A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.
BACKGROUND
Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells.


METHODS
We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment.


RESULTS
Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing.


CONCLUSIONS
We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.
Hovatta
Outi
O
Department of Clinical Science, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden. Outi.Hovatta@klinvet.ki.se
Mikkola
Milla
M
Gertow
Karin
K
Strömberg
Anne-Marie
AM
Inzunza
José
J
Hreinsson
Julius
J
Rozell
Björn
B
Blennow
Elisabeth
E
Andäng
Michael
M
Ahrlund-Richter
Lars
L
D000818:Animals; D001757:Blastomeres / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D007621:Karyotyping; D008297:Male; D051379:Mice; D016513:Mice, SCID; D012867:Skin / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D013724:Teratoma