Dynamic culture improves cell reprogramming efficiency

Abstract

Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

“…However, we found that reprogramming efficiency decreased when the total vector concentration was too high. Previous studies have shown that cell density is critical for reprogramming (Zhao et al 2015 ; Sia et al 2016 ). Thus, to optimize cell density, we plated BJ cells transfected with 9 µg episomal vectors onto Matrigel coated 6-well dishes at a density ranging from 1.0 × 10 4 to 3.0 × 10 5 cells per well.…”

Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche

“…Reprogramming was more rapid and more efficient in the engineered hydrogels when compared with traditional 2D culture (167). Nutrient transport within cultures has also been demonstrated to play a significant role in reprogramming efficiency (168).…”

Biophysical regulation of cell reprogramming

“…In fact, Sia et al . conversely reported enhancement in the reprogramming efficiency of mouse iPSC with dynamic culture applied at mid to late phase of reprogramming [31]. The authors attribute this to convective mixing that prevents cell cycle arrest as cells reached confluency.…”