Kenichi Kagawa

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.

Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to differentiate. Here we show the differentiation potential and growth rate of ESC correlates positively with the expression level of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). When ESCs are maintained in feeder-free conditions and single cell seeding, ESC KhES-1 having 4520 copies or more of CHD7 in 5 ng total RNA show differentiation potential, but this is lost when the CHD7 copy number is reduced in KhES-1 to less than 696 by alternative culture conditions. Introduction of siCHD7 reduced differentiation potential and growth rate of KhES-1. Interestingly, KhES-1 underwent spontaneous differentiation when mCHD7 was introduced and we could not obtain CHD7-overexpressing ESC in culture. These data suggest that CHD7 drives differentiation, and there is a lower limit for CHD7 to initiate differentiation and an upper limit for CHD7 if maintained in undifferentiated state, and such upper limit varies depending on culture condition. As CHD7 drives cell growth, ESC with the highest permissible CHD7 level in the given culture become dominant in a couple of passages. Thus, we can select differentiation resistance-free cell clones by optimizing the culture system using CHD7 as an index.