SCIENTISTS CLONE RHESUS MONKEY TO PRODUCE STEM CELLS

Scientists Clone Rhesus Monkey to Produce Stem Cells

Scientists Clone Rhesus Monkey to Produce Stem Cells

Introduction

The extra cellular matrices in tissues are likely to be the first molecular components with which stem cells interact. Stem cell niches are considered to be significant for programming their differentiation. These complex extra cellular matrices function in tissues to provide support and store growth factors and cytokines as well as biological signals to promote and maintain cell differentiation. The effect of individual Matrix components as well as complex matrices on differentiation of diverse units has been well documented utilising in vitro assays, but their effect on stem cell differentiation is less well studied. The basement membrane matrix, which underlies epithelial and endothelial cells and associates glossy sinew, fat, and peripheral cheek cells, is highly enriched in development factors and has been shown to encourage the differentiation and formation of tissue-like structures of many cell types. In specific, endothelial cells attached to a convoluted blend of basement membrane components, termed Matrigel or EHS gel, pattern capillary-like organisations with a lumen. Likewise, recently isolated Sertoli cells pattern chord-like organisations, and salivary gland cells form acinar-like organisations on Matrigel. (Asakura , 2005)

Materials and Methods

Reagents

Polyclonal antibodies to laminin and monoclonal antibodies to class III tubulin ß were obtained from Chemicon (Temecula, CA). Monoclonal antibodies to vementin (V9) were got from Vector laboratories (Burlingame, CA), and monoclonal antibodies to cytokeratin (AE1/AE3) were obtained from DAKO (Carpenteria, CA). Basement membrane Matrigel at 11-13 mg/ml, Collagen I at 3 mg/ml, and Laminin-1 at 1 mg/ml were obtained from Collaborative Biomedicals, Inc. (Bedford, MA). Using essentially the same protocol as for Matrigel preparation, Cartrigel was prepared from calf knee cartilage obtained from a slaughterhouse. Cartilage was slash into small parts, iced in liquid nitrogen, and pulverized with a mortar and pestle. The fragments were cleaned in high salt and extracted overnight with 2 M urea, as has been done for Matrigel groundwork or with 2 M guanidine. The final Cartrigel preparation was dialyzed against Dulbecco's modified Eagle's medium (DMEM), aliquotted, and stored at -20°C.

Cell Culture

Acloned rhesus monkey ES cell line (R366.4) derived from blastocysts was got from WiCell organisation (Madison, WI) . ES cells were cocultured with mitomycin C-treated (0.8 mg/ml for 2 hours) (Sigma, St. Louis) murine embryonic fibroblasts (MEFs) (Cell fundamental elements, Inc., Boston) in gelatin-coated six-well plates (Nalge Nunc, Inc., Naperville, IL) to prevent spontaneous differentiation. These cultures were grown in ES culture medium that contained knockout DMEM supplemented with 20% defined fetal bovine serum (Hyclone, Logan, UT), 1% Nonessential amino acids (Invitrogen, Rockville, MD), 1 mM L-glutamine (Invitrogen), and 0.1 mM ß-mercaptoethanol (Invitrogen). ES cells were divide by issuing them from the culture plate with 0.8 mg/ml collagenase IV (Invitrogen) and seeded onto a new mitomycin C-treated MEF feeder level in a gelatin-coated six-well plate.(Asakura , 2005)

Extra cellular Matrix Studies

ES cells were released from the six-well plate with collagenase IV (0.8 mg/ml) and suspended in knockout DMEM development ...