A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Abstract

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.

Long‐Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation

“…Furthermore, we could quantify the doubling time for each generation of cells with a mean value of 22.8 h ± 10. ( Table 1 ), which lies between the doubling times that were previously reported for ES cells cultured in 2i medium in adherent conditions (≈13 h) and in suspension (30 h ± 5) . We conclude that single mES cells can be encapsulated in agarose beads and propagated long‐term (68 h) under perfusion conditions, forming monoclonal stem cell spheroids, while remaining undifferentiated.…”

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells

“…4B) of cyst-forming cells, providing evidence for the presence of TLCs as suggested by previous studies. 4,5,25,26 A comparative immunostaining of EB for CDX2 turned out negative for the trophoblast marker (Fig. 4C), suggesting that the cysts were distinctively dissimilar to EBs in both structure and expression (compare Fig.…”

“…[4][5][6][7][8] BMP4 is a member of the transforming growth factor beta superfamily, which controls numerous events of embryonic, fetal, and even adult development in all vertebrates. 9 However, although widely used to derive TLCs from hESCs, this model has not been successful with hiPSCs, as illustrated by the fact that only very few studies have succeeded in converting hiPSCs into TLCs by BMP4 treatment.…”

Serum Inter-α-inhibitor Activates the Yes Tyrosine Kinase and YAP/TEAD Transcriptional Complex in Mouse Embryonic Stem Cells

“…4, A and B). Interestingly, the 2i inhibitor mixture containing CHIR99021 and PD0325901, which is known to stimulate stem cell factors and has been described to accomplish lower grades of spontaneous differentiation in culture, also increased YAP nuclear localization, further linking YAP nuclear localization to stem cell maintenance and selfrenewal (19,38).…”