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1.  Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin 
Developmental biology  2013;385(2):366-379.
Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5Cre-driven GFP expression, we identified in addition to the expected GFP+ myogenic cells (presumably satellite cells), a second dominant GFP+ population distinguished as being Sca1+, non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5Cre-driven reporter when compared to the MyoDCre driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5Cre-driven GFP+ cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP+ cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5Cre-driven GFP+ cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.
PMCID: PMC3921074  PMID: 23969310
Extraocular muscles; satellite cells; fibro/adipogenic progenitors; pericytes and vascular smooth muscle cells; Myf5; MyoD; Pax3; XLacZ4; Wnt1; Sca-1
2.  Adeno-associated viral (AAV) vectors do not efficiently target muscle satellite cells 
Adeno-associated viral (AAV) vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Several recombinant AAV vectors (rAAV) have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular dystrophies. However, most studies have focused on the ability to transduce mature muscle cells, and have not examined the ability to target myogenic stem cells such as skeletal muscle satellite cells. Here we examined the relative ability of rAAV vectors derived from AAV6 to target myoblasts, myocytes and myotubes in culture and satellite cells and myofibers in vivo. AAV vectors are able to transduce proliferating myoblasts in culture, albeit with reduced efficiency relative to post-mitotic myocytes and myotubes. In contrast, quiescent satellite cells are refractory to transduction in adult mice. These results suggest that while muscle disorders characterized by myofiber regeneration can be slowed or halted by AAV transduction, little if any vector transduction can be obtained in myogenic stems cells that might other wise support ongoing muscle regeneration.
PMCID: PMC4288464  PMID: 25580445
3.  Decline in muscle strength and running endurance in klotho deficient C57BL/6 mice 
Biogerontology  2013;14(6):10.1007/s10522-013-9447-2.
Alpha klotho (known as klotho) is a multifunctional protein that may be linked to age-associated decline in tissue homeostasis. The original klotho hypomorphic (klothohm) mouse, produced on a mixed C57BL/6 and C3H background, is short lived and exhibits extensive aging-like deterioration of several body systems. Differently, klothohm mice on a pure C57BL/6 background do not appear sickly nor die young, which has permitted us to gain insight into the effect of klotho deficiency in adult life. First, analyzing klotho transcript levels in the kidney, the main site of klotho production, we demonstrated a 71-fold decline in klothohm females compared to wildtype females versus only a 4-fold decline in mutant males. We then examined the effect of klotho deficiency on muscle-related attributes in adult mice, focusing on 7–11 month old females. Body weight and forelimb grip strength were significantly reduced in klothohm mice compared to wildtype and klotho overexpressing mice. The female mice were also subjected to voluntary wheel running for a period of 6 days. Running endurance was markedly reduced in klothohm mice, which exhibited a sporadic running pattern that may be characteristic of repeated bouts of exhaustions. When actually running, klothohm females ran at the same speed as wildtype and klotho overexpressing mice, but spent about 65 % less time running compared to the other two groups. Our novel results suggest an important link between klotho deficiency and muscle performance. This study provides a foundation for further research on klotho involvement as a potential inhibitor of age-associated muscle deterioration.
PMCID: PMC3851892  PMID: 24030242
Aging; Alpha klotho; EFmKL46 transgene; Hypomorph; Klotho; Muscle strength; Running endurance; Sarcopenia; Skeletal muscle
4.  Moderate-intensity treadmill running promotes expansion of the satellite cell pool in young and old mice 
The FEBS journal  2013;280(17):4063-4073.
Satellite cells, myogenic progenitors located at the myofiber surface, are essential for repair of adult skeletal muscle. There is ample evidence for age-linked decline in satellite cell numbers and performance in limb muscles. Hence, effective means to activate and expand the satellite cell pool may enhance muscle maintenance and reduce the impact of age-associated muscle deterioration (sarcopenia). Toward this aim, we explored the potential beneficial effect of endurance exercise on satellite cells in young and old mice. Animals were subjected to an 8-week moderate-intensity treadmill running approach that does not inflict apparent muscle damage (0° inclination, 11.5 meter/min, 30 min/day, 6 days/week). Myofibers of extensor digitorum longus muscles were then isolated from exercised and sedentary mice and used for monitoring satellite cell numbers and for harvesting individual satellite cells for clonal growth assays. We specifically focused on satellite cell pools of single myofibers, with the view that daily ware of muscles is likely inflicting individual myofibers rather than causing overall muscle damage. We found an expansion of the satellite cell pool in the exercised groups compared with the sedentary groups, with the same increase factor (~1.6) in both age groups. Current results accord with our findings with rat gastrocnemius, attesting for the consistent effect of exercise running on satellite cell expansion in limb muscles. The experimental paradigm established here is useful for studying satellite cell dynamic at the myofiber niche and for broader investigation of the impact of physiologically and pathologically relevant factors on adult myogenesis.
PMCID: PMC3711960  PMID: 23464362
Skeletal muscle; myofibers; satellite cells; aging; endurance exercise; Nestin-GFP
5.  Discrimination of Myogenic and Nonmyogenic Cells From Embryonic Skeletal Muscle by 90° Light Scattering1 
Cytometry  1988;9(2):121-125.
A myogenic cell suspension was isolated from the breast muscles of 10-day-old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblast like cells present. The Percoll-isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90° light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120–200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.
PMCID: PMC4128021  PMID: 3359891
Myoblasts; fibroblasts; cell sorting; tissue culture
6.  Isolation and Clonal Analysis of Satellite Cells from Chicken Pectoralis Muscle 
Developmental biology  1987;119(1):252-259.
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 × 105 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.
PMCID: PMC4128172  PMID: 3025033
7.  Temporal Expression of Regulatory and Structural Muscle Proteins during Myogenesis of Satellite Cells on Isolated Adult Rat Fibers 
Developmental biology  1994;164(2):588-603.
Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we analyzed myogenesis of satellite cells on intact fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. In the present study patterns of satellite cell proliferation, expression of myogenic regulatory factor proteins, and expression of differentiation-specific, cytoskeletal proteins were determined, via immunohistochemistry of cultured fibers. The temporal appearance and the numbers of cells positive for proliferating cell nuclear antigen (PCNA) or for MyoD were similar, suggesting that MyoD is present in detectable amounts in proliferating but not quiescent satellite cells. Satellite cells positive for myogenin, α-smooth muscle actin (αSMactin), or developmental sarcomeric myosin (DEVmyosin) appeared following the decline in PCNA and MyoD expression. However, expression of myogenin and αSMactin was transient, while DEVmyosin expression was continuously maintained. Moreover, the number of DEVmyosin+ cells was only half of the number of myogenin+ or αSMactin+ cells—indicating, perhaps, that only 50% of the satellite cell descendants entered the phase of terminal differentiation. We further determined that the number of proliferating satellite cells can be modulated by basic FGF but the overall schedule of cell cycle entry, proliferation, differentiation, and temporal expression of regulatory and structural proteins was unaffected. We thus conclude that satellite cells conform to a highly coordinated program when undergoing myogenesis at their native position along the muscle fiber.
PMCID: PMC4128087  PMID: 7913900
8.  Characterization of myosin isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior muscles 
Satellite cells were isolated by enzymatic dissociation and Percoll gradient centrifugation from adult rat diaphragm, soleus, and tibialis anterior muscles with fairly reproducible yields. Diaphragm and soleus muscle yielded approximately five times more satellite cells than tibialis anterior muscle. According to light microscopic criteria, no morphological differences existed between the satellite cell cultures of different origin. Contrary to the donor muscles, myotubes from the 10-day-cultured satellite cells contained a uniform myosin heavy chain (MHC) pattern with predominance of an immunochemically identified embryonic heavy chain. The three types of cultures displayed a typical embryonic light chain (LC) pattern with LC1emb, LC1f, LC2f, and traces of LC3f. The isomyosin pattern was characterized by four embryonic isomyosins, eMl–eM4, with similar distributions in the three cultures. In summary, these myosin analyses provide no evidence for the existence of satellite cell diversity among three rat muscles of different fiber-type composition, at least not under the applied in vitro conditions.
PMCID: PMC4096307  PMID: 2090520
We describe a simple technique for maintaining highly contractile long-term chicken myogenic cultures on Matrigel, a gel composed of basement membrane components extracted from the Engelbreth-Holm-Swarm mouse tumor. Cultures grown on Matrigel consist of three-dimensional multilayers of cylindrical, contracting myotubes which endure for at least 60 d without myotube detachment. A Matrigel substrate increases the initial plating efficiency but does not effect cell proliferation. Large-scale differentiation in cultures maintained on Matrigel is delayed by 1 to 2 d, compared to cultures grown on gelatin-coated dishes. Long-term maintenance on Matrigel also results in increased expression of the neonatal and adult fast myosin heavy chain isoforms. Culturing of cells on a Matrigel substrate could thus facilitate the study of later events of in vitro myogenesis.
PMCID: PMC4096309  PMID: 2243058
myogenesis; basement membrane; Matrigel; myosin isoforms; chicken embryo
10.  Influence of PDGF-BB on Proliferation and Transition Through the MyoD-myogenin-MEF2A Expression Program During Myogenesis in Mouse C2 Myoblasts 
We have previously demonstrated that PDGF-BB enhances proliferation of C2 myoblasts. This has led us to examine whether the mitogenic influence of PDGF-BB in the C2 model correlates with modulation of specific steps associated with myogenic differentiation. C2 myoblasts transiting through these differentiation specific steps were monitored via immunocytochemistry. We show that the influence of PDGF on enhancing cell proliferation correlates with a delay in the emergence of cells positive for sarcomeric myosin. We further monitored the influence of PDGF-BB on differentiation steps preceding the emergence of myosin+ cells. We demonstrate that mononucleated C2 cells first express MyoD (MyoD+/myogenin− cells) and subsequently, myogenin. Cells negative for both MyoD and myogenin (the phenotype preceding the MyoD+ state) were present at all times in culture and comprised the majority, if not all, of the cells which responded mitogenically to PDGF. Additionally, the frequency of the MyoD+/myogenin+ cell phenotype was reduced in cultures receiving PDGF, suggesting that PDGF can modulate the transition of the cells into the myogenin+ state. We determined that many of the myogenin+ cells subsequently become MEF2A+ and this phenomenon is not influenced by PDGF-BB. FGF-2 also enhanced the proliferation of C2 myoblasts and suppressed the appearance of the myogenin+ cells, but did not influence the subsequent transition into the MEF2A+ state. The study raises the possibility that PDGF-BB and FGF-2 might delay the transition of the C2 cells into the MyoD+/myogenin+ state by depressing a paracrine signal that enhances differentiation.
PMCID: PMC4096310  PMID: 9401815
PDGF; FGF; C2 myoblasts; myogenic regulatory factors; myogenic enhancer factor 2; cyclin A; desmin p21
11.  Isolation and Distribution of Elongation Factor 2 in Eggs and Embryos of Sea Urchins† 
Biochemistry  1983;22(22):5205-5212.
The subcellular distribution of elongation factor 2 (EF-2) in eggs and early embryos of the sea urchin, Strongylocentrotus purpuratus, was studied by employing the diphtheria toxin dependent ADP-ribosylation of EF-2. When egg and embryo homogenates were fractionated by sedimentation, EF-2 was found associated with a low-speed pellet containing yolk, nuclei, and mitochondria. It also sedimented at 80 S and 5 S. No significant amounts of EF-2 were found on polyribosomes. The 5S form of EF-2 probably represents a monomeric unit of the factor as EF-2 had a molecular weight of 95 000 on sodium dodecyl sulfate–polyacrylamide gels. EF-2 could only be isolated intact if soybean trypsin inhibitor or EGTA was present. The total amount of EF-2 was similar in eggs and embryos. However, the distributions of the factor between the various fractions were substantially different for eggs and embryos. Also, a marked difference in the physical association of EF-2 with material in the low-speed pellet occurred after fertilization. Specifically, in eggs, 23% of the EF-2 was associated with the low-speed pellet; in cleavage-stage embryos, only 11% of the EF-2 was associated with the pellet. In eggs, 65% of the EF-2 sedimented as 80 S; by the 16-cell stage, this amount decreased to 44%. Concomitantly, the amount of EF-2 in the 5S fraction increased from about 8% in eggs to 44% in the 16-cell embryos. In addition, Triton X-100 was required for the extraction of EF-2 from the low-speed pellet of eggs, but not of embryos. We suggest that a redistribution of EF-2 after fertilization either may account for the increase in EF-2 activity observed by Felicetti et al. (1972) [Felicetti, L., Metafora, S., Gambino, R., & Di Matteo, G. (1972) Cell Differ. 1, 265–277] and, thus, be important in mediating the observed 2.5-fold increase in elongation rates after fertilization or may allow the activity of elongation factors to keep pace with the 50-fold increased rate of translation that occurs by the 2-cell stage.
PMCID: PMC4096313  PMID: 6652061
12.  Development and Postnatal Regulation of Adult Myoblasts 
Microscopy research and technique  1995;30(5):366-380.
The myogenic precursor cells of postnatal and adult skeletal muscle are situated underneath the basement membrane of the myofibers. It is because of their unique positions that these precursor cells are often referred to as satellite cells. Such defined satellite cells can first be detected following the formation of a distinct basement membrane around the fiber, which takes place in late stages of embryogenesis. Like myoblasts found during development, satellite cells can proliferate, differentiate, and fuse into myofibers. However, in the normal, uninjured adult muscle, satellite cells are mitotically quiescent. In recent years several important questions concerning the biology of satellite cells have been asked. One aspect has been the relationship between satellite cells and myoblasts found in the developing muscle: are these myogenie populations identical or different? Another aspect has been the physiological cues that control the quiescent, proliferative, and differentiative states of these myogenie precursors: what are the growth regulators and how do they function? These issues are discussed, referring to previous work by others and further emphasizing our own studies on avian and rodent satellite cells. Collectively, the studies presented indicate that satellite cells represent a distinct myogenie population that becomes dominant in late stages of embryogenesis. Moreover, although satellite cells are already destined to be myogenie precursors, they do not express any of the four known myogenie regulatory genes unless their activation is induced in the animal or in culture. Furthermore, multiple growth factors are important regulators of satellite cell proliferation and differentiation. Our work on the role of one of these growth factors [platelet-derived growth factor (PDGF)] during proliferation of adult myoblasts is further discussed with greater detail and the possibility that PDGF is involved in the transition from fetal to adult myoblasts in late embryogenesis is brought forward.
PMCID: PMC4082319  PMID: 7787236
Myogenesis; Myosin; MyoD; Myogenin; PDGF; FGF; Transferrin; Chicken; Rat; C2 cells
13.  The Emergence of the Endothelial Cell Lineage in the Chick Embryo Can Be Detected by Uptake of Acetylated Low Density Lipoprotein and the Presence of a von Willebrand-like Factor 
Developmental biology  1989;132(1):230-240.
Vascular endothelial cells from 3- to 10-day-old chicken embryos were identified by the uptake of acetylated low density lipoprotein (Ac-LDL) and the presence of a von Willebrand-like factor. These were determined on cross sections of aortic arches as well as in cell cultures prepared from the arches. To visualize the uptake of Ac-LDL, the fluorescent probe l,r-dioctadecyl-l-3,3,3’,3’-tetramethyl-indo-carbocyanine perchlorate-Ac-LDL (DiI-Ac-LDL) was used. Following injection of the DiI-Ac-LDL probe into the embryonic heart, the endothelium of the aortic arches became specifically labeled. Also, following the administration of the probe to cell cultures, about 5–10% of the cells became DiI-positive. Indirect immunofluorescence with an antibody against von Willebrand (vW) factor also revealed specific staining of the endothelium of the aortic arches as well as of a subset of cells in cultures from aortic arches. These two histochemical markers were further used to identify the emergence of the endothelial cell lineage in the chicken blastodisc. Cultured cells from embryos incubated in ovo for 16 hr exhibited both uptake of DiI-Ac-LDL and expression of a vW-like factor. The proportion of these cells was about 30% of the total cultured cells and increased to over 50% in cultures of embryos incubated in ovo for 20 hr. However, cells positive for uptake of DiI-Ac-LDL and expression of vW-like factor were lacking in cultures of unincubated eggs or eggs incubated for 6–10 hr. We conclude that the very early endothelial cells in the chick blastodisc are already capable of expressing characteristic properties of vascular endothelium.
PMCID: PMC4080922  PMID: 2783918
14.  Skeletal Muscle Satellite Cells Appear during Late Chicken Embryogenesis 
Developmental biology  1992;153(2):206-216.
The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult (12–16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.
PMCID: PMC4075331  PMID: 1397678
15.  Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program 
Anatomy and embryology  1998;197(6):421-437.
Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocyto-chemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (αSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for αSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.
PMCID: PMC4046509  PMID: 9682974
Aortic arch-derived arteries; Vascular system; Chick embryo; Cytoskeletal proteins; Basement membrane
16.  Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle 
Desmin expression by myoblasts cultured from embryonic and adult chicken breast muscle was examined employing indirect immunofluorescence. The study was performed in conjunction with [3H]thymidine autoradiography and analysis of skeletal myosin expression in order to determine whether the desmin-expressing cells were terminally differentiated. Following 2 h of labeling with [3H]thymidine, 0.55%, 2.60%, and 15.10% of the cells in mass cultures from 10-day-old embryos, 18-day-old embryos and adults, respectively, incorporated [3H]thymidine and were desmin-positive but did not express skeletal-muscle-specific myosin. Using the same approach we determined that 0.07%, 1.25%, and 7.59% of the mononucleated cells in myogenic clones from 10-day-old embryos, 18-day-old embryos and adults, respectively, were desmin-positive, myosin-negative, [3H]thymidine-positive. We suggest that these desmin-positive, myosin-negative myoblasts are proliferating cells, and we conclude that the progeny of adult myoblasts exhibit more desmin-expressing cells of this type than embryonic myoblasts do.
PMCID: PMC4038325  PMID: 2292359
17.  Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells 
Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.
PMCID: PMC3633469  PMID: 23179849
Skeletal muscle; satellite cells; stem cells; collagen; Matrigel; myofiber isolation; flexor digitorum brevis; extensor digitorum longus; diaphragm; masseter; extraocular; mouse; immunostaining; Pax7
18.  Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells 
Myofibers are the functional contractile units of skeletal muscle. Mononuclear satellite cells located between the basal lamina and the plasmalemma of the myofiber are the primary source of myogenic precursor cells in postnatal muscle. This chapter describes protocols used in our laboratory for isolation, culturing and immunostaining of single myofibers from mouse skeletal muscle. The isolated myofibers are intact and retain their associated satellite cells underneath the basal lamina. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. Myofibers are cultured in dishes coated with Vitrogen collagen and satellite cells remain associated with the myofibers undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL). Different from the FDB myofibers, the longer EDL myofibers tend to tangle and break if cultured together; therefore, EDL myofibers are cultured individually. These myofibers are cultured in dishes coated with Matrigel. The satellite cells initially remain associated with the myofiber and later migrate away to its vicinity, resulting in extensive cell proliferation and differentiation. These culture protocols allow studies on the interplay between the myofiber and its associated satellite cells.
PMCID: PMC3523695  PMID: 15361669
Satellite cells; skeletal muscle; myofiber isolation; single myofiber culture; flexor digitorum brevis; extensor digitorum longus; mouse; Vitrogen collagen; Matrigel
19.  Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System 
Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. While emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells.
PMCID: PMC3325159  PMID: 22130829
Skeletal muscle; satellite cell; stem cell; myogenesis; Pronase; gelatin; Matrigel; Pax7; MyoD; myogenin
20.  Reflections on lineage potential of skeletal muscle satellite cells: Do they sometimes go MAD? 
Postnatal muscle growth and repair is supported by satellite cells - myogenic progenitors positioned between the myofiber basal lamina and plasma membrane. In adult muscles, satellite cells are quiescent but become activated and contribute differentiated progeny when myofiber repair is needed. The development of cells expressing osteogenic and adipogenic genes alongside myoblasts in myofiber cultures, raised the hypothesis that satellite cells possess mesenchymal plasticity. Clonal studies of myofiber-associated cells further suggested that satellite cell myogeneity and diversion into Mesencymal Alternative Differentiation (MAD) occur in vitro by a stochastic mechanism. However, in vivo this potential may be executed only when myogenic signals are impaired and the muscle tissue is compromised. Such a mechanism may contribute to the increased adipocity of aging muscles. Alternatively, it is possible that mesenchymal interstitial cells (sometimes co-isolated with myofibers), rather than satellite cells, account for the nonmyogenic cells observed in myogenic cultures. Herein, we first elaborate on the myogenic potential of satellite cells. We then introduce definitions of adult stem-cell unipotency, multipotency and plasticity, and elaborate on recent studies that established the status of satellite cells as myogenic stem cells. Lastly, we highlight evidence in favor of satellite cell plasticity and emerging hurdles restraining this hypothesis.
PMCID: PMC3276064  PMID: 17341181
Mesenchymal stem cell; myoblast; adipocyte; myogenesis; adipogenesis; osteogenesis; multipotential
21.  FGFR4 and its novel splice form in myogenic cells: interplay of glycosylation and tyrosine phosphorylation 
Journal of Cellular Physiology  2008;215(3):803-817.
The family of fibroblast growth factor receptors (FGFRs) is encoded by four distinct genes. FGFR1 and FGFR4 are both expressed during myogenesis, but whereas the function of FGFR1 in myoblast proliferation has been documented, the role of FGFR4 remains unknown. Here we report on a new splice form of FGFR4 cloned from primary cultures of mouse satellite cells. This form, named FGFR4(−16), lacks the entire exon 16, resulting in a deletion within the FGFR kinase domain. Expression of FGFR4(−16) coincided with that of wildtype FGFR4 in all FGFR4-expressing tissues examined. Moreover, expression of both FGFR4 forms correlated with the onset of myogenic differentiation, as determined in mouse C2C12 cells and in the inducible myogenic system of 10T½-MyoD-ER cell line. Both endogenous and overexpressed forms of FGFR4 exhibited N-glycosylation. In contrast to FGFR1, induced homodimerization of FGFR4 proteins did not result in receptor tyrosine phosphorylation. Surprisingly, coexpression of FGFR4 forms and a chimeric FGFR1 protein resulted in FGFR4 tyrosine phosphorylation, raising the possibility that FGFR4 phosphorylation might be enabled by a heterologous tyrosine kinase activity. Collectively, the present study reveals novel characteristics of mouse FGFR4 gene products and delineates their expression pattern during myogenesis. Our findings suggest that FGFR4 functions in a distinctly different manner than the prototype FGFR during myogenic differentiation.
PMCID: PMC3276070  PMID: 18186042
Fibroblast growth factor receptor; FGFR4; alternative splicing; N-glycosylation; tyrosine phosphorylation; myogenesis; satellite cells; SU5402; AP20187
22.  The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny 
Developmental biology  2010;340(2):330-343.
Satellite cells are myogenic progenitors residing on the myofiber surface that support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin-GFP/Myf5nLacZ/+ mice reveals a decline with age in the number of satellite cells that express detectable levels of βgal. Nestin-GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7+ reserve population. Clonal analysis of sorted GFP+ satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.
PMCID: PMC2854302  PMID: 20079729
Stem cells; satellite cells; reserve cells; aging; myogenesis; skeletal muscle; nestin-GFP; Myf5nLacZ/+; MLC3F-nLacZ; myostatin; Pax7; α7 integrin; Myf5
23.  Reduced Satellite Cell Numbers and Myogenic Capacity in Aging Can Be Alleviated by Endurance Exercise 
PLoS ONE  2010;5(10):e13307.
Muscle regeneration depends on satellite cells, myogenic stem cells that reside on the myofiber surface. Reduced numbers and/or decreased myogenic aptitude of these cells may impede proper maintenance and contribute to the age-associated decline in muscle mass and repair capacity. Endurance exercise was shown to improve muscle performance; however, the direct impact on satellite cells in aging was not yet thoroughly determined. Here, we focused on characterizing the effect of moderate-intensity endurance exercise on satellite cell, as possible means to attenuate adverse effects of aging. Young and old rats of both genders underwent 13 weeks of treadmill-running or remained sedentary.
Gastrocnemius muscles were assessed for the effect of age, gender and exercise on satellite-cell numbers and myogenic capacity. Satellite cells were identified in freshly isolated myofibers based on Pax7 immunostaining (i.e., ex-vivo). The capacity of individual myofiber-associated cells to produce myogenic progeny was determined in clonal assays (in-vitro). We show an age-associated decrease in satellite-cell numbers and in the percent of myogenic clones in old sedentary rats. Upon exercise, there was an increase in myofibers that contain higher numbers of satellite cells in both young and old rats, and an increase in the percent of myogenic clones derived from old rats. Changes at the satellite cell level in old rats were accompanied with positive effects on the lean-to-fat Gast muscle composition and on spontaneous locomotion levels. The significance of these data is that they suggest that the endurance exercise-mediated boost in both satellite numbers and myogenic properties may improve myofiber maintenance in aging.
PMCID: PMC2953499  PMID: 20967266
24.  LRP5 Is Required for Vascular Development in Deeper Layers of the Retina 
PLoS ONE  2010;5(7):e11676.
The low-density lipoprotein receptor-related protein 5 (LRP5) plays an important role in the development of retinal vasculature. LRP5 loss-of-function mutations cause incomplete development of retinal vessel network in humans as well as in mice. To understand the underlying mechanism for how LRP5 mutations lead to retinal vascular abnormalities, we have determined the retinal cell types that express LRP5 and investigated specific molecular and cellular functions that may be regulated by LRP5 signaling in the retina.
Methods and Findings
We characterized the development of retinal vasculature in LRP5 mutant mice using specific retinal cell makers and a GFP transgene expressed in retinal endothelial cells. Our data revealed that retinal vascular endothelial cells predominantly formed cell clusters in the inner-plexiform layer of LRP5 mutant retina rather than sprouting out or migrating into deeper layers to form normal vascular network in the retina. The IRES-β-galactosidase (LacZ) report gene under the control of the endogenous LRP5 promoter was highly expressed in Müller cells and was also weakly detected in endothelial cells of the retinal surface vasculature. Moreover, the LRP5 mutant mice had a reduction of a Müller cell-specific glutamine transporter, Slc38a5, and showed a decrease in b-wave amplitude of electroretinogram.
LRP5 is not only essential for vascular endothelial cells to sprout, migrate and/or anastomose in the deeper plexus during retinal vasculature development but is also important for the functions of Müller cells and retinal interneurons. Müller cells may utilize LRP5-mediated signaling pathway to regulate vascular development in deeper layers and to maintain the function of retinal interneurons.
PMCID: PMC2907392  PMID: 20652025
25.  Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury 
PLoS ONE  2010;5(6):e10920.
Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described.
Methodology/Principal Findings
In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with α7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury.
Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration.
PMCID: PMC2880004  PMID: 20532193

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