2
figure7

FC analysis of hiPSC-derived cells for the early neuroectoderm marker Pax6. The results represent the mean of the percentage of positively stained cells analyzed by FC, calculated from n-replicates. The darker area in the FC graphs represents the isotype control and the lighter grey area represents the positively stained cells. (JPEG 139 kb)

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Rodrigues, G.M.C., Matos, A.F.S., Fernandes, T.G. et al. Integrated Platform for Production and Purification of Human Pluripotent Stem Cell-Derived Neural Precursors. Stem Cell Rev and Rep 10, 151–161 (2014). https://doi.org/10.1007/s12015-013-9482-z

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Keywords

  • Human pluripotent stem cells
  • Neural commitment
  • Magnetic activated cell sorting (MACS)
  • Neural precursors
  • Cell purification