Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting.
Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm-committed cells in high numbers in a 4-day treatment protocol. Second, a method for the purification of CXCR4-positive endoderm cells by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm-derived somatic cells. © 2017 by John Wiley & Sons, Inc.
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Ulf
U
Diekmann
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Claudia
C
Davenport
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Jasmin
J
Kresse
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Ortwin
O
Naujok
D002452:Cell Count; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004707:Endoderm / Q000166:cytology*; D061252:Feeder Cells / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008280:Magnetics / Q000379:methods*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D019718:Receptors, CXCR4 / Q000378:metabolism
C527276:CXCR4 protein, human; D019718:Receptors, CXCR4
FACS; MACS; cell sorting; definitive endoderm; differentiation; human pluripotent stem cells; purification