Developmental Biology and Molecular Pathology, W7, University of Bielefeld, D-33501 Bielefeld, Germany
* Author for correspondence (e-mail: h.jockusch{at}uni-bielefeld.de)
Accepted 10 January 2003
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Summary |
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Key words: Stem cells, Migration, Muscle transplantation, GFP, nLacZ, Nude mouse
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Introduction |
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Here we use a new type of marker of origin, a combination of an enhanced
GFP transgene (Okabe et al.,
1997), driven by the ß-actin promotor, a CMV enhancer and a
nuclear-localized bacterial ß-galactosidase (nLacZ) knock-in, driven by
the endogenous desmin promoter (Li et al.,
1996
). In addition, we used a novel fixation method for GFP
(Jockusch et al., 2003
). The
ß-actin promotor would be expected to lack cell-type specificity, whereas
the desmin promotor conveys specificity of expression for smooth, cardiac and
skeletal muscle. Although the latter expression pattern was confirmed
(Li et al., 1996
), the GFP
pattern was not homogeneous (Jockusch et
al., 2003
), and levels of expression were especially high in
smooth, cardiac and skeletal muscle. In skeletal muscle, mature muscle fibres
had a higher GFP level than myotubes, and among mature fibres the oxidative
ones were richer in GFP than the glycolytic fibres. In cross-striated muscle
all fibres expressed GFP in the transgenic mouse, and the fluorescence level
was always well above any autofluorescence levels
(Jockusch et al., 2003
).
In conjunction with muscle grafting, we have studied the migratory behaviour of resident myogenic stem cells, in both directions, from host to graft and vice versa. Owing to the sensitivity of the GFP-labelling method we visualized hitherto unrecognised migratory activities of myogenic stem cells through solid muscle tissue.
Preliminary reports on these experiments have been published elsewhere
(Jockusch et al., 2000;
Eberhard and Jockusch,
2002
).
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Materials and Methods |
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We bred the eGFP (designated GFP) and nLacZ transgenes into the NMRI-nu strain to obtain nude mice as recipients, either GFP or GFP and nLacZ (GFP/nLacZ) labelled. The absence of a transgene (i.e. wildtype) is indicated by `0'.
Surgery, organ preparation and histochemistry
Ages of donors ranged from 103 to 172 days (average 117 days), and those of
recipients from 47 to 74 days (average 70 days). Transplantation of anterior
tibial muscles with the attached extensor digitorum longus was performed as
described previously (Füchtbauer et
al., 1988) but with ketasel-5/xylazin (Selectavert,
Weyarn-Holzolling, Germany; Animedica, Senden-Bösensell, Germany)
anaesthesia and without bupivacaine pretreatment of the graft. Animals gained
weight and the operated leg was used normally after two weeks. All animal
experiments were performed according to the German laws for the protection of
animals, and a permit was obtained from the local authorities.
Between 4 and 11 weeks after operation (p.o.), animals were killed by
cervical dislocation, and the grafted anterior tibial muscle (TA) with the
adjacent M. gastrocnemius and M. soleus were removed in one piece, but without
the tibia, and mounted either as proximal or distal halves supported by a
chunk of hard-boiled egg white for cross sectioning (starting with the cut
surface) or, in some cases, longitudinal sectioning. The blocks were shock
frozen in liquefied propane (190°C) followed by liquid
N2. Frozen sections were cut on a Leitz cryostat microtome to 8 to
10 µm and processed for the subsequent staining. Owing to its extreme
solubility, GFP cannot be fixed with conventional aqueous fixatives in frozen
cross-sections of the muscle. We have developed a fixation method that avoids
loss of the highly soluble GFP. In brief, this consisted of the exposure of
the section to formaldehyde (FA) vapour from a pad soaked with 37% FA at
20°C for at least 2 hours
(Jockusch et al., 2003).
The FA-fixed sections were counterstained with Hoechst stain (Sigma, B2261)
to visualize nuclei. Parallel sections (conventionally fixed or unfixed) were
stained for ß-galactosidase and subsequently with eosin. The immuno- and
enzyme-histochemical methods used here
(Jockusch et al., 2003) worked
only on unfixed sections so that muscles could not be fixed as a whole. For
GFP fluorescence, sections were embedded in Elvanol (10 g Mowiol 4-88 + 40 ml
PBS + 20 ml glycerol); conventionally stained sections were embedded in
Entellan (Merck, Darmstadt, Germany).
Evaluation and documentation
Serial sections of grafts with neighbouring gastrocnemius and soleus
muscles were photographed within a few days after fixation on a Zeiss Axiophot
microscope equipped with UV (and Nomarski) optics using an Olympus digital
camera DP10.
Processing of images was done with Photoshop 5.5 (Adobe Systems Inc., San José).
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Results |
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In general the lateral border between the host and the graft appeared to be sharp, with a separation of the grafted TA and the host gastrocnemius by a thin lining of connective tissue, the perimysium. At some places, however, some intermingling was observed, and this lead to co-fusion between host and graft myogenic cells as demonstrated by GFP-positive fibres with LacZ-positive nuclei (Fig. 1C,D).
Immigration of myogenic stem cells from host to graft
Following longer regeneration periods for 0 to GFP transplantations,
GFP-positive fibers appeared in the graft, often in small groups of two to
five (Fig. 2). Parallel
staining with standard methods of muscle histochemistry revealed nothing
special for the GFP-positive fibres in comparison to their GFP-negative
neighbours in the graft. With the host double labelled, nLacZ-positive nuclei
in GFP-positive fibres were usually found close to the host. The finding that
GFP-positive fibres in the graft contained only a minority of host nuclei was
corroborated by a complementary labelling experiment of the type nLacZ to GFP
(Fig. 2E,F). At distances of
>0.25 mm from the host muscle, blue nuclei were present in GFP-positive
fibres at apparently the same density as in GFP-negative fibres.
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In total, 10 transplantation experiments of the type 0 to GFP have been performed, in various combinations with the nLacZ label. Cross-sections of grafted TAs proximal and distal from the centre were quantitatively evaluated by counting GFP-positive fibres as a function of the distance from host muscle (Fig. 3). A maximal number of GFP-positive fibres (single or in small groups) was observed at distances between 0.25 and 1.5 mm from the host muscle, with maximal distances of 2 mm.
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Emigration of myogenic cells from graft to host
In experiments of the type GFP to 0, migration of graft cells into host
muscle can be monitored after 7 weeks of regeneration
(Fig. 4). Central nuclei, which
would indicate previous regeneration, were not observed in GFP-positive host
fibres with Hoechst fluorescence or in HE-stained parallel sections
(Fig. 4A,B). Oxidative fibres
of the host were found to be preferentially converted to GFP-positive ones
(Fig. 4C,D). In the
contralateral leg, no GFP fluorescence or green autofluorescence was found in
corresponding regions of the gastrocnemius and soleus muscles
(Fig. 4C,D insets).
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Quantitative evaluation of the colonization of adjacent host muscle by graft myogenic cells shows gradients of GFP-positive fibres from the graft border extending nearly 2 mm into the host (Fig. 5). Surprisingly, with grafts from neonatal mice, extremely poor regeneration and no migration of myogenic cells from graft to host was observed (data not shown).
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Initial stages of the emigration of graft cells could be observed in GFP/nLacZ to 0/0 grafts (Fig. 6). Apparently donor myogenic cells, as recognized by both the GFP and the nLacZ label, detach from the graft and migrate along the endomysial connective tissue into the adjacent muscle, over distances of several hundred microns (Fig. 6A-D). In one case, an nLacZ-positive, GFP-negative cell was observed; this was probably a very immature muscle precursor cell in which the GFP expression was below the threshold of detection. In longitudinal sections, regenerated immature fibres can be distinguished from mature fibres by their lower GFP content and very high ß-galactosidase activity. As in the complementary case 0 to GFP, single GFP-positive fibres are seen in a GFP-negative environment (Fig. 6E,F), but, in contrast to the myogenic cells at initial stages of emigration, nLacZ-positive nuclei were not observed, indicating that few myogenic precursor cells had fused into pre-existing host muscle fibres so that the nuclei of the latter were in excess of the donor nuclei, which provided the GFP gene and its highly diffusible product.
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Discussion |
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In most experiments with transgenic labels permanent cell lines have been
used as donors, often in combination with short (a few days) regeneration
times. In such experiments, the role of matrix metalloproteinases during the
migration of myogenic cells has been demonstrated
(El Fahime et al., 2000).
However, the use of grafted muscle tissue creates a situation closer to
natural conditions for cell migration (cf.
Fan et al., 1996
). In previous
experiments using muscle grafts no or very low migratory activity of myogenic
precursor cells has been observed (Watt et
al., 1987
; Moens et al.,
1996
). This difference in our results may not only be due to the
use of markers of origin with a low diffusion coefficient, like dystrophin,
but also to a lack of an initial traumatic stimulus (which was provided by
replacing one muscle by another in our experiments). The induction of
degeneration/regeneration and elimination of host myogenic cells by
irradiation has proved to be essential to obtain even low yields of
circulating stem cells that integrated into skeletal recipient muscle
(Ferrari et al., 1998
). In more
recent experiments, Smythe and Grounds transplanted longitudinally sliced EDL
muscles into a slit of the anterior tibial muscle of the mouse
(Smythe and Grounds, 2001
) but
did not pharmacologically induce muscle degeneration or irradiate the host to
suppress its contribution to regeneration. Here, the `double wounding', that
is, local destruction of both host and donor perimysium, would have provided a
strong wound stimulus and removed an important barrier to migration. Using the
Y chromosome as a marker, Smythe and Grounds showed that immigration of graft
cells into host muscle was enhanced by a delayed differentiation owing to the
inactivation of the MyoD gene.
Origin of myogenic cells
The appearance of donor markers in host tissue and vice versa may be due to
circulating pluripotent stem cells (Ferrari
et al., 1998; Galli et al.,
2000
). However, in the present transplantation experiments the
evidence points to a local and muscular origin of migrant myogenic cells (cf.
Grounds et al., 2002
). Host
cells in a graft, and graft cells in a host, appear only after a delay of
about 7 weeks. Graft cells in the host are distributed in a gradient from
their presumed origin, the adjacent graft muscle tissue. In double labelling
experiments, single myogenic cells have been observed that seem on their way
from one muscle to the other. In the reverse experiments, most fibres with the
host label are found near the centre of the cross section of the graft where
the highest level of ischemia causes the most intense degeneration and
regeneration (Carlson,
1986
).
The migration of myogenic cells from intact host muscle into the
regenerating graft may be stimulated by `wound hormones', that is.
chemoattractants diffusing from the graft into the neighbourhood
(Watt et al., 1994;
Grounds and Davies, 1996
;
Bischoff, 1997
). On the other
hand, the degeneration/regeneration processes within a muscle graft
disconnected from its original blood and nervous supply lead to a massive
activation of satellite cell proliferation
(Carlson, 1986
) and may also
stimulate a long distance emigration of muscle precursor cells. At a low
level, migration of juvenile and adult myogenic cells may take place even in
the absence of a wound stimulus. This would constitute a potentially
continuous repair mechanism. In GFP <->0 chimeras, patches of GFP-free
skeletal muscle fibres are extremely rare and only observed in special cases
such as extraocular muscles and in chimeras with very low GFP-positive
contributions (D. Eberhard and H.J., unpublished). This suggests an extensive
migration and mixing of myogenic cells, at least during organogenesis.
The observation of a long-term exchange of myogenic cells across borders of
individual muscles may have a bearing on the possibility of cell therapy of
diseased muscle. Solid transplanted muscle tissue
(Füchtbauer et al., 1988;
Fan et al., 1996
;
Smythe and Grounds, 2001
) may
act as a source of muscle precursor cells for an extended period of time and
may thus circumvent the low yields in myoblast and stem cell colonization of
muscle (Hodgetts et al., 2000
;
Ferrari et al., 2001
;
Partridge, 2002
) as a route
for therapeutical improvement.
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Acknowledgments |
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