Feeder-free growth of undifferentiated human embryonic stem cells

Abstract

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

A novel xeno-free and feeder-cell-free system for human pluripotent stem cell culture

“…Each preparation typically generates about 214 mg matrix proteins from 100 g placental tissue. We tested the growth of H7 cells on FCF matrix with MEF-conditioned medium (MEF-CM), and found that about 10% of the concentration of Matrigel typically used (Xu et al, 2001) is sufficient for maintaining the self-renewing capacity of H7 hESCs.…”

“…The widely-used hESC culture system includes Matrigel (MG) as the ECM and MEF-CM (MEF-CM/MG) (Xu et al, 2001). We compared the capacity of the MEF-CM/MG culture system with our novel XF/FCF culture system for maintaining the self-renewing and pluripotent potential of hESCs.…”

“…Later, Xu et al replaced MEF with Matrigel and MEF-CM (Xu et al, 2001). The major constituents of Matrigel are laminin, collagen, and heparan sulphate proteoglycans.…”

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine

“…To avoid those shortcomings, several human feeder, feeder-free, recombinant laminin, and artificially-formulated defined culture systems have been developed for derivation and maintenance of hESCs, though the elements for sustaining prolonged stable undifferentiated growth in those culture systems remain unsolved [33–39]. These exogenous feeder cells and molecules help maintain the long-term stable growth of undifferentiated hESCs while mask their ability to respond to differentiation inducing signals and molecules.…”

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

“…Human counterparts do not have the same response to LIF [4,5], suggesting that mouse ES cells and human ES (hES) cells require different signals to maintain self-renewal and pluripotency. Recently feeder-free system has been adapted especially for culturing hES cells to eliminate the potential issues of infection and cross-species contamination [6,7]. Some feeder-free systems support the culture of undifferentiated hES cells more efficiently than others [8,9], however, porcine and bovine epiblast and ICM cells cultured on a feeder free system did not grow continuously and started senescence or differentiation [10][11][12][13].…”