Isolation and clonal analysis of satellite cells from chicken pectoralis muscle

Abstract

Satellite cells, liberated from the breast muscle of young adult chickens by sequential treatment with collagenase and trypsin, were fractionated by Percoll density centrifugation to remove myofibril fragments and cell debris which otherwise heavily contaminate the preparation. This procedure allowed direct measurements of cell yields (1.5-4 X 10(5) cells/g tissue), plating efficiencies (27-40%) and identification of single cells in culture. In mass cultures, satellite cells gave rise to myotubes on the fifth day, and the progeny of these cells were sequentially passaged several times without losing myogenic traits. In clonal studies, over 90% of the satellite cells gave rise to large clones of which more than 99% were myogenic as demonstrated by the appearance of myotubes. The results obtained with satellite cells differ from observations made using embryonic muscle cell preparation from chicks. In the embryonic system massive formation of myotubes was observed following the third day of culture; sequential subculturing led to overgrowth of fibroblast-like cells following the first passage; and cells gave rise to both small myogenic clones (up to 16 terminally differentiated cells per clone) and non-myogenic clones in addition to large myogenic clones. We conclude that the isolated satellite cells represent a homogeneous cell population and reside in a stem cell compartment.

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

“…Since more than 90% of the isolated cells express Pax7, no further purification is required (Figures 6-8). This avoids extra purification steps in other methods such as pre-plating on uncoated dishes 14,37,38 , fractionation on Percoll 39,40 , or fluorescent-or magnetic cell sorting 41,43 . For trituration it is essential to induce shear between the tissue fragments and the opening of the pipette tip as this allows the mechanical release of the SCs.…”

“…Mincing, enzymatic digestion, and trituration are generally required to release SCs from their niche. SCs can be purified by pre-plating on uncoated dishes 14,37,38 , fractionation on Percoll 39,40 , or fluorescent-or magnetic cell sorting [41][42][43] . Here we present a new economic and rapid protocol for the isolation of satellite cells from branchiomeric head muscles of young adult rats.…”

A combinatorial role for NFAT5 in both myoblast migration and differentiation during skeletal muscle myogenesis

“…Three days following injury, the muscles were dissected and enzymatically dissociated as described previously (Mitchell and Pavlath, 2004). Following differential centrifugation through an isotonic Percoll gradient (Yablonka-Reuveni et al, 1987), cells were isolated, quantified and resuspended in cold PBS containing 0.25% BSA. Cells were incubated with either FITC-conjugated anti-CD11b or isotype control IgG2b (BD Pharmingen) at a concentration of 2.35 g/10 6 cells for 20 minutes.…”

Muscle reconstitution by muscle satellite cell descendants with stem cell-like properties

“…After transplantation, myoblasts fuse with host myofibers expressing the same type of myosin heavy chain . In addition, clonal analysis suggests that satellite cells within the same muscle proliferate at different rates in vitro (Molnar et al, 1996;Schultz et al, 1985;Yablonka-Reuveni et al, 1987). However, whether multipotentiality is a common property of all satellite cells or is preserved in only a limited fraction of satellite cells remains unknown.…”