Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders.
OBJECTIVE
To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stem cell niche in situ.


METHODS
We isolated and characterized human placental fibroblast lines from individual donors and tested their ability to support growth of federally registered hESC lines. Moreover, we performed extensive pathogen testing to ensure their suitability as feeders for the derivation of therapy-grade hESCs.


RESULTS
Human placental fibroblasts were comparable or superior to mouse embryo fibroblasts as hESC feeders. We used these qualified placental fibroblasts to derive two new hESC lines in knockout Dulbecco's modified Eagle's medium with serum-free 20% knockout serum replacement. The cells, which had a normal karyotype, were grown for more than 25 passages, expressed markers of stemness including Oct-3/4, Tra 1-60, Tra 1-80, and SSEA-4, exhibited high telomerase activity, and differentiated in vitro and in vivo into cells derived from all three germ layers, confirming their pluripotency. Additionally, newly derived hESCs were adapted to growth on a human placental laminin substrate in a defined medium.


CONCLUSIONS
To our knowledge, this is the first report of hESC derivation in the absence of serum on qualified pathogen-free human feeders.
Genbacev
Olga
O
Department of Cell and Tissue Biology, Program in Human Stem Cell Biology, and Developmental and Stem Cell Biology Program, University of California San Francisco, San Francisco, California 94143-0512, USA.
Krtolica
Ana
A
Zdravkovic
Tamara
T
Brunette
Elisa
E
Powell
Sandra
S
Nath
Aneel
A
Caceres
Eduardo
E
McMaster
Michael
M
McDonagh
Susan
S
Li
Yan
Y
Mandalam
Ramkumar
R
Lebkowski
Jane
J
Fisher
Susan J
SJ
D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D010920:Placenta / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D011247:Pregnancy; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D046509:Tissue Culture Techniques / Q000379:methods*
D016895:Culture Media, Serum-Free