The iPS cell laboratory routinely cultures human embryonic stem cells (hESCs) and human iPS cells in a standardized feeder-free mode (geltrex, Essential E8 medium).
To generate new patient-specific iPS cells from primary cell cultures (e.g. fibroblast), we use a polycistronic lentiviral vector in which codon-optimized reprogramming factors (OCT4, KLF4, SOX2 and cMYC) are co-expressed with the fluorescent marker dTomato under the control of a retroviral promoter (SFFV) which is rapidly silenced in iPS cells. iPSCs are characterized using an enzymatic assay to measure alkaline phosphatase activity, immunocytochemistry and RT-PCR to study expression of the pluripotent genes, like Nanog, Tra-1-60, SSEA3, OCT4, SOX2, KLF4 and cMYC. Additionally we perform karyotype analysis, study embryoid body differentiation and analyze germ layer marker expression. To generate the specific neural cell types (oligodendrocytes, astrocytes and neurons) existing protocol have been optimized. The iPS-derived neural cell types are characterized with immunofluorescence and RT-PCR for known markers of the neural cell lineages, and functionally tested in collaboration with others.
The iPS center can assist in the generation of new iPS cells, the differentiation towards and functional analyses of specific neural cell types.
The iPS cell laboratory is an initiative of Prof. dr. M.S. van der Knaap, department of Pediatrics and Child Neurology, VU University Medical Center.