Fluorescence-activated single cell sorting of human embryonic stem cells.
Human embryonic stem cells (hESC) are the subject of intense investigation for use in regenerative medicine, in toxicity testing, and as models for the study of human development. Automated cell sorting will enhance the isolation of homogenous pools of differentiated hESCs both for basic studies and for therapeutic applications. Sorting could also be used to deplete undifferentiated, potentially tumourigenic cells. However, hESCs are sensitive to single cell disaggregation and recover poorly when plated at clonal density. Here we report a method for successful semi-automated single cell sorting of hESCs. This method utilizes an ES-specific promoter-transgene construct and automated FACS-based single cell sorting and plating. Clonal recovery in physiologic oxygen (2%) was increased fourfold over room oxygen (21%; p < 0.01). This automated protocol will help to realize proposed hESC strategies that are hampered by low throughput and poor yields.
Hewitt
Z
Z
Gene Function and Development, Roslin Institute, Roslin, Midlothian, United Kingdom.
Forsyth
N R
NR
Waterfall
M
M
Wojtacha
D
D
Thomson
A J
AJ
McWhir
J
J
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase