Integrated Platform for Production and Purification of Human Pluripotent Stem Cell-Derived Neural Precursors Gonçalo M. C. Rodrigues & Andreia F. S. Matos & Tiago G. Fernandes & Carlos A. V. Rodrigues & Michael Peitz & Simone Haupt & Maria Margarida Diogo & Oliver Brüstle & Joaquim M. S. Cabral # Springer Science+Business Media New York 2013 Abstract Human pluripotent stem cells (hPSCs) are a prom- ising source of cells for clinical applications, such as trans- plantation 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 tumor- igenic 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 proto- col paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC- derived neural cells for different applications. Keywords Human pluripotent stem cells . Neural commitment . Magnetic activated cell sorting (MACS) . Neural precursors . Cell purification Introduction Human pluripotent stem cells (hPSCs) hold great promise in a wide range of potential applications – including regenerative medicine [1, 2], disease modeling [3, 4] and drug screening tests [5, 6] - due to their unique self-renewal and differentia- tion capabilities [7, 8]. Current differentiation protocols are able to derive clinically relevant cell populations, such as dopaminergic neurons [9], cardiomyocytes [10] and insulin- producing cells [11], from hPSCs. However, many pluripotent stem cells (PSCs), which are potentially tumorigenic, do not differentiate and remain in the differentiated populations. Moreover, in advanced phases of the differentiation protocols, these cells are undesired because they can alter the results of 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 The online version of this article (doi:10.1007/s12015-013-9482-z) contains supplementary material, which is available to authorized users. G. M. C. Rodrigues : A. F. S. Matos : T. G. Fernandes : C. A. V. Rodrigues : M. M. Diogo (*) : J. M. S. Cabral Department of Bioengineering and IBB - Institute for Biotechnology and Bioengineering, Instituto Superior Técnico, University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon, Portugal e-mail: margarida.diogo@ist.utl.pt M. Peitz : S. Haupt : O. Brüstle Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany S. Haupt : O. Brüstle LIFE & BRAIN GmbH, Bonn, Germany Stem Cell Rev and Rep DOI 10.1007/s12015-013-9482-z