Abstract
In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells — bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.
Highlights
Human embryonic stem cells are pluripotent cells derived from the inner cell mass of blastocyst-stage human embryos [1,2]
In guiding Human embryonic stem (hES) cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. hES cells are most commonly maintained on inactivated mouse embryonic fibroblasts (MEFs) feeders in medium supplemented with knockout serum-replacement (KSR) together with basic fibroblast growth factors
We found that H9 hES cells colonies grown on human fetal liver stromal cells (hFLSCs) were dense and compact, and they exhibited the typical morphology of hES cells (Fig. 1C)
Summary
Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage human embryos [1,2]. They can be expanded indefinitely as undifferentiated cells for extended periods of time, and possess the capacity to generate all cell types in the body. HES cells are most commonly maintained on inactivated mouse embryonic fibroblasts (MEFs) feeders in medium supplemented with knockout serum-replacement (KSR) together with basic fibroblast growth factors (bFGF, or FGF 2). These xeno-support systems run the risk of crosstransfer of animal pathogens from the animal feeder. One example is hES cells cultured with animal products that express Neu5Gc, as a nonhuman sialic acid that triggers an immunogenic response [5]
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