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Title: Quantitative and semi-quantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

Author
item Talbot, Neil
item Sparks, Wendy
item Powell, Anne
item KAHL, STANISLAW - US Department Of Agriculture (USDA)
item Caperna, Thomas

Submitted to: In Vitro Cellular and Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2011
Publication Date: 1/1/2012
Citation: Talbot, N.C., Sparks, W.O., Powell, A.M., Kahl, S., Caperna, T.J. 2012. Quantitative and semi-quantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells. In Vitro Cellular and Developmental Biology. 48(1):1-11.

Interpretive Summary: A research study was performed to define better the in vitro culture conditions commonly used for the growth and maintenance of embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and various fastidious cells of the body. Feeder-cells are cells which support the in vitro culture of ESC, iPSC, and other hard to grow and maintain cell types, and the study examined the cell growth and survival molecules elaborated from feeder-cells. Quantitative and semi-quantitative immunoassays were performed to identify these molecules and two widely used types of feeders-cells were included in the study. The most abundant growth factors found to be expressed by the feeder-cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor-1 and -2 (IGF1, IGF2), IGF-binding protein-6 (IGFBP6), macrophage-colony stimulating factor (M-CSF), and pigment epithelium-derived factor (PEDF). CF-1 cells expressed 10x more activin A than STO cells, and also produced larger amounts of IL-6 and IGFBP-2, -3, -4, and -5. Conversely, STO cell produced almost 10x more HGF and 5x more SCF than CF-1 cells. Another class of cell growth and maintenance factors, referred to as chemokines, were also produced by both feeder-cells and included, fractalkine (CX3CL1), IP-10 (CXCL10), JE (CCL2/MCP-1), KC (CXCL1; GROa), NOV (CCN3, IGFBP-9), SDF-1 (CXCL12), and serpine 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), MIP-1a (CCL3), MIP-1ß (CCL4), pentraxin-3 (TSG-14), and platelet factor (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 (ESM-1), endostatin (collagen XVIII), and MMP-3 were expressed more from CF-1 cells. TIMP-1 was robustly expressed by both feeder-cells. Other proteins primarily detected from CF-1 cells included RBP4 and FGF21, while STO cells secreted more IFN-'. Both feeder-cells produced no or low amounts of LIF, TNF-a, VEGF, VEGF-B, prolactin, various interleukins, FGF-1, -2, -7, EGF, HB-EGF, and amphiregulin. The results will help in devising a better in vitro culture environment for the growth and maintenance of farm animal ESC and iPSC.

Technical 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 semi-quantitative immunoassays were performed to identify what cytokines and chemokines 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 and -2 (IGF1, IGF2), IGF-binding protein-6 (IGFBP6), macrophage-colony stimulating factor (M-CSF), and pigment epithelium-derived factor (PEDF). CF-1 cells expressed 10x more activin A than STO cells, and also produced larger amounts of IL-6 and IGFBP-2, -3, -4, and -5. Conversely, STO cell produced almost 10x more HGF and 5x more SCF than CF-1 cells. Assayed semi-quantitatively, relatively large amounts of chemokines were produced by both feeder-cells and included, fractalkine (CX3CL1), IP-10 (CXCL10), JE (CCL2/MCP-1), KC (CXCL1; GROa), NOV (CCN3, IGFBP-9), SDF-1 (CXCL12), and serpine 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), MIP-1a (CCL3), MIP-1ß (CCL4), pentraxin-3 (TSG-14), and platelet factor (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 (ESM-1), endostatin (collagen XVIII), and MMP-3 were expressed more by CF-1 cells. TIMP-1 was robustly expressed by both feeder-cells. Other proteins primarily detected from CF-1 cells included RBP4 and FGF21, while STO cells secreted more IFN-'. Both feeder-cells produced no or low amounts of LIF, TNF-a, VEGF, VEGF-B, prolactin, various interleukins, FGF-1, -2, -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.