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Migration and Differentiation of Myogenic Precursors Following Transplantation into the Developing Rat Brain JAN STEFFEL,a,b MARIUS WERNIG,a ULRICH KNAUF,c SANTHOSH KUMAR,d OTMAR D. WIESTLER,b ANTON WERNIG,c OLIVER BRÜSTLEa a
Institute of Reconstructive Neurobiology and Departments of bNeuropathology and cPhysiology, University of Bonn, Bonn, Germany; dInstitute for Molecular Pathology, Vienna, Austria Key Words. Transplantation · Myoblasts · Neural differentiation · Endothelium · Transdifferentiation
A BSTRACT There is increasing evidence that muscle-derived precursor cells can, under appropriate conditions, give rise to other than myogenic cell types. Transplantation into the embryonic ventricular zone provides a unique opportunity to study the migration and differentiation of non-neural somatic progenitor cells in response to instructive cues within the developing neuroepithelium. Here, we demonstrate that myogenic cell lines grafted into the ventricles of rat embryos showed widespread migration into several host brain compartments. In contrast to incorporation patterns observed after transplantation of neural cells, grafted myoblasts incorporated virtually exclusively
along endogenous blood vessels. Preferential incorporation sites included cortex, olfactory bulb, hippocampus, striatum, thalamus, hypothalamus, and tectum. While the engrafted myoblasts showed no evidence of neural differentiation, a fraction exhibited pronounced coexpression of endothelial marker antigens. These findings support the concept of a close developmental relationship between the myogenic and the endothelial lineages. Used as a delivery system, transfected myoblasts may be exploited for widespread gene transfer to the perivascular compartment of the perinatal central nervous system. Stem Cells 2003;21: 181-189
INTRODUCTION The results of several recent studies indicate that the differentiation potential of adult stem cells is broader than previously assumed. Following bone marrow transplantation in mice, donor-derived cells have been found to enter the central nervous system (CNS) and express markers of glia [1] and neurons [2, 3]. A single bone marrow stem cell was recently shown to give rise to skin, lung, and gastrointestinal epithelium [4]. Juvenile and adult rodent skin cells can be differentiated in vitro into cells bearing antigens
typically found in neurons, glia, smooth muscle cells, and adipocytes [5]. Human bone marrow stem cells derived from male donors and transplanted into female patients were reported to give rise to hepatocytes and epithelial cells of the skin and gastrointestinal tract [6]. These examples suggest that adult stem cells, if exposed to a new microenvironment, can adopt identities distinct from their tissue of origin. Recent findings have challenged this view, attributing at least some of the observed “transdifferentiation” events to cell fusion [7, 8].
Correspondence: Oliver Brüstle, M.D., Institute of Reconstructive Neurobiology, University of Bonn Medical Center, Sigmund-Freud-Str. 25, D - 53105 Bonn, Germany. Telephone: 49-228-287-9508; Fax 49-228-287-9883; e-mail: brustle @uni-bonn.de Received August 9, 2002; accepted for publication September 30, 2002. ©AlphaMed Press 10665099/2003/$5.00/0
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The limited access to fetal neural tissue makes adult stem cells an attractive donor source for neural transplantation. Myogenic progenitors represent particularly interesting candidates; they are expandable in vitro and can be obtained in an autologous manner. Under physiological conditions, myogenic progenitors reside as ‘satellite cells’ attached to skeletal muscle fibers. During muscle growth and regeneration, they give rise to new muscle tissue. Upon transplantation, they form new muscle fibers or fuse with existing ones [9-11]. In lethally irradiated mice, cells derived from muscle tissue have been reported to contribute to blood cell lineages [12, 13]. Upon implantation into the heart, satellite cells from adult muscle acquired features of cardiomyocytes [14, 15]. Transdifferentiation of myogenic cells into osteocytes [16, 17] and adipocytes [18, 19] has also been observed. To explore the migration and differentiation of myogenic progenitors in response to neurogenic cues, the clonal myogenic cell line C2C12 and an expanded primary culture from mouse skeletal muscle (i28 cell line) were implanted into the ventricles of rat embryos. In this model, the grafted cells were directly exposed to the developing neuroepithelium, which, at this stage, should provide ultimate exposure to instructive cues regulating neuro- and gliogenesis. Previous studies had shown that neural cells transplanted in this manner populated large areas of the CNS and underwent region-specific differentiation irrespective of their site of origin [20-22]. In contrast, cells remaining in the ventricle, and thus, not exposed to the new environment, failed to adopt local traits [23]. Our data demonstrate that the i28 and C2C12 cell lines incorporated across large areas of the developing brain but failed to undergo neural differentiation. Instead, the grafted cells tightly associated with endogenous blood vessels and partially coexpressed endothelial marker antigens.
Myoblast Differentiation in the Developing Brain Northern Analysis For analysis of transcripts, total RNA was isolated from myoblasts and myotubes of both C2C12 and i28 cells using TRI Reagent™ (Sigma; St. Louis, MO; http://www.sigmaaldrich.com). Ten micrograms of RNA per lane were loaded on a 1.4% agarose/18% formaldehyde gel, transferred to nylon membrane, and hybridized to 32P-labeled DNA probes. All hybridizations were normalized to β-actin expression on the same blot. Radioactivity of specific bands was measured and evaluated by the ImageQuant software (Molecular Dynamics/Ammersham Bioscience; Sunnyvale, CA; http://www.mdyn.com). Preparation of the DNA probes was performed via polymerase chain reaction (PCR) using cDNA from mouse aorta as a template and the following primers: von Willebrand factor (vWF) 5′-AGGCACC TAACCTGGTCTACA-3′, 5′-CCTGCTGATCCTCTCCA GTTC-3′; Flk-1 (an antibody to vascular endothelial growth factor receptor-2) 5′-TCGCCTATGGATTCCTACCAG-3′, 5′-GCTTGGATGACCAGCGTACTT-3′; and β-actin 5′ATCTGGCACCACACCTTCTAC-3′; 5′-GTCAGGCAGCT CATAGCTCTT-3′. PCR products were checked for correct size, and an aliquot was sequenced to verify specific amplification of target DNA (data not shown). In Utero Transplantation Timed pregnant Sprague-Dawley rats (embryonic day 16.5 [E16.5]) were anesthetized, their abdominal cavities were opened, and the uterine horns were exposed. The telencephalic vesicles of the embryos were identified under transillumination. Using a glass capillary, 7 × 104 - 3 × 105 cells (concentrated to 1.5 - 6 × 104 cells/µl Hanks’ balanced salt solution) were injected into the telencephalic vesicle of each embryo as previously described [29]. The transplanted embryos were placed back into the abdominal cavity for spontaneous delivery. All animal experiments were carried out in accordance with German animal protection laws.
MATERIALS AND METHODS Cell Culture Two different myogenic cell preparations derived from mouse skeletal muscle were used: one clonal cell line (C2C12) and one cell line derived from an expanded primary culture (line i28 [9, 10, 24-26]). The C2C12 cell line was stably transfected with the lacZ gene [27, 28]. Myogenic precursors were proliferated in F10 medium containing 20% fetal calf serum. Upon serum withdrawal, both cell populations formed characteristic myotubes (Fig. 1). Before transplantation, the cells were enzymatically dissociated (1× trypsin/EDTA in phosphate-buffered saline [PBS] for 2-5 minutes at 37°C) and gently triturated to a single-cell suspension.
DNA-DNA Fluorescence In Situ Hybridization and Immunofluorescence Three weeks after transplantation, live-born recipients were deeply anesthetized and perfused with 4% paraformaldehyde in PBS. Their brains were removed and processed for vibratome sectioning. Fifty-micrometer sections were hybridized with a digoxigenin end-labeled oligonucleotide probe to mouse satellite DNA, as described previously [20]. Hybridized cells were double labeled with antibodies to glial fibrillary acid protein (GFAP; DAKO; Hamburg, Germany; http://www.dako.com; diluted 1:200 or ICN; Aurora, OH; http://www.icnbiomed.com; diluted 1:100), S100-β (Swant; Bellinzona, Switzerland; http://www. swant.com; diluted 1:2,000), microtubule-associated
Steffel, Wernig, Knauf et al. protein (MAP)2abc and MAP2ab (both from Sigma; diluted 1:250), β-III-tubulin (Covance Research Products; Berkeley, CA; http://www.crpinc.com; diluted 1:500), neuronal nuclei (NeuN; Chemicon; Temecula, CA; http://www.chemicon.com; diluted 1:50), nestin (a gift from R.D. McKay; diluted 1:1,000), desmin (DAKO; diluted 1:100), vWF (DAKO; diluted 1:500), platelet endothelial cell adhesion molecule-1 (PECAM-1; Pharmingen; San Diego, CA; http://www.pharmingen.com; diluted 1:100), or Flk-1 (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.scbt.com; diluted 1:1,000) and with lycopersicon esculentum lectin (LEL; Sigma; diluted 1:200). Antigens were visualized with appropriate fluorophore-conjugated secondary antibodies. In some cases, destruction of the antigen during the in situ hybridization process precluded subsequent multilabeling analyses. In those cases, biotinylated tyramide (NEN; Perkin Elmer; Shelton, CT; http://www.perkinelmer.com; diluted 1:100) was deposited in direct association with the antigen prior to the in situ hybridization according to the manufacturer’s recommendations. Following hybridization, the tyramide was visualized using avidin-fluorescein isothiocyanate (FITC) or avidin-Texas Red conjugates (Vector; Burlingame, CA; http://www.vectorlabs.com; diluted 1:250). Antibodies to Flk-1, vWF, and desmin were also used for in vitro characterization of the donor cells. Immunolabeling of untransplanted rat and mouse tissue and omission of the first antibody served as controls. Double and triple labeling of the transplanted cells were confirmed by confocal laser scanning microscopy with subsequent computer-aided three-dimensional (3D) reconstruction. Sections were analyzed on Zeiss Axiovert 2 and LSM 510 laser scanning microscopes. Detection of β-Galactosidase Recipients scheduled for X-gal histochemistry were perfused with 2% paraformaldehyde, 2 mM MgCl2, and 5 mM EGTA in 0.1 M piperazine-N,N’-bis(2-ethane sulfonic acid) (PIPES) buffer. Fifty-
Figure 1. In vitro differentiation of murine myogenic precursor cells. Proliferating myoblasts expressed desmin (A: line C2C12) and nestin (B: line i28). Following serum withdrawal, both cell lines formed characteristic multinucleated myotubes retaining expression of both antigens (C: i28, D: C2C12). Scale bars = 40 µm.
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micrometer vibratome sections were permeabilized in PBS, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.01% Nonidet P-40 at 4°C overnight. They were subsequently incubated in 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mg/ml X-gal (Sigma) in PBS at 37°C for 1 hour. X-gal histochemistry typically produced strongly labeled nuclei; in some cells, the cytoplasm was weakly stained as well. Alternatively, cells were visualized with an antibody to bacterial β-galactosidase (Abcam; Cambridge, UK; http://www.abcam.com; diluted 1:500). Immunofluorescence analysis produced the same staining pattern as the X-gal histochemistry. If combined with fluorescence in situ hybridization, anti β-galactosidase immunoreactivity was restricted to hybridized cells. RESULTS Myogenic Precursor Cells Express Muscle- and EndothelialSpecific Antigens In Vitro C2C12 and i28 myoblasts were proliferated in medium containing 20% serum. Upon serum withdrawal, they formed characteristic multinucleated myotubes. Immunofluorescence analyses revealed that both proliferating and differentiated cells expressed desmin and nestin (Fig. 1). In addition, myoblasts and differentiated myotubes showed weak immunoreactivity to vWF. Flk-1 immunoreactivity was weak in myoblasts and greater upon differentiation into myotubes (data not shown). Expressions of vWF and Flk-1 were confirmed by Northern analysis. Specific signals for Flk-1 and vWF were detected in RNA preparations from C2C12 and i28 myoblasts and myotubes (Fig. 2).
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myogenic cells showed virtually no integration into the brain parenchyma but incorporated nondisruptively along endogenous blood vessels (Fig. 3). Large numbers of donor cells were found in tight association with host capillaries, yielding a ‘string of pearls’ appearance. However, detailed confocal microscopic analyses provided no evidence for donor cell incorporation into blood vessel walls. No formation of tumors or space-occupying lesions was observed.
vWF
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Figure 2. Expression of vWF and Flk-1 in C2C12 and i28 cell lines. Northern analysis of C2C12 myoblasts (lane 1), C2C12 myotubes (lane 2), i28 myoblasts (lane 3), and i28 myotubes (lane 4). A β-actin probe served as internal loading control.
Widespread Blood Vessel-Associated Integration upon Transplantation into the Developing Brain Following transplantation into the ventricles of E16.5 rat embryos, myogenic precursors showed extensive incorporation into a variety of host brain regions. DNA in situ hybridization and/or detection of β-galactosidase performed at 2 weeks of age revealed incorporated cells in cortex, hippocampus, striatum, hypothalamus, and tectum (Fig. 3 and 4). The number of incorporated cells showed pronounced inter- and intraindividual variations (Fig. 4B). A particularly high degree of chimerism was noted in hippocampus and tectum. In contrast to grafted neural precursors [20-22], the
Figure 3. Migration of myogenic precursors upon transplantation into the developing rat brain. Grafted myoblasts exhibited widespread nondisruptive incorporation along endogenous blood vessels. Donor cells were identified in a variety of fore- and midbrain areas, including hippocampus (A and B: X-gal histochemistry), frontal cortex (C: antiβ-galactosidase immunofluorescence), and inferior colliculus (D: fluorescence in situ hybridization). Note the close association of the grafted cells with endogenous blood vessels. Scale bars = 400 µm (A), 20 µm (B), and 100 µm (C, D).
Sustained Transgene Expression after Transplantation C2C12 cells transfected with the lacZ gene retained strong transgene expression up to at least 3 weeks, i.e., the longest postoperative period studied (Fig. 3A, 3B, and 3C). Double labeling of hybridized cells with an antibody to βgalactosidase revealed transgene expression in more than 98% of the incorporated cells. Grafted Myoblasts Show No Signs of Neural Differentiation but Partially Express Endothelial Marker Antigens To analyze the differentiation of the grafted myoblasts, donor cells visualized by in situ hybridization or immunohistochemical detection of β-galactosidase were double labeled with antibodies to glial-, neural-, endothelial-, and muscle-specific markers. The majority of the transplanted cells retained immunoreactivity to desmin and nestin (Fig. 5A and 5B), markers typically expressed in myogenic precursors. Many of these cells displayed an elongated shape, spanning up to 100 µm and connecting to other desmin- and nestin-positive donor cells. Remarkably, the donor cells retained a mononucleated phenotype. Multinucleated
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Figure 4. Widespread distribution of engrafted donor cells. A) Schematic representation of frequent incorporation sites. Following intraventricular transplantation, myoblasts engrafted bilaterally into telencephalic, diencephalic, and mesencephalic brain regions. B) Quantitative assessment of donor cell incorporation. The table shows average numbers of incorporated cells per 50-µm section, based on five sections per region. Each column represents one transplanted animal. All animals were transplanted at embryonic day 16.5 (E16.5) and analyzed at postnatal day 14 (P14). Cells were identified by donor-cell-specific in situ hybridization. Note the pronounced inter- and intraindividual variations and the preferential incorporation into tectum and hippocampus.
elongated myotubes characteristic of differentiated muscle tissue could not be detected. A small subset of the transplanted cells was found to be immunopositive for vWF. While most vWF+ cells were located around blood vessels (Fig. 5C, 5D, and 6A), some were also detected within the host brain parenchyma without obvious contact with capillaries (Fig. 6B). Intraparenchymal vWF+ donor cells frequently exhibited a rounded shape without processes. Furthermore, a small fraction of the hybridized or β-galactosidase-labeled donor cells bound LEL (Fig. 5G, 5H, and 6C) and
Flk-1 (Fig. 5E and 5F), i.e., markers characteristic for endothelial differentiation. Triple labeling revealed that all Flk-1+ cells had retained desmin expression (Fig. 6D). No expression of PECAM-1 was found in any of the incorporated cells. Extensive double immunofluorescence analyses with antibodies to MAP2, β-III tubulin, NeuN, GFAP, and S100β were used to assess the neural transdifferentiation capacity of the grafted cells. While some confocal scans had suggested potential labeling of individual cells, careful correlation with the Hoechst staining failed to corroborate any
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Figure 5. Differentiation of myogenic progenitors following incorporation into the rat brain. Following transplantation, the majority of the donor cells retained immunoreactivity to desmin (A) and nestin (B) without forming polynucleated muscle fibers. Arrowheads indicate individual immunonegative cells. A subset of the transplanted cells expressed endothelial marker antigens such as vWF (red; C and D: temporal cortex) and Flk-1 (red; E and F: hippocampus) and bound LEL (green; G and H: inferior colliculus). In contrast, no expression of PECAM-1 (CD31) was detected in any of the grafted cells (data not shown). Donor cells were identified by in situ hybridization (DAPI/green; A, B, D, and F) or an antibody to β-galactosidase (red; H). Scale bars = 20 µm.
expression of neuronal or glial marker antigens in the donor cells. DISCUSSION Angiotropic Incorporation and Endothelial Marker Expression—A Lineage Relationship Between Myogenic and Endothelial Cells? Our data demonstrate that myogenic precursors transplanted into the ventricular system of the embryonic CNS incorporate in large numbers into a variety of host brain regions. In contrast to neural precursors, muscle-derived cells rarely invaded the host brain parenchyma but integrated mainly along endogenous blood vessels. Yet, they showed a preferential incorporation into the same regions populated by neural donor cells [20]. Whereas cortex, hippocampus, and inferior colliculus contained large numbers of engrafted cells, other areas, such as cerebellum and brain stem, were consistently devoid of donor cells. This incorporation pattern might be attributable to local differences in the neurogenic activity of the host ventricular zone. Indeed, all brain regions showing high incorporation rates are areas with prolonged neurogenesis until late gestation, suggesting facilitated donor cell entry through mitotically active segments of the ventricular zone.
Following incorporation, the vast majority of the cells retained expression of the myogenic marker antigens desmin and nestin but remained mononucleated. Some of the donor cells bound LEL and exhibited prominent expression of vWF and Flk-1, i.e., properties characteristic of endothelial cells. The close association of the grafted cells with the host vasculature and the pronounced expression of endothelial marker antigens could suggest that the donor cells participated in host angiogenesis. However, confocal laser scanning microscopy and subsequent 3D reconstruction
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Figure 6. Confocal laser scanning microscopy confirming the presence of endothelial markers in grafted myoblasts. (A and B) Hybridized donor cells (green) expressing vWF (red) were found both with (A) and without (B) close association to host vessels. (C) Transplanted cell in the inferior colliculus, expressing β-galactosidase (red) and binding LEL (green). (D) β-galactosidase-positive donor cell (red) in the hippocampus, coexpressing Flk-1 (blue) and desmin (green). All Flk-1+ donor cells also stained positive for desmin. Double labelings were confirmed by digital reconstruction in the x-z and y-z planes.
provided no evidence for an integration of donor cells into hostderived blood vessels. These data suggest that the donor cells showed a tropism for host capillaries but did not undergo endothelial differentiation. This interpretation is supported by the observation that the engrafted Flk1+ cells coexpressed desmin (Fig. 6D). Furthermore, the incorporated donor cells were negative for PECAM-1, a marker typically found in endothelial cells [30]. Thus, instead of indicating a potential transdifferentiation event, vascular tropism and endothelial marker expression may rather point to a close developmental relationship between the myogenic and endothelial lineages. Such an interpretation is supported by recent studies showing that endothelial cells can take on characteristics of cardiomyocytes and skeletal muscle cells [31, 32]. De Angelis et al. reported that clonable skeletal myogenic cells were present in the embryonic aorta of mouse embryos. Both aorta-derived myogenic cells and postnatal muscle satellite cells were found to express the endothelial marker antigens vascular endothelial-cadherin, Flk-1, αM-integrin, β3-integrin, P-selectin, smooth muscle actin, and PECAM [32]. In contrast to our study, their myoblast preparations were vWF– and expressed PECAM-1 only after differentiation into multinucleated myotubes. However, these subtle differences might be due to the fact that cell lines rather than primary cell preparations were used in our experiments. Interestingly, the engrafted myoblasts did not appear to undergo cell fusion and myotube formation. Similar observations have been made following transplantation of primary myoblasts into the adult rat brain [33]. In contrast,
C2C12 and i28 cells readily form multinucleated muscle fibers upon intramuscular [34] and subcutaneous transplantation [35]. These observations could suggest that extrinsic cues within the CNS inhibit myoblast fusion and muscle fiber formation. Myoblast Transplantation as a Potential Route for Widespread Gene Delivery to the Perivascular Compartment of the Perinatal Brain The potential to incorporate myoblasts in a nondisruptive manner across large areas of the neonatal brain provides interesting perspectives for cell-mediated gene transfer. In the past, encapsulated C2C12 cells were used to deliver ciliary neurotrophic factor and human growth hormone to the adult rodent brain [36, 37]. A key advantage of primary myoblast transplantation is the possibility of deriving the donor cells in an autologous manner. Several studies have indicated that transfected primary myoblasts grafted into the CNS survive and maintain transgene expression up to several weeks [33, 38]. Tyrosine-hydroxylase (TH)-expressing myoblasts transplanted into the striata of 6-OHDA-lesioned rats were reported to result in pronounced TH expression as well as long-term amelioration of Parkinsonian symptoms
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[39]. While these studies document the applicability of myoblast transplantation for localized gene delivery, widespread cell-based gene transfer to the CNS has remained a challenge. Lisovoski et al. reported poor survival of primary myoblasts upon transplantation into the ventricle of adult rats [33]. In contrast, myoblasts grafted into the ventricle of latestage rat embryos showed widespread nondisruptive incorporation. No cluster or tumor formation was observed within the 3-week postoperative period studied. Considering that C2C12 cells have been reported to form tumorous masses upon intramuscular transplantation [10, 34], this observation could point to extrinsic signals preventing myoblast aggregation and proliferation in the neonatal brain. The sustained transgene expression observed in the recipients’ brains might eventually be exploited for gene transfer in the perinatal CNS. The close association of the grafted cells with the host blood vessels makes this approach particularly interesting for inducible gene delivery systems controlled by systemically applied agents. Neural Chimeras in the Study of Adult Stem Cell Differentiation The generation of neural chimeras by intrauterine transplantation into the cerebral ventricles represents a versatile experimental platform for studying the migration and differentiation of non-neural stem cells in response to environmental cues derived from the developing brain. Following
Myoblast Differentiation in the Developing Brain delivery to the ventricular system, the cells are directly exposed to the ventricular zone. This exposure to an area of active neurogenesis should provide the grafted cells with the ultimate stimuli required for neural differentiation. In contrast to postnatal grafts into the brain parenchyma, intraventricular grafting into the developing CNS causes no traumatic or reactive alterations, which otherwise complicate the interpretation of transplant-based transdifferentiation studies. A key advantage of this model is the possibility of analyzing allogeneic and xenogeneic cells without the need for immunosuppression. Previous studies have shown that intrauterine transplantation permits widespread incorporation of murine and human donor cells without eliciting inflammatory changes [20, 40, 41]. This approach should, therefore, be particularly useful for studying the neuropoietic potential of other non-neural human stem cell populations. ACKNOWLEDGMENTS This study was supported by the Hertie Foundation, the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 246), the Innovationsprogramm Forschung des Landes Nordrhein-Westfalen, the Helga-Ravenstein Stiftung, and the BMBF (grant 01GN0122 to A.W.). The authors thank Frau Margit Zweyer for technical assistance. The C2C12 cells were a gift from S. Hughes (London/Stanford).
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