title: Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.
abstract: Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.
authors.affiliation: Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Farhan
authors.initials: F
authors.lastname: Chowdhury
authors.forename: Yanzhen
authors.initials: Y
authors.lastname: Li
authors.forename: Yeh-Chuin
authors.initials: YC
authors.lastname: Poh
authors.forename: Tamaki
authors.initials: T
authors.lastname: Yokohama-Tamaki
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.forename: Tetsuya S
authors.initials: TS
authors.lastname: Tanaka
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001703:Biophysics / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D015536:Down-Regulation; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology; D013724:Teratoma / Q000378:metabolism
chemical_list: D053605:Leukemia Inhibitory Factor; D000469:Alkaline Phosphatase