Culture and expansion of the human embryonic stem cell line HS181, evaluated in a double-color system.
An approach of using RFP-transfected human foreskin fibroblasts (hFS-RFP) to support the growth of GFP expressing human embryonic stem cells (hES; HS181-GFP) is reported. The two-color system was applied to detect interactions between hFS and human embryonic stem cells (hES). After overnight culture, the hES cell colonies showed a behavior of "pushing away" the underlying feeder cells. This phenomenon occurred with both a low and high density of feeders. The density of the feeder cell layer, however, influenced the growth pattern of hES cell colonies. At a high feeder cell density, the hES colonies were more pointed and aligned with the direction of the fibroblasts, whereas less dense feeder layers allowed a more rounded and flat hES colony formation. Not surprisingly, a small fraction of mitotically inactivated feeder cells reattached after passage and remained viable in the cultures for up to four subsequent passages. The prospect of using the two-color system for detection of possible fusion events between hES cells and feeder cells was assessed by screening a large number of cell cultures for double RFP/EGFP expressing cells. The results indicate that fusion events are extremely rare (<10(-6)), or alternatively that after fusion the dual expression of both EGFP and RFP is not easily detected for other reasons. In summary, a two-color system allows analysis of colony formation and also helps to identify and follow the differentiation of cells.
Imreh
Marta P
MP
Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden. marta.imreh@crc.ki.se
Wolbank
Susanne
S
Unger
Christian
C
Gertow
Karin
K
Aints
Alar
A
Szeles
Anna
A
Imreh
Stefan
S
Hovatta
Outi
O
Fried
Gabriel
G
Dilber
Sirac
S
Ahrlund-Richter
Lars
L
D001483:Base Sequence; D002450:Cell Communication; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D017931:DNA Primers; D004622:Embryo, Mammalian; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology; D014162:Transfection
D017931:DNA Primers; D049452:Green Fluorescent Proteins