Reprogramming human somatic cells into the pluripotent state is a subject of thousands of publications since their discovery in 2007. Originally these studies were pioneered with a set of four genes found to be expressed in native human embryonic stem cells; Oct4, Sox2, KLF4, and c-Myc (Lin28 and Nanog), delivered using genetically modifying methods such as retrovirus, and cultured on non-defined matrices.
In the 13 years since these seminal studies many improvements were made such as (1) replacing genetically modifying methods with non-genome altering safer alternatives; (2) replacement of c-Myc oncogene with non-transforming family member, L-Myc; (3) inclusion of small molecules to boost efficiency of reprogramming; (4) optimizing culture conditions which includes the use of low O2
tension (4–5 %); and (5) use of clinically relevant defined media and matrices.
In this novel study, we combined many of the improvements to demonstrate the successful reprogramming of human foreskin fibroblasts with low O2
conditions in the CellXpert® C170i
incubator. We observed that the tested growth substrates offered stable cell adhesion and spreading following electroporation of fibroblasts, which in turn led to a robust production of karyotypically normal iPSC colonies capable of robust expansion in defined media and ability to differentiate into neural and cardiac lineages.Materials and methods
The reagents and procedures used in this Application Note* are similar to that described previously. When indicated, culture vessels were coated with 5 μg/mL of Vitronectin and used in comparison with a synthetic substrate. Once iPSC colonies appeared in Reprogramming Media, they were transitioned into Animal Free Low Protein hESC media for expansion. iPSC expansion and early steps in neuronal differentiation was carried out with 6-well plates, whereas cells analyzed for immunostaining or terminally differentiated to motor neurons or cardiomyocytes were spin seeded onto 24-well culture plates. Neurons were developed as described previously.
Results and discussion
Expansion and characterization of iPSCs under hypoxic conditions
Rudimentary iPSC colonies gave rise to more mature colonies at day 21–30 post modification and were passaged using gentle non-enzymatic methods. In order to purify the culture of Vitronectin derived cells, manual picking of colonies was used, whereas iPSCs grown on synthetic substrates outgrew any minor number of fibroblasts that were dislodged during the non-enzymatic passaging.
After 7 passages, the episomal plasmid reprogrammed line from the synthetic coating was analyzed for karyotype and found to be normal. Similar cultures were spin seeded onto 24-well dishes and analyzed for routine pluripotency or differentiation markers (Fig. 1). At passage 8, the cultures were found to lack any appreciable expression of SSEA1 and robustly expressed both SSEA4 and Oct4. Likewise, these cells coexpressed Lin28, Nanog, and Tra-1-60.