Integrated platform for production and purification of human pluripotent stem cell-derived neural precursors.
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.
Department of Bioengineering and IBB - Institute for Biotechnology and Bioengineering, Instituto Superior Técnico, University of Lisbon, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
Gonçalo M C
GM
Rodrigues
Andreia F S
AF
Matos
Tiago G
TG
Fernandes
Carlos A V
CA
Rodrigues
Michael
M
Peitz
Simone
S
Haupt
Maria Margarida
MM
Diogo
Oliver
O
Brüstle
Joaquim M S
JM
Cabral
D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002469:Cell Separation; D005434:Flow Cytometry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D058953:Neural Stem Cells / Q000502:physiology*