title: 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.
authors.affiliation: Department of Bioengineering, University of California, Berkeley, USA; UC Berkeley & UCSF Joint Graduate Program in Bioengineering, Berkeley, San Francisco, USA. Electronic address: siajunren@gmail.com.
authors.forename: Junren
authors.initials: J
authors.lastname: Sia
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, USA.
authors.forename: Raymond
authors.initials: R
authors.lastname: Sun
authors.affiliation: Department of Bioengineering, University of California, Berkeley, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Chu
authors.affiliation: Department of Bioengineering, University of California, Berkeley, USA; UC Berkeley & UCSF Joint Graduate Program in Bioengineering, Berkeley, San Francisco, USA. Electronic address: song_li@berkeley.edu.
authors.forename: Song
authors.initials: S
authors.lastname: Li
mesh_terms: D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle; D065150:Cellular Reprogramming; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D008822:Mice, Transgenic; D010750:Phosphoproteins / Q000378:metabolism; D033081:Shear Strength; D015398:Signal Transduction; D013997:Time Factors; D051176:beta Catenin / Q000378:metabolism
chemical_list: C493965:Cdkn1c protein, mouse; D050761:Cyclin-Dependent Kinase Inhibitor p57; D010750:Phosphoproteins; D051176:beta Catenin
keywords: Cell proliferation; Cell reprogramming; Induced pluripotent stem cell (iPSC)