In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.     

Isolation and characterization of porcine amniotic fluid-derived multipotent stem cells

The aim of this study was to isolate and characterize porcine amniotic fluid-derived multipotent stem cells (pAF-MSC). The porcine amniotic fluid (AF) from the amniotic cavity of pregnant gilts in the early stages of gestation (at E35) was collected and centrifuged for 5-10 min at 400 g to pellet cells. The primary culture of AF showed the multiple cell types, including the epithelial-like cells and fibroblast-like cells. By culturing in AMM medium for 6 to 8 days, the epithelial-like cells disappeared and the remaining cells presented the fibroblastoid morphology. The doubling time of pAF-MSCs was about 34.6 h, and the cells had been continually cultured over 60 passages in vitro. The flow cytometry results showed that pAF-MSCs were positive for CD44, CD117 and CD166, but negative for CD34, CD45 and CD54. Meanwhile, pAF-MSCs expressed ES cell markers, such as Oct4, Nanog, SSEA4, Tra-1-60 and Tra-1-81. The ratio of CD117(+) CD44(+) cells accounted for 98% of pAF-MSCs population. Three germ layer markers, including FGF5 (ectodermal marker), AFP (endodermal marker) and Bra (mesodermal marker), were detected in embryoid bodies derived from pAF-MSCs. Under the different induction conditions, the pAF-MSCs were capable of differentiating into neurocytes, adipocytes and beating cardiomyocytes. Furthermore, the pAF-MSCs didn't form teratoma when injected into immunodeficiency mice. These optimal features of pAF-MSCs provide an excellent alternative stem cell resource for potential cell therapy in regenerative medicine and transgenic animals.     

Myokine IL-15 regulates the crosstalk of co-cultured porcine skeletal muscle satellite cells and preadipocytes

The present study was carried out to preliminarily reveal the underlying mechanisms of the co-culture system between porcine muscle satellite cells (SCs) and stromal-vascular cells (SVs). The two cell types were co-cultured to assess both proliferation and differentiation. Desmin and Pref-1 immunofluorescence staining technique were taken to identify the two types of isolated cells. The expression of specific marker genes Myogenin was up-regulated in SCs (P < 0.05) and the differentiation of SCs could be promoted when co-cultured with preadipocytes compared with the single-cultured control, while expression of c/EBPβ in SVs was down-regulated (P < 0.05) and the differentiation of preadipocytes could be inhibited. Furthermore, secretion of myokine IL-15 was markedly increased, as well as its gene and protein expression levels in co-culture supernatants. However, the secretion of adipokine leptin was significantly decreased. These findings demonstrate that myokines like IL-15 could facilitate the SCs’ differentiation while inhibit the SVs differentiation, and act as an important regulator of co-culture between muscle cells and adipocytes.     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

Interaction between satellite cells and skeletal muscle fibers

Single myofibers with attached satellite cells isolated from adult rats were used to study the influence of the mature myofiber on the proliferation of satellite cells. The satellite cells remain quiescent when cultured in serum containing medium but proliferate when exposed to mitogen from an extract of crushed adult muscle. The response of satellite cells to mitogen was measured under three situations with respect to cell contact: (1) in contact with a viable myofiber and its basal lamina, (2) detached from the myofiber by centrifugal force and deposited on the substratum and (3) beneath the basal lamina of a Marcaine killed myofiber. The results show that satellite cells in contact with the plasmalemma of a viable myofiber have reduced mitogenic response. Since inhibiting growth may induce differentiation, I tested whether satellite cells proliferating on the surface of a myofiber would fuse. Although the satellite cell progeny were fusion competent, they did not fuse with the myofiber. To determine whether fusion competence of the myofiber changes with time in culture, embryonic myoblasts were challenged to fuse with myofibers that had been stripped of satellite cells and cultured for several days. The myoblasts fused with pseudopodial sprouts growing from the ends of the myofiber, but did not fuse with the original myofiber surface. These results indicate that contact with the surface of a mature myofiber suppresses proliferation of myogenic cells but the cells do not fuse with the myofiber.     

Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population

In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. We describe a multipotent Pax7⁺ satellite cell population located within the skeletal muscle of the salamander limb. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation.     

The satellite cells of normal anuran skeletal muscle

Population counts and size measurements of satellite cell nuclei and myonuclei were carried out on the normal gastrocnemius muscles of adult Rana pipiens and Rana clamitans. Satellite cell profiles occurred with an observed frequency of about 1.3% in the muscles of the R. pipiens, and with an observed frequency of about 1.6% in the muscles of the R. clamitans. These frequencies were not found to be significantly different. The observed frequencies were corrected for the sampling bias introduced by the difference in the mean size of the satellite cell nuclei and myonuclei. This correction suggests that R. pipiens and R. clamitans both have a true satellite cell frequency of approx 2.7%. Analysis of these data indicates that the satellite cells of the normal anuran gastrocnemius occur in sufficient numbers to account for the regeneration seen after injury to this muscle.     

Isolation of three creatine kinases MM from porcine skeletal muscle

The purified creatine kinase MM of porcine skeletal muscle [Takasawa, T. & Shiokawa, H. (1981) J. Biochem. 90, 195-204] was separated into three distinct fractions by isoelectric focusing (IEF) in a sucrose gradient column, and the three active fractions were isolated by repeated IEF. There were one major fraction with isoelectric point (pI) 6.57 and two minor fractions with pI 6.74 and pI 6.34, respectively. No differences were observed in the IEF pattern of the enzyme in the presence and absence of dithiothreitol throughout the column. There was no interconversion from one form to another during IEF. The distribution of the three forms on IEF was not affected by adding protease inhibitor to the extraction medium. Of the three fractions, the major fraction had the highest specific activity. The three fractions differed from one another in their amino acid compositions. Not only porcine muscle but also rabbit muscle creatine kinase displayed this type of heterogeneity. Such microheterogeneities may occur widely in muscle creatine kinases.     

Regulation of satellite cells during skeletal muscle growth and development

Satellite cells are myogenic cells attributed with the role of postnatal growth and regeneration in skeletal muscle. Following proliferation and subsequent differentiation, these cells will fuse with one another or with the adjacent muscle fiber, thereby increasing myonuclei numbers for fiber growth and repair. The potential factors which could regulate this process are many, including exercise, trauma, passive stretch, innervation, and soluble growth factors. Three classes of growth factors in particular (fibroblast growth factor, insulin-like growth factor, and transforming growth factor-beta) have been studied extensively with respect to their effects on satellite cell proliferation and differentiation in culture. Fibroblast growth factor has been shown to stimulate proliferation but depress differentiation. Insulin-like growth factor stimulates both proliferation and differentiation, although the latter to a much greater degree. Transforming growth factor-beta slightly depresses proliferation but inhibits differentiation. When administered in combination, these factors can induce satellite cell activities in culture which mimic those typical of satellite cells found in vivo in growing, regenerating, or healthy mature muscle. Alterations in the concentrations of these growth factors in the muscle environment as well as alterations in the cell's sensitivity or responsiveness to these factors represent potential mechanisms for regulating satellite cell activity in situ.     

Oxygen-mediated regulation of skeletal muscle satellite cell proliferation and adipogenesis in culture

Major problems in stem cell biology revolve around defining the developmental potential of cell populations and understanding how their potential is maintained or progressively restricted. Oxygen (O(2)) is an obvious environmental factor which has received little attention in culturing skeletal muscle progenitor cells. In this work, we examine the effects of O(2) levels on the developmental potential, proliferative capacity, and phenotype of the adult skeletal muscle fiber progenitor population (satellite cells), and cell lines that model multipotential embryonic paraxial mesoderm from which skeletal muscle develops. Both satellite cell proliferation and survival of mature fibers increased in physiologic (6%) O(2) vs. non-physiologic 20% O(2) used in virtually all traditional cell culture. Six percent O(2) conditions also accelerated the up-regulation of multiple MyoD family myogenic regulatory factors (MRFs). An unexpected finding was that fiber-adherent satellite cells could assume a non-myogenic phenotype. By the criteria of molecular markers and gross lipid accumulation, satellite cells were found to assume an adipocyte phenotype, and did so more prominently in 20% O(2) than in physiologic O(2). Selection of the adipogenic fate and execution of adipogenesis by multipotential mesenchymal cell lines was also dramatically higher in traditional 20 vs. 6% O(2), and decreased adipogenesis in physiologic O(2) was associated with significantly less expression of the adipogenic regulator, PPAR gamma. These results suggest that regulatory pathways affected by O(2) are important for satellite cell proliferation, execution of cell fate, and parent muscle survival in culture, and so may play a role in vivo under normal or pathologic conditions.     

Isolation and mapping of two porcine skeletal muscle myosin heavy chain isoforms

The present paper describes the isolation and linkage mapping of two isoforms of skeletal muscle myosin heavy chain in pig. Two partial cDNAs (pAZMY4 and pAZMY7), coding for the porcine myosin heavy chain-2B and -β respectively, have been isolated from a pig skeletal muscle cDNA library. Four RFLPs were detected with the putative porcine skeletal myosin heavy chain-2B probe (pAZMY4) and one RFLP was identified with the putative myosin heavy chain-β probe (pAZMY7). Two myosin heavy chain loci were mapped by linkage analysis performed with the five RFLPs against the PiGMaP linkage consortium ResPig database: the MYH1 locus, which identifies the fast skeletal muscle myosin heavy chain gene cluster, was located at the end of the map of porcine chromosome 12, while the MYH7 locus, which identifies the myosin heavy chain-α/-β gene cluster, was assigned to the long arm of porcine chromosome 7.     

Conditions for isolation and culture of porcine myogenic satellite cells.

Myogenic satellite cells were isolated from semimembranosus muscles of 4-8 week-old pigs. Muscles were ground and incubated in 0.8 mg/ml Pronase solution for 40 min at 37 degrees C. Following enzymatic digestion, cells were separated from muscle debris by differential centrifugation and sequential filtering through 500 and 53 microns nylon mesh. Primary cultures grown in 16 mm diameter cell culture wells were used to evaluate five sera, media, and substrata for their ability to promote satellite cell proliferation and differentiation. Porcine satellite cell proliferation and myotube formation were optimized in cultures grown on gelatin-coated substratum in the presence of Minimum Essential Medium-alpha supplemented with 10% fetal bovine serum (FBS) (P less than 0.01). Maximum fusion was induced by 48 hr exposure to 2% FBS, horse serum, or lamb serum. These data 1) document the first evidence that myogenic satellite cells can be isolated from porcine skeletal muscle, and 2) identify culture conditions which optimize proliferation and myotube formation of porcine satellite cells.     

Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010. © 2010 Wiley‐Liss, Inc.     

Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD

Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD^iCre knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (β-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature.     

Isolation and culture of embryonic stem cells from porcine blastocysts

This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7-9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent.     

On the non-equivalence of skeletal muscle satellite cells and embryonic myoblasts

Postnatal satellite cells, isolated from normal or previously denervated skeletal muscles of juvenile quails, were tested as to their capacity to participate in embryonic muscle ontogeny. They were grafted into 2-day chick embryo hosts, in place of a piece of brachial somitic mesoderm. Satellite cell implants were prepared from pellets either of freshly isolated cells or of cells precultured in vitro under proliferative conditions. Myogenic capacity of the implanted cells was attested by their ability to fuse into myotubes when cultured under differentiation conditions. In no case did the implanted satellite cells invade the adjacent wing bud or participate in wing muscle morphogenesis. They did not either give rise to myotubes at the site of implantation, nor did they even survive longer than 3 days in the embryonic environment. These negative results indicate that postnatal satellite cells, unlike embryonic myoblasts, are unable to take part in muscle embryogenesis. Although they derive from the same somitic myogenic cell line as the embryonic myoblasts, they therefore represent a differentiated non-totipotent type of myogenic cell.     

大鼠骨骼肌卫星细胞诱导分化为胰岛素生成细胞的研究

目的探讨大鼠骨骼肌卫星细胞（MDSCs）定向诱导分化为胰岛素生成细胞（IPCs）,为1型糖尿病的干细胞治疗提供一种新的研究思路。 方法通过二次酶消化法和差速贴壁培养法分离、培养大鼠MDSCs,利用不同的诱导培养液使MDSCs定向分化为IPCs,并对诱导后细胞进行形态观察,通过双硫腙染色和免疫组化染色对MDSCs-IPCs形态进行鉴定,采用Q-PCR和Western Blot方法检测MDSCs-IPCs中C-peptide和Insulin的表达,通过胰岛素释放实验检测MDSCs-IPCs的生物学功能,β细胞和MDSCs-IPCs两组间比较采用t检验。 结果MDSCs在接种4 h后开始贴壁部分细胞伸出小的突起,48 h后绝大多数细胞贴壁呈梭形、胞浆丰富、折光度高。随着培养时间的延长,细胞的梭形形状更为明显且生长迅速。免疫组化结果显示细胞表达Desmin、α-Sarcomeric Actinin、MyoD1、Myf5和PAX7。成胰诱导后MDSCs形成胰岛样的圆形细胞团,双硫腙染色呈猩红色,Insulin免疫组化染色阳性。Q-PCR结果显示MDSCs-IPCs中C-peptide和Insulin mRNA表达量分别是β细胞的0.73倍（P > 0.05）和0.79倍（P > 0.05）。胰岛素释放实验显示,5.6 mmol/L和16.7 nmlol/L葡萄糖刺激培养2 h后,β细胞和MDSCs-IPCs分泌胰岛素量分别为[（20.3±4.2）mU/L]、[（16.1±3.7）mU/L]、[（60.5±9.3）mU/L]和[（40.9±7.3）mU/L],葡萄糖可调节MDSCs-IPCs胰岛素的分泌。 结论MDSCs易于分离培养、增殖能力强,体外可诱导分化为有功能的IPCs,适合作为再生医学的种子细胞。