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Address correspondence to Mindy George-Weinstein, Department of Anatomy, Philadelphia College of Osteopathic Medicine, 4170 City Ave., Philadelphia, PA 19131. Tel.: (215) 871-6541. Fax: (215) 871-6540. email: mindyw{at}pcom.edu
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Abstract |
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Key Words: embryonic stem cells; skeletal myogenesis; bone morphogenetic protein; noggin; cadherins
D. Beckmann's present address is Duke University, Durham, NC 27708.
Abbreviations used in this paper: BMP, bone morphogenetic protein; DMEM, Dulbecco's minimal essential medium; ES cell, embryonic stem cell; G8pos, G8 positive; G8neg, G8 negative; HGF/SF, hepatocyte growth factor/scatter factor; MyoDpos, MyoD positive; N-cadherinpos, N-cadherin positive.
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Introduction |
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We use an in vitro system comprised of epiblast cells from the chick embryo to dissect the steps involved in commitment and differentiation. Chick epiblast cells differentiate when cultured at high density in medium that lacks exogenous growth factors (George-Weinstein et al., 1996a). Although some neurons, notochord, and cardiac muscle cells emerge under these conditions, the preferred pathway of differentiation is skeletal myogenesis (George-Weinstein et al., 1996a).
The precise factors that direct the majority of chick epiblast cells to the skeletal muscle lineage in vitro are unknown; however, a change in the expression of cellcell adhesion molecules appears to be required for terminal stages of differentiation. Cultured epiblast cells recapitulate the switch in expression from E- to N-cadherin that occurs in vivo when they enter the primitive streak and mesoderm (Edelman et al., 1983; Hatta and Takeichi, 1986; George-Weinstein et al., 1997). Although both E- and N-cadherin support the synthesis of the skeletal muscle specific transcription factor MyoD (Holt et al., 1994; George-Weinstein et al., 1997), adhesions via N-cadherin are necessary for the accumulation of sarcomeric proteins (George-Weinstein et al., 1997).
Alterations in cell adhesion also accompany skeletal myogenesis in vivo. Muscle differentiation is initiated in the embryonic somites, structures that form as a result of compaction and epithelialization of mesoderm cells (Christ and Ordahl, 1995; Pourquie, 2001). Members of the Wnt family produced in the neural tube and presomitic mesoderm, and sonic hedgehog released from the notochord, promote the expression of members of the MyoD family and the onset of differentiation in somites (Pownall et al., 2002). Wnts may play a dual role in regulating myogenesis in somites because they enhance the stability of ß-catenin, a protein that serves as both a transcription factor and a link between cadherins and the actin cytoskeleton (Gumbiner, 2000). Somite cells from N-cadherin null mice are able to undergo myogenesis; however, they appear to express another cadherin (Radice et al., 1997).
Although skeletal myogenesis is regulated by factors in the environment of somites, MyoD mRNA is present in a small number of cells in the epiblast before somite formation (Gerhart et al., 2000). The following experiments were designed to explore the significance of this early expression of MyoD in the epiblast and the potential of these cells to regulate the fate of pluripotent cells. These experiments demonstrate that epiblast cells expressing MyoD mRNA in vivo differentiate into skeletal muscle in vitro. The MyoD positive (MyoDpos) cells promote the expression of N-cadherin in pluripotent cells and their recruitment to the skeletal muscle lineage. The recruitment process involves blocking the bone morphogenetic protein (BMP) signaling pathway.
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Results |
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Differentiation of nonskeletal muscle cell types in epiblast cultures
The ability of epiblast cells to differentiate into nonskeletal muscle cell types in the absence of the MyoD/G8pos cells was investigated with cell typespecific antibodies. Although the majority of cells in unsorted cultures formed skeletal muscle, some chondroblasts, cardiac muscle cells, and neurons began to emerge after the first day in culture (George-Weinstein et al., 1996a; Tables I and II). The sum of the percentages of these four cell types equaled >90%, indicating that in unsorted cultures few undifferentiated cells or those of other lineages were present by the fifth day in culture.
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The effect of medium conditioned by G8pos cells on G8neg cells
The effects of the G8pos cells on G8neg cells in mixed cultures were at least partially mediated by soluble factors because transfer of conditioned medium from 48-h G8pos cultures stimulated skeletal myogenesis in G8neg cultures (Table III and Fig. 1 G). Medium from unsorted 48-h cultures had a slight stimulatory effect on muscle differentiation in G8neg cultures. When conditioned medium from G8pos cultures was added to G8neg cells plated at low density, only a small percentage of cells not in contact with other cells synthesized detectable levels of the 12101 antigen (10 ± 4, n = 4). Therefore, cellcell contacts facilitate the effect of conditioned medium on differentiation. By contrast, most G8pos cells plated at low density were stained with the 12101 mAb (92% ± 2, n = 5; Fig. 1 C). This demonstrates that the differentiation of epiblast cells expressing MyoD mRNA before removal from the embryo is independent of direct cellcell interactions.
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We had shown previously that treatment of epiblast cells from pregastrulating embryos with hepatocyte growth factor/scatter factor (HGF/SF) resulted in a moderate increase in muscle differentiation (DeLuca et al., 1999). G8neg cells from gastrulating embryos did not form skeletal muscle in response to HGF/SF, Wnt-1 (George-Weinstein et al., 1998), or Wnt-3A; however, they did respond to Noggin (Table III and Fig. 1 H). A dose response experiment revealed that 50 ng of human recombinant Noggin per milliliter of medium produced maximal stimulation of myogenesis.
Because Noggin is an inhibitor of BMPs (for review see Balemans and Van Hul, 2002), and BMP-4 is a regulator of myogenesis in the somites (Pourquie et al., 1996; Dietrich et al., 1998; Sela-Donenfeld and Kalcheim, 2002), BMP-4 was added to G8pos-conditioned medium at the time of its addition to G8neg cultures. BMP-4 blocked the stimulatory effect of G8pos-conditioned medium on myogenesis (Table III). Maximal inhibition was produced with a dose of 40 ng/ml.
Further evidence for involvement of BMPs in the inhibition of G8neg differentiation is that a soluble form of the BMP receptor-IA, a known competitor of the endogenous receptor for ligand binding, stimulated myogenesis in G8neg cultures (Table III and Fig. 1 I). The optimal dose for this effect was 80 ng/ml. These results suggest that BMP signaling represses myogenesis in G8neg cultures and G8pos cells produce a BMP antagonist.
Expression of cadherins in G8pos and G8neg epiblast cultures
Previous experiments demonstrated that epiblast cells switch from E- to N-cadherin as they ingress through the primitive streak (Edelman et al., 1983; Hatta and Takeichi, 1986; unpublished data), and down-regulation of E-cadherin and adhesion via N-cadherin are required for epiblast cells to form skeletal muscle in vitro (George-Weinstein et al., 1997). 3 h after plating, most G8pos cells continued to express E-cadherin (67 ± 17, n = 5); however, some were synthesizing N-cadherin (21 ± 11, n = 5). After 24 h in culture, most G8pos were stained with antibodies to both N-cadherin (70 ± 19, n = 6) and E-cadherin (74 ± 12, n = 6). By the fifth day, most cells in G8pos cultures expressed N-cadherin, but not E-cadherin (Table IV; Fig. 2, A and B).
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Discussion |
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Skeletal myogenesis is the preferred pathway of differentiation when the entire population of chick epiblast cells is grown in vitro (George-Weinstein et al., 1996a). This phenomenon is dependent on several factors including separating the epiblast from the mesoderm, disrupting cellcell contacts within the epithelium, plating cells on a substrate of denatured collagen and fibronectin to promote their attachment and survival, and culturing them in serum-free medium (George-Weinstein et al., 1996a). In addition, most unsorted epiblast cells must be plated at high density in order to differentiate (George-Weinstein et al., 1996b). This reflects a requirement for cadherin-mediated adhesions to support the synthesis of MyoD and sarcomeric myosin (George-Weinstein et al., 1997). Our conclusion from these earlier experiments was that the emergence of skeletal muscle as the dominant phenotype in epiblast cultures was the result of a "community effect" (Gurdon, 1988) in which equivalent cells were induced to differentiate along the same pathway (George-Weinstein et al., 1996a). In these cultures, the community effect was mediated by cellcell adhesions via N-cadherin (George-Weinstein et al., 1997).
We have now determined the epiblast is a heterogeneous tissue comprised of both pluripotent cells and those that are biased, and possibly committed, to differentiate into skeletal muscle. Furthermore, it would seem that the single most important factor for the widespread induction of skeletal myogenesis in epiblast cultures is the release of factors from cells that are preprogrammed to form skeletal muscle within the embryo. Despite the fact that the culture conditions overwhelmingly support skeletal myogenesis, most pluripotent cells cannot form skeletal muscle in the absence of epiblast cells that express MyoD mRNA and the G8 antigen in vivo.
The MyoDpos/G8pos epiblast cells also affect cadherin expression. In unsorted and G8pos cultures, most cells down-regulated E-cadherin and up-regulated N-cadherin during skeletal muscle differentiation. In addition, previous experiments with unsorted epiblast cells revealed that both E- and N-cadherin supported MyoD protein synthesis; however, only those cells with N-cadherin but not E-cadherin differentiated (George-Weinstein et al., 1997). The G8neg cells differed from unsorted and G8pos cells in three ways. First, G8neg cells were impaired in their ability to switch cadherins. Second, neither cadherin appeared to support the synthesis of MyoD protein in more than a minor population of G8neg cells. Finally, the percentage of differentiated skeletal muscle cells was significantly less than the percentage of N-cadherinpos/E-cadherin negative cells. The lack of skeletal myogenesis could reflect, in part, reduced homotypic N-cadherinmediated adhesions due to the presence of cells that continued to express E-cadherin alone or both cadherins.
The precise factors produced by MyoDpos/G8pos cells that stimulate myogenesis and N-cadherin expressions remain to be identified; however, the effect of the G8pos cells appears to involve antagonism of the BMP signaling pathway. Noggin, an inhibitor of BMPs-4, -6, and -7, and growth and differentiation factor-5 (for review see Balemans and Van Hul, 2002), stimulated N-cadherin synthesis and skeletal myogenesis when added to G8neg cultures. Furthermore, BMP-4 blocked the stimulatory effect of G8pos-conditioned medium, and a soluble form of BMP receptor-IA known to compete with the endogenous receptor for ligand binding, increased muscle differentiation in G8neg cultures in the absence of G8pos-conditioned medium. Releasing the G8neg cells from the inhibitory effects of BMPs may be sufficient to trigger myogenesis, although it is possible that these cells produce stimulatory factors that are also required for skeletal muscle differentiation.
These experiments provide additional evidence that skeletal myogenesis is the preferred pathway of chick epiblast cells under these culture conditions. They also complement experiments of the regulation of muscle differentiation in the somite. Expression of Noggin in the presomitic and somitic mesoderm releases cells from BMP-mediated repression of MyoD and Myf5 activation (Pourquie et al., 1996; Hirsinger et al., 1997; Marcelle et al., 1997; Dietrich et al., 1998; Reshef et al., 1998; Amthor et al., 1999; Sela-Donenfeld and Kalcheim, 2002; Linker et al., 2003). Noggin is also involved in the down-regulation of E-cadherin during epithelial bud formation in combination with Wnt3a (Jamora et al., 2003). At this time it is unclear whether G8pos cells directly affect cadherin synthesis, or if the expression of N-cadherin follows the recruitment of G8neg cells to the skeletal muscle lineage.
The results of our cell sorting experiments support the theory proposed decades ago by Holtzer and colleagues (Holtzer et al., 1983) that founder cells for the myogenic lineage exist in the early embryo (Choi et al., 1989). Testing the role of these cells in vivo could be accomplished by ablating the MyoD population at early times in development. There are several possible outcomes of this type of experiment including a complete or partial disruption of muscle differentiation, or no effect due to a stimulation of myogenic transcription factor expression in other cells. Molecules downstream in the sonic hedgehog pathway can directly regulate the promoter of the MyoD family member, myf5 (Gustafsson et al., 2002), suggesting that signaling from the notochord may compensate for the loss of MyoDpos epiblast cells. Alternatively, release of factors from the original population of MyoD expressing cells could prime surrounding pluripotent cells to respond to molecules produced by the notochord and neural tube. In this regard, a subpopulation of cells within the presomitic mesoderm secretes Wnt5b which stimulates MyoD expression (Linker et al., 2003). It is possible that the Wnt5b producing mesoderm cells are derived from cells that express MyoD within the epiblast.
Apart from any role that the MyoDpos epiblast cells may play in regulating muscle differentiation in vivo, their ability to recruit pluripotent cells to the myogenic lineage in vitro has important implications in the field of stem cell biology. We suggested previously that implanting a mixture of committed and pluripotent stem cells into diseased tissues might facilitate regeneration (Gerhart et al., 2001). Our results now suggest that exposure of stem cells to committed precursors or the factors they produce before implantation may preprogram them to follow the desired pathway in vivo. Asakura et al. (2002) demonstrated that neonatal myoblasts can induce stem cells derived from adult muscle to undergo skeletal myogenesis; however, the rate of conversion of these stem cells to the muscle lineage was low (4%). Furthermore, conditioned medium from the neonatal myoblast cultures did not stimulate the stem cells to differentiate. This suggests that stem cells from adult muscle are more resistant to conversion to the myogenic lineage than pluripotent epiblast cells and/or neonatal myoblasts are less effective recruiters than MyoDpos epiblast cells. A third possibility is that the culture conditions for the neonatal myoblasts and adult stem cells were less permissive for the recruitment process than the epiblast culture system. Modifications in the method for pretreating adult stem cells with committed cells may enhance the extent of their conversion to the myogenic lineage and their regenerative potential in vivo.
An equally important consideration for the use of stem cells therapeutically is the role of the community effect in facilitating differentiation (Gurdon, 1988; Cossu et al., 1995). The molecular mechanism for the community effect involves cadherin-mediated adhesion (Holt et al., 1994; George-Weinstein et al., 1997). Although the G8pos cells release soluble factors that stimulate skeletal myogenesis in G8neg cultures, the factors are more effective when G8neg cells are plated at high density. Ensuring that the appropriate cadherin is expressed in the stem cell population may be a critical component of a strategy to regenerate skeletal muscle and other tissues.
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Materials and methods |
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Magnetic cell sorting
Epiblasts were isolated from stages 3 to 5 embryos and dissociated in 0.25% trypsin-EDTA (Invitrogen) at 37°C for 10 min (George-Weinstein et al., 1996a). Cells were centrifuged and resuspended in Dulbecco's minimal essential medium (DMEM) containing 5% FBS, 5% horse serum, 100 U of penicillin and streptomycin (Invitrogen Corp.), and 5% chick embryo extract (Myo medium). The G8 mAb was added to the cell suspension at a concentration of 2 µg/ml. After incubation at 37°C for 45 min, cells were centrifuged and resuspended in 80 µl PBS. 20 µl of rat antimouse IgM microbeads (Miltenyi Biotec) were added for 15 min at RT. Cells were centrifuged, resuspended in 500 µl PBS, and added to the MiniMACS column attached to the magnetic sorting separation unit (Miltenyi Biotec). G8neg cells were eluted by rinsing the column with PBS. The column was then removed from the separation unit and the G8pos cells eluted in PBS with gentle plunging. The purity of the sorts was determined by labeling an aliquot of the positive and negative populations with a fluorescent secondary antibody, and calculating the percentage of the total cells that was fluorescent. G8pos and G8neg cells were centrifuged, resuspended in Myo medium, and placed in culture.
Preparation of epiblast cell cultures
Sorted and unsorted epiblast cells were cultured under conditions shown previously to promote myogenesis (George-Weinstein et al., 1994, 1996a). 20,000 cells in 15 µl Myo medium were plated in the center of 35-mm tissue culture dishes coated with gelatin and human serum fibronectin (Invitrogen). After the cells had attached to the dish (90 min), they were flooded with 1 ml serum and hormone-free DMEM/F12 medium (Invitrogen). Low density cultures were prepared by plating 40,000 cells in 1 ml of Myo medium over the entire surface of a coated 35-mm dish. 90 min after plating, the medium was replaced with DMEM/F12.
G8neg cells were treated with conditioned medium by removing their medium after 48 h in culture and replacing it with 48-h medium obtained from G8pos, other G8neg, or unsorted cultures. The medium was centrifuged to remove cells before its addition to the G8neg cultures. Medium in 48-h G8neg cultures was replaced with 1 ml DMEM/F12 medium containing recombinant human Noggin (PeproTech), soluble BMP receptor-IA/Fc chimeric protein, HGF/SF, or 1 ml G8pos-conditioned medium containing recombinant human BMP-4 (all purchased from Sigma-Aldrich).
Immunofluorescence localization
Cells were fixed in 2% formaldehyde, permeabilized with 0.5% Triton X-100, and labeled with primary and fluorescent secondary antibodies as described previously (George-Weinstein et al., 1994, 1996a). Labeling with antibodies to the cadherins required fixation in methanol. The skeletal muscle specific 12101 mAb (Kitner and Brockes, 1984), MF20 mAb to sarcomeric myosin heavy chain (Bader et al., 1982), mAb to LCAM/E-cadherin (Gallin et al., 1983), CIIC1 mAb to type II collagen (Holmdahl et al., 1986), and 3A10 mAb to neurofilament associated antigen (Furley et al., 1990) were obtained from the Developmental Studies Hybridoma Bank. The AB-1 mAb to cardiac muscle specific troponin T (Neomarkers) stained cardiomyocytes in vivo but did not bind skeletal muscle cells in fetal pectoralis muscle cultures or G8pos cultures (Table IV; Fig. 1 E). The mAb 5B2H5 to cadherin 11 was purchased from Zymed Laboratories. A rabbit polyclonal antiserum to N-cadherin was generated as described previously (George-Weinstein et al., 1997). Secondary antibodies were affinity purified, goat antimouse F(ab')2 fragments conjugated with rhodamine or Cy3 (Jackson ImmunoResearch Laboratories), and goat antirabbit IgG conjugated with Cy2 (CHEMICON International, Inc.). Cells that had been prelabeled with the G8 IgM mAb before being placed in culture were later stained with IgG primary mAbs and a goat antimouse IgG secondary antibody conjugated with rhodamine (CHEMICON International, Inc.). Nuclei were counterstained with bis-benzamide. The anti-IgG secondary antibody did not recognize the G8 IgM mAb.
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Acknowledgments |
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These studies were supported by the National Institutes of Health (HD043157 to M. George-Weinstein, GM61702 to K.A. Knudsen).
Submitted: 25 September 2003
Accepted: 9 January 2004
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References |
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