Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

Abstract

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.

MicroRNAs 221 and 222 regulate the undifferentiated state in mammalian male germ cells

“…An intriguing possibility is that upregulation of KIT expression, which occurred after miR-221/222 inhibition, allowed for activation of signaling from KIT ligand (KITL), thereby causing irreversible loss of stem cell potential. Maintenance of THY1 + undifferentiated spermatogonia as a proliferative stem cellcontaining population in vitro requires co-culture with feeder cell monolayers and in this study we utilized STO feeder cells, which are known to secrete KITL (Lim and Bodnar, 2002;Talbot et al, 2012). These results indicate that the stem cell capacity of spermatogonia is linked to miR-221/222 abundance.…”

“…Here, we used qRT-PCR analysis to measure changes in the abundance of miR-221 and miR-222 in cultured THY1 + undifferentiated spermatogonia following individual exposure to extrinsic factors. The maintenance of these cells as a proliferative stem cell-containing population in vitro requires supplementation of culture media with GDNF and FGF2, and co-culture with feeder cells that secrete other factors including CSF-1 (Lim and Bodnar, 2002;Talbot et al, 2012). Therefore, we removed the germ cell clumps from feeder cell monolayers and…”

First steps to define murine amniotic fluid stem cell microenvironment

“…Amniotic membrane is rich in stromal cells and secretes cytokines and growth factors, such as SCF, HGF and IGF, that are produced by cell lines (i.e. STO and CF-1) usually employed as feeder layer in vitro for ESC expansion52. Moreover, Bashamboo et al 53 showed that in vitro ESC differentiation is dependent on the SCF-KIT pathway, and Chen et al 54 demonstrated how a signalling by vitamin A/retinol promotes the self-renew of ESC through the activation of PI3K/Akt signalling via IGF-1 receptor.…”

Feeder Cell Sources and Feeder-Free Methods for Human iPS Cell Culture

“…It should be noted that the mitotic inactivation or irradiation of MEFs stimulates the expression of several signaling proteins, such as Wnt-3 [16], which may participate in the molecular mechanisms underlying the maintenance of pluripotency in co-cultured human ESCs/iPSCs. The outbred mouse CF-1 strain may be the most widely used donor for MEF feeder cells for ESC and iPSC culture [13,17], as CF-1 MEFs have been shown to produce TGF-β1, activin A, BMP-4, gremlin, and noggin [14,18,19] but not bFGF [14]. One disadvantage of using primary MEFs is their limited proliferation capacity, which requires repeated isolation from embryonic mice to supply feeder cells [20].…”

“…Proteome analyses have revealed that STO cells produce unique protein species, such as insulin-like growth factor binding protein 4 (IGFBP-4), pigment epithelium-derived factor (PEDF) and secreted protein acidic and rich in cysteine (SPARC, also known as osteonectin), which may be associated with differentiation and cell growth [26]. Talbot et al [17] showed from quantitative immunoassays that STO cells express lower levels of activin A, interleukin-6, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 than CF-1 cells but higher levels of hepatocyte growth factor (HGF) and stem cell factor (c-kit ligand). The difference in the growth factor production among feeder cell types may result in different abilities to support the growth of undifferentiated human ESCs/iPSCs.…”