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1.  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.
doi:10.1002/jemt.1070300504
PMCID: PMC4082319  PMID: 7787236
Myogenesis; Myosin; MyoD; Myogenin; PDGF; FGF; Transferrin; Chicken; Rat; C2 cells
2.  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
3.  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
4.  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
5.  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
6.  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.
doi:10.1007/978-1-62703-128-8_28
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
7.  Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells 
Summary
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
8.  Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System 
Summary
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.
doi:10.1007/978-1-61779-343-1_2
PMCID: PMC3325159  PMID: 22130829
Skeletal muscle; satellite cell; stem cell; myogenesis; Pronase; gelatin; Matrigel; Pax7; MyoD; myogenin
9.  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
10.  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.
doi:10.1002/jcp.21365
PMCID: PMC3276070  PMID: 18186042
Fibroblast growth factor receptor; FGFR4; alternative splicing; N-glycosylation; tyrosine phosphorylation; myogenesis; satellite cells; SU5402; AP20187
11.  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.
doi:10.1016/j.ydbio.2010.01.006
PMCID: PMC2854302  PMID: 20079729
Stem cells; satellite cells; reserve cells; aging; myogenesis; skeletal muscle; nestin-GFP; Myf5nLacZ/+; MLC3F-nLacZ; myostatin; Pax7; α7 integrin; Myf5
12.  Reduced Satellite Cell Numbers and Myogenic Capacity in Aging Can Be Alleviated by Endurance Exercise 
PLoS ONE  2010;5(10):e13307.
Background
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.
Methodology
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.
doi:10.1371/journal.pone.0013307
PMCID: PMC2953499  PMID: 20967266
13.  LRP5 Is Required for Vascular Development in Deeper Layers of the Retina 
PLoS ONE  2010;5(7):e11676.
Background
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.
Conclusions
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.
doi:10.1371/journal.pone.0011676
PMCID: PMC2907392  PMID: 20652025
14.  Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury 
PLoS ONE  2010;5(6):e10920.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0010920
PMCID: PMC2880004  PMID: 20532193
15.  A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis 
Satellite cells are skeletal muscle stem cells that provide myogenic progeny for myofiber growth and repair. Temporal expression of muscle regulatory factors (MRFs) and the paired box transcription factor Pax7 defines characteristic phases of proliferation (Pax7+/MyoD+/myogenin−) and differentiation (Pax7−/MyoD+/myogenin+) during myogenesis of satellite cells. Here, using bromodeoxyuridine (BrdU) labeling and triple immunodetection, we analyzed expression patterns of Pax7 and the MRFs MyoD, Myf5, or myogenin within S phase myoblasts prepared from posthatch chicken muscle. Essentially all BrdU incorporation was restricted to Pax7+ cells, of which the majority also expressed MyoD. The presence of a minor BrdU+/Pax7+/myogenin+ population in proliferation stage cultures suggests that myogenin upregulation is alone insufficient for terminal differentiation. Myf5 was detected strictly within Pax7+ cells and decreased during S phase while MyoD presence persisted in cycling cells. This study provides novel data in support of a unique role for Myf5 during posthatch myogenesis.
doi:10.1002/dvdy.21903
PMCID: PMC2799193  PMID: 19301399
16.  Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle 
Developmental biology  2006;294(1):50-66.
The deteriorating in vivo environment is thought to play a major role in reduced stem cell function with age. Stem cell capacity to support tissue maintenance depends not only on their response to cues from the surrounding niche, but also on their abundance. Here we investigate satellite cell (myogenic stem cell) pool size and its potential to participate in muscle maintenance through old age. The numbers and performance of mouse satellite cells have been analyzed using molecular markers that exclusively characterize quiescent satellite cells and their progeny as they transit through proliferation, differentiation and generation of reserve cells. The study establishes that abundance of resident satellite cells declines with age in myofibers from both fast- and slow-twitch muscles. Nevertheless, the inherent myogenic potential of satellite cells does not diminish with age. Furthermore, the aging satellite cell niche retains the capacity to support effective myogenesis upon enrichment of the mitogenic milieu with FGF. Altogether, satellite cell abundance, but not myogenic potential, deteriorates with age. This study suggests that the population of satellite cells that participate in myofiber maintenance during routine muscle utilization is not fully replenished throughout life.
doi:10.1016/j.ydbio.2006.02.022
PMCID: PMC2710453  PMID: 16554047
Myogenesis; satellite cell; cell renewal; stem cell; myofiber; myoblast; Pax7; MyoD; FGF; aging; sarcopenia; mouse
17.  Myogenic reprogramming of retina-derived cells following their spontaneous fusion with myotubes 
Developmental biology  2007;311(2):449-463.
Satellite cells are recognized as the main source for myoblasts in postnatal muscle. The possible participation of other cell types in myofiber maintenance remains a subject of debate. Here, we investigated the potential of vascular preparations from mouse retina to undergo myogenesis when cultured alone or with differentiated primary myogenic cultures. The choice of retina, an organ richly supplied with capillary network and anatomically separated from skeletal muscles, ensures that the vasculature preparation is devoid of satellite cells. We demonstrate that retinal-derived cells spontaneously fuse with preexisting myotubes and contribute additional myonuclei, some of which initiate expression of muscle-specific genes after fusion. Myogenic differentiation of retinal cells prior to their fusion with preexisting myotubes was not detected. Although originating from vasculature preparations, nuclei undergoing myogenic reprogramming were contributed by cells that were neither endothelial nor blood borne. Our results suggest smooth muscle/pericytes as the possible source, and that myogenic reprogramming depends on the muscle specific transcription factor MyoD. Our studies provide insights into a novel avenue for myofiber maintenance, relying on nuclei of non-myogenic origin that undergo fusion and subsequent myogenic conversion within host myofibers. This process may support ongoing myofiber maintenance throughout life.
doi:10.1016/j.ydbio.2007.08.056
PMCID: PMC2233612  PMID: 17919536
Retina; vasculature; endothelial cells; pericytes; skeletal muscle; myogenesis; satellite cells; Sca-1; MyoD; Myf5
18.  Nestin-GFP reporter expression defines the quiescent state of skeletal muscle satellite cells 
Developmental biology  2006;304(1):246-259.
Repair of adult skeletal muscle depends on satellite cells, quiescent myogenic stem cells located beneath the myofiber basal lamina. Satellite cell numbers and performance decline with age and disease, yet the intrinsic molecular changes accompanying these conditions are unknown. We identified expression of GFP driven by regulatory elements of the nestin (NES) gene within mouse satellite cells, which permitted characterization of these cells in their niche. Sorted NES-GFP+ cells exclusively acquired a myogenic fate, even when supplemented with media supporting non-myogenic development. Mutual and unique gene expression by NES-GFP+ cells from hindlimb and diaphragm muscles demonstrated intra- and inter-muscular heterogeneity of satellite cells. NES-GFP expression declined following satellite cell activation and was reacquired in late stage myogenic cultures by non-proliferating Pax7+ progeny. The dynamics of this expression pattern reflect the cycle of satellite cell self-renewal. The NES-GFP model reveals unique transcriptional activity within quiescent satellite cells and permits novel insight into the heterogeneity of their molecular signatures.
doi:10.1016/j.ydbio.2006.12.026
PMCID: PMC1888564  PMID: 17239845
Satellite cells; self-renewal; stem cells; endothelial cells; nestin; desmin; Myf5; Pax3; Pax7; Sca1; CD31; GFP; Sca1-GFP; nestin-GFP

Results 1-18 (18)