Rapid isolation of undifferentiated human pluripotent stem cells from extremely differentiated colonies.
Conventionally, researchers remove spontaneously differentiated areas in human pluripotent stem cell (hPSC) colonies by using a finely drawn glass pipette or a commercially available syringe needle. However, when extreme differentiation occurs, it is inefficient to purify the remaining undifferentiated cells, as these undifferentiated areas are too small to be isolated completely with the mechanical method. Antibodies can be utilized to purify the rare undifferentiated cells; however, this type of purification cannot be used in xeno-free culture systems. To avoid the loss of valuable hPSCs, we developed a novel method to isolate undifferentiated hPSCs from extremely differentiated colonies that could be easily adapted to xeno-free culture conditions. This protocol involves dissecting away differentiated areas, dissociating the remaining colony into clumps, seeding small clumps into new dishes, and picking undifferentiated colonies for expansion. Using this method, we routinely achieve completely undifferentiated colonies in one passage without the use of antibody-based purification.
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada.
Guoliang
G
Meng
Shiying
S
Liu
Derrick E
DE
Rancourt
D000943:Antigens, Differentiation / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques; D058732:Embryoid Bodies / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007621:Karyotyping; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism
D000943:Antigens, Differentiation; D000954:Antigens, Surface; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C519803:TRA-1-81 antigen, human; C066584:stage-specific embryonic antigen-4