title: Efficient culturing and genetic manipulation of human pluripotent stem cells.
abstract: Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Schinzel
authors.forename: Tim
authors.initials: T
authors.lastname: Ahfeldt
authors.forename: Frank H
authors.initials: FH
authors.lastname: Lau
authors.forename: Youn-Kyoung
authors.initials: YK
authors.lastname: Lee
authors.forename: Alicia
authors.initials: A
authors.lastname: Cowley
authors.forename: Tony
authors.initials: T
authors.lastname: Shen
authors.forename: Derek
authors.initials: D
authors.lastname: Peters
authors.forename: David H
authors.initials: DH
authors.lastname: Lum
authors.forename: Chad A
authors.initials: CA
authors.lastname: Cowan
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D005821:Genetic Techniques; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*