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    Phase-contrast photomicrographs of canine MSCs (A and B) and fibroblasts (C and D) before (A and C) and after (B and D) induction of neural differentiation. Notice that induction caused transformation from fibroblastic to neuronlike morphology in both MSCs and fibroblasts. Bar = 100 μm.

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    Representative results of flow cytometric analysis of canine MSCs (black line) after incubation with primary antibodies against CD34 (A), CD90 (B), CD45 (C), MHC-I (D), and MHC-II (E). Isotype control samples are also included in each panel (gray line). Notice that MSCs had negative results for CD34, CD45, and MHC-II and positive results for CD90 and MHC-I. PE = Phycoerythrin. APC = Allophycocyanin.

  • View in gallery

    Immunofluorescent photomicrographs of canine MSCs stained with primary antibodies against β III-tubulin (A and D), GFAP (B and E), or both β III-tubulin and GFAP (C and F) before (A, B, and C) and after (D, E, and F) induction of neural differentiation. In panel C, notice that some cells only express GFAP (those in the upper right corner) and others have coexpression of β III-tubulin and GFAP (double-stained yellow cells). Also, notice that the expression of β III-tubulin and GFAP increased after induction of neural differentiation, partly as a result of condensed protein localization. Panels C and F were counterstained with 4,6-diamidino-2-phenylindole. Bar in panels A, B, D, and E = 100 μm and in panels C and F = 50 μm.

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    Western blots of glial (GFAP; top row), neuronal (β III-tubulin; middle row), and β-actin (bottom row) proteins in samples of spinal cord lysate (positive control sample; A), fibroblasts before neural induction (B), fibroblasts after neural induction (C), MSCs before neural induction (D), and MSCs after neural induction (E).

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Expression of neural markers on bone marrow–derived canine mesenchymal stem cells

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  • 1 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.
  • | 2 Department of Anatomy and Cell Biology, Program in Stem Cell Biology and Regenerative Medicine, College of Medicine, University of Florida, Gainesville, FL 32610.
  • | 3 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.
  • | 4 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.
  • | 5 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

Abstract

Objective—To evaluate cell surface markers of bone marrow–derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells.

Sample Population—Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs.

Procedures—Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (β III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation.

Results—Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed β III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in β III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions.

Conclusions and Clinical Relevance—Canine bone marrow–derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.

Abstract

Objective—To evaluate cell surface markers of bone marrow–derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells.

Sample Population—Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs.

Procedures—Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (β III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation.

Results—Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed β III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in β III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions.

Conclusions and Clinical Relevance—Canine bone marrow–derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.

Bone marrow–derived MSCs, also referred to as bone marrow stromal cells, have gained considerable attention as a source for cellular transplantation treatments for animals with a number of diseases because of their proliferative capacity, accessibility, and multipotentiality. For specific conditions in vitro and in vivo, MSCs give rise to various cell types of mesodermal origin, such as osteoblasts, chondrocytes, adiopocytes, myoblasts, and tendocytes.1–4 Osteogenic properties of MSCs have been used by many researchers to regenerate osseous tissues for abnormal conditions, such as those that result from osteogenesis imperfecta,5,6 or to reconstruct large bone defects.7,8 Regeneration of muscular tissues, particularly cardiomyocytes, by use of MSCs or bone marrow–derived mononuclear cells (which contain MSCs) has been described in experimental studies9–11 in dogs and has been attempted successfully in human patients with myocardial infarcts.12

Furthermore, regeneration of damaged tissues by MSCs does not seem to be restricted in mesodermal tissues because studies4,13–15 have revealed the ability of MSCs to differentiate into cell types of endodermal and ectodermal origins. These findings have expanded the possibility of the use of MSCs for clinical applications, particularly for use in regeneration of tissues with a limited capacity to regenerate naturally, such as those in the CNS.

Neural differentiation of MSCs isolated from humans, rats, and mice has been reported16–19 by several investigators. In vitro studies of human and murine MSCs revealed that MSCs constitutively express neuronal and glial markers without induction, which suggests the intrinsic neural properties of MSCs. In some studies16–19 with specific culture conditions, MSCs derived from human and rodent bone marrow assumed neuronlike morphologic characteristics that coincided with an increased amount of expression of several neural markers, although their results varied depending on the neural induction used and the number of passages for the MSCs isolated for the assays.

However, the importance of some of the morphologic changes observed has been questioned by other investigators, who have attributed them to shrinkage of the actin cytoskeleton, rather than genuine neurofilament extension.20–22 As such, interpretation of neural differentiation of MSCs in vitro requires great caution and systematic approaches, including assays at the molecular level. Although it is still debated whether neurogenesis of MSCs represents genuine transdifferentiation or an epiphenomenon caused by induced chemical stress, functionalities of MSC-derived neurons (such as their abilities to secrete neurotransmitters or trigger action potentials) have been reported23–26 by several groups. These in vitro studies have raised tremendous interest that MSCs may have the potential to differentiate into functional neural cells and can be used in regenerative medicine to treat patients with neurologic diseases. In line with this concept, other in vivo studies27–29 have revealed that transplanted MSCs could migrate in the CNS and adopt neural fates in response to the host microenvironment. Most importantly, functional or behavioral improvements have been reported29–31 after transplantation of MSCs in rodents with experimental spinal cord injuries and ischemic brain lesions.

On the basis of findings in studies of human and rodent MSCs, we hypothesized that canine MSCs can be expanded in culture while maintaining their multipotentiality and that these cells have neural properties. Therefore, the objectives of the study reported here were to evaluate the cell surface markers and homogeneity of culture-expanded canine MSCs by use of flow cytometry and evaluate whether these cells express neuronal and glial-specific proteins, as determined by use of immunocytochemical and western blot analyses. Rapid neural differentiation was induced by culturing canine MSCs in a defined medium known to increase concentrations of intracellular cAMP, and changes in expression of neural proteins were evaluated.

Materials and Methods

Sample population—Samples of bone marrow were collected from 5 cadavers of adult mixed-breed dogs that were euthanized at a local animal shelter. Exact ages of the dogs were unknown; however, we collected samples only from dogs that had a complete set of adult teeth with minimal deposition of dental calculus. Thus, all dogs included were considered young adults. The protocol for the study was approved by the Institutional Animal Care and Use Committee of the University of Florida.

Collection of bone marrow and culture of MSC—Bone marrow (5 mL) was aseptically aspirated by use of 16-gauge Jamshidi needles from the iliac crests of the cadaveric dogs into sterilized 10-mL syringes containing 2,000 units of heparin. All bone marrow aspirates were obtained within 30 minutes after the dogs were euthanized.

The MSCs were isolated and cultured by use of techniques described elsewhere32 with modifications. Briefly, the marrow was washed with Hank's balanced salt solution and mixed with 2 volumes of culture medium consisting of 20% fetal bovine serum and antimicrobials (100 U of penicillin G/mL, 100 μg of streptomycin sulfate/mL, and 0.25 μgof amphotericin B/mL) in low-glucose (1g/L) Dulbecco's modified Eagle's medium (ie, complete medium). Aliquots of bone marrow suspension were layered over ficolla and centrifuged at 400 × g for 30 minutes to enrich mononuclear cells. Enriched mononuclear cells were plated in T-75 culture flasks (1.5 × l05 cells/cm2 in 15 mL of complete medium for each cadaver) and incubated at 37°C in a humidified 5% carbon dioxide environment. After 48 hours, flasks were washed with PBS solution to remove nonadherent cells. Medium was changed twice weekly.

Cells were passaged on reaching approximately 80% confluence. Cells were released by use of 0.05% trypsin-0.53mM EDTA. Released cells were collected by centrifugation at 800 × g for 5 minutes, washed twice with Hank's balanced salt solution, counted by use of a hematocytometer, and replated at 8 × l03 cells/cm2 for subsequent passages. This process was repeated 3 times to obtain third-passage cultures in which expanded MSCs appeared morphologically homogeneous. These third-passage MSCs were used in assays.

FACS analysis of canine MSCs—To evaluate cell surface markers and homogeneity of canine MSCs, third-passage MSCs were analyzed by use of flow cytometry. Culture medium was removed, and attached cells were washed 3 times with PBS solution. Cells were detached from the flasks by incubation with 0.05% Trypsin-0.53mM EDTA. Detached cells were collected by centrifugation, washed with FACS buffer (PBS solution containing 0.5% bovine serum albumin and 0.1% sodium azide), and counted.

Aliquots containing 1 × 106 cells were incubated with primary antibodies for 30 minutes on ice. Primary antibodies used were R-phycoerythrin–conjugated mouse anti-canine CD34 (1:1),b FITC-conjugated rat anti-canine CD45 (1:15),c mouse anti-canine CD90 (1:80),d mouse anti-canine MHC-I (1:133),e and mouse anti-canine MHC-II (1:133).f Allophycocyanin-conjugated goat anti-mouse IgM (1:5)g was used to label anti-CD90 antibodies, and FITC-conjugated rat anti-mouse IgG2a (1:25)h was used to label anti-MHC I and anti-MHC II antibodies by incubation for 30 minutes on ice. Aliquots containing an equal number of cells were incubated with respective isotype control antibodies under the same conditions. Cells without antibodies were used as a negative control sample. Labeled cells were then washed with FACS buffer, pelleted, and fixed in 0.5% paraformaldehyde and 0.1% sodium azide in PBS solution. Data were analyzed by recording 10,000 events on an FACS cytometeri by use of commercially available software.j

Neural differentiation of canine MSCs—Neural differentiation of MSCs was induced by replacing the complete medium with neural induction medium. The neural induction medium consisted of 0.5mM IBMX and 1mM dbcAMP in Dulbecco's modified Eagle's medium–F12; this medium has been used in neural differentiation of human bone marrow stromal cells.16 To support growth of neural cells, the neural induction medium included 1% N-2 supplements.k

For immunocytochemical analysis, 1 × 104 third-passage MSCs were suspended in 100 μL of complete medium and plated on coverslips placed in each well of 12-well culture plates. After incubation for 12 hours, 900 μL of complete medium was added and cells were cultured until reaching 70% confluency. Subsequently, medium was removed, and 1 mL of neural induction medium was added. Plates were then cultured at 37°C in a humidified 5% carbon dioxide environment for 5 hours. For western blot analysis, third-passage MSCs were plated at 8,000 cells/cm2 in complete medium on 60-mm culture dishes. On reaching 70% confluency, cells were washed with PBS solution and cultured in neural induction medium for 5 hours. Cells were also cultured in complete media and served as a control sample. We also used canine fibroblast cultures (also from the third passage)l as a control sample for several assays.

Immunocytochemical analysis for neural-specific markers—Immunocytochemical analysis was performed to evaluate whether canine MSCs express neuronal (β III-tubulin), astrocyte-specific (GFAP), and oligodendrocyte-specific (MBP) proteins. Cells on coverslips cultured in complete medium or neural differentiation medium were gently washed with PBS solution and fixed in ice-cold 4% paraformaldehyde for 30 minutes at 23°C. Fixed cells were permeabilized by incubation in 1.0% Triton × −100 in PBS solution for 10 minutes at 23°C. Nonspecific binding was blocked by incubation with a blocking solution (1.0% bovine serum albumin in PBS solution) for 30 minutes at 23°C. Cells were then incubated with primary antibodies (anti-β III-tubulinm [1:1,000]; anti-GFAPn [1:1,000]; and anti-MBPo[1:50,000]) for 1 hour at 23°C. Primary antibodies were removed, and cells were washed with PBS solution and incubated with one of the secondary antibodies (Cy2-conjugated goat anti-mouse IgG1p [1:400]; rhodamine-conjugated goat anti-mouse IgG2bq [1:100]; or FITC-conjugated goat anti-mouse IgG2ar [1:400]) for 1 hour in darkness at 23°C. Cells were also incubated without primary antibodies to control for nonspecific staining by secondary antibodies. Cells were washed with PBS solution and mounted by use of a mounting medium containing 4,6-diamidino-2-phenylindole.s Stained cells were observed by use of a fluorescent microscopet with appropriate filters.

Western blotting and densitometric analyses—To confirm expression of neuronal (β III-tubulin) and glial (GFAP and MBP) markers on canine MSCs, cultures were detached by use of 0.05% trypsin-0.53mM EDTA for use in western blot analyses. Cells were lysed with a lysis solution (0.18M Tris-HCl, 40% glycerol, 4% SDS, 0.04% bromophenol blue, and 0.05M threo-1,4-dimercapto-2,3-butanediol). Canine spinal cord lysate, obtained by use of the same lysis solution, was used as a positive control sample. Total protein concentrations of each sample were determined by use of a protein assay kit.u Samples containing 20 μg of protein were loaded in each lane of 12% polyacrylamide gels and electrophoretically separated. Separated proteins were transferred to nitro-cellulose membranes, blocked by incubation with TBS solution containing 5% nonfat dry milk and 0.1% Tween-20 for 1 hour at 23°C, and incubated with primary antibodies (β III-tubulin [1:500], GFAP [1:4,000], or MBP [1:50,000]) overnight at 4°C. Membranes were also probed with β-actin (1:1,000)v as loading control samples.

After washing the membranes 3 times with 0.05% Tween-20 in TBS solution, the membranes were incubated with alkaline phosphatase–conjugated secondary antibodyw(1:5,000) for 1 hour at 23°C. The membranes were washed 3 times in 0.05% Tween-20 in TBS solution and developed in a solution containing nitrotetrazolium blue and 5-bromo-4-chloro-3-indolyl phosphate. Photographs of the membranes were taken by use of a molecular imager,x and densitometric analyses were performed by use of a software program.y Western blotting and densitometric analyses were performed in duplicate for all samples.

Results

MSC culture and FACS analysis—Approximately 5 days after plating of cells, we observed that cells started to attach to the flask and proliferate in colonies. Initially, cells with differing morphologic characteristics were observed in the culture, mainly consisting of loosely attached rounded cells and tightly attached spindle-shaped cells. During culture expansion, rounded cells were removed during washing and spindle-shaped cells became predominant. These spindle-shaped cells further flattened to assume a fibroblastic appearance during the third passage.

For each culture passage, the cell number was multiplied approximately 10-fold. Expanded cells in the third passage appeared morphologically homogeneous (Figure 1). Cell surface markers of these cells were comparable to those reported in human and rodent MSCs. Analysis by use of flow cytometry revealed that these cells had positive results for CD90 and MHC-I and negative results for MHC-II (Figure 2). Lack of expression for CD34 and CD45 indicated that cells of hematopoietic origin had been excluded during the cell expansion process.

Figure 1—
Figure 1—

Phase-contrast photomicrographs of canine MSCs (A and B) and fibroblasts (C and D) before (A and C) and after (B and D) induction of neural differentiation. Notice that induction caused transformation from fibroblastic to neuronlike morphology in both MSCs and fibroblasts. Bar = 100 μm.

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1921

Figure 2—
Figure 2—

Representative results of flow cytometric analysis of canine MSCs (black line) after incubation with primary antibodies against CD34 (A), CD90 (B), CD45 (C), MHC-I (D), and MHC-II (E). Isotype control samples are also included in each panel (gray line). Notice that MSCs had negative results for CD34, CD45, and MHC-II and positive results for CD90 and MHC-I. PE = Phycoerythrin. APC = Allophycocyanin.

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1921

Neural differentiation of canine MSCs— Induction of neural differentiation of canine MSCs caused rapid changes in their appearance, from fibroblastic to neuronlike characteristics with multiple branching processes of various lengths in some cells (Figure 1). Cells with neuronlike characteristics also had refractile cell bodies that resembled cultured neurons. During induction of neural differentiation, cells became less adherent to the culture flask. As a result, some of the cells detached during flask manipulation. Changes in cell morphology were, however, not specific to MSCs because similarly treated fibroblasts assumed neuronlike characteristics after incubation with induction agents for 5 hours.

Immunocytochemical analysis—Results of immunocytochemical analysis revealed that canine MSCs constitutively expressed neuronal (β III-tubulin) and astrocyte-specific (GFAP) proteins. Almost all MSCs were immunoreactive against GFAP at a relatively low degree, whereas a subset of these cells (approximately 75% of all MSCs) was strongly positive when tested for β III-tubulin (Figure 3). Double staining of cells revealed that MSCs positive for GFAP also expressed β III-tubulin. After induction of neural differentiation, expression of β III-tubulin and GFAP appeared to be pronounced, although these changes may have resulted from condensed localization of these proteins that accompanied the morphologic changes of the cells. In contrast, MSCs with positive results when tested for MBP were not found for our culture conditions before or after induction of neural differentiation. Canine fibroblasts used as a negative control sample did not have immunoreactivity against any neuronal or glial proteins.

Figure 3—
Figure 3—

Immunofluorescent photomicrographs of canine MSCs stained with primary antibodies against β III-tubulin (A and D), GFAP (B and E), or both β III-tubulin and GFAP (C and F) before (A, B, and C) and after (D, E, and F) induction of neural differentiation. In panel C, notice that some cells only express GFAP (those in the upper right corner) and others have coexpression of β III-tubulin and GFAP (double-stained yellow cells). Also, notice that the expression of β III-tubulin and GFAP increased after induction of neural differentiation, partly as a result of condensed protein localization. Panels C and F were counterstained with 4,6-diamidino-2-phenylindole. Bar in panels A, B, D, and E = 100 μm and in panels C and F = 50 μm.

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1921

Western blot analysis—Western blot analyses were used to confirm expression of neuronal and glial proteins for canine MSCs and to evaluate the amount of expression before and after induction of neural differentiation. Distinct bands corresponding to β III-tubulin and GFAP were observed in untreated canine MSCs from all samples (Figure 4). Densitometric analyses confirmed that the amount of expression of GFAP in MSCs consistently increased after induction of neural differentiation (mean ± SD increase of 39.0 ± 20.6% relative to β-actin). On the other hand, the expression of β III-tubulin in MSCs did not differ significantly before and after induction of neural differentiation (mean ± SD increase of 9.6 ± 18.5% relative to β-actin). Bands corresponding to MBP were not detected in MSCs before or after induction of neural differentiation. Expression of these proteins was not detectable in fibroblasts.

Figure 4—
Figure 4—

Western blots of glial (GFAP; top row), neuronal (β III-tubulin; middle row), and β-actin (bottom row) proteins in samples of spinal cord lysate (positive control sample; A), fibroblasts before neural induction (B), fibroblasts after neural induction (C), MSCs before neural induction (D), and MSCs after neural induction (E).

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1921

Discussion

In the study reported here, a population of cells was isolated from canine bone marrow and expanded in vitro. The technique used for isolation and expansion of these cells was adopted and modified from standard methods commonly used in studies3,32,33 of human, rodent, and canine MSCs. Centrifugation on a ficoll cushion followed by cultivation of plastic-adherent cells yielded morphologically uniform fibroblastic cells in the third-passage culture. Results of flow cytometric analysis revealed that the expanded cells expressed neither CD34 nor CD45, which therefore confirmed a lack of cells of the hematopoietic lineage in the culture. The expression of CD90 and MHC-I and the lack of MHCII were in agreement with other reports34,35 for human MSCs. On the basis of the morphologic characteristics and results of flow cytometric analysis, we considered these cells to be canine MSCs.

We selectively expanded canine MSCs solely on the basis of their physical characteristics of adhesion to plastic; however, magnetically labeled antibodies have been commonly used to eliminate undesirable cell types from primary culture for expansion of human and rodent MSCs.34,36 These techniques may also be valuable to yield purified canine MSCs from a small amount of bone marrow within a shorter culture period.

For our culture conditions, canine MSCs constitutively expressed neuronal and astrocyte-specific markers. Immunocytochemical and western blot analyses revealed that untreated canine MSCs strongly expressed β III-tubulin. This observation was consistent with reports16,34 for human MSCs. Because β III-tubulin can be found on early neurons, canine MSCs that are positive for β III-tubulin may have the potential to differentiate into neuronal cells under appropriate conditions.

We found that GFAP, a marker for mature astrocytes, was also expressed on untreated canine MSCs. Reports on constitutive expression of GFAP in untreated human MSCs have been conflicting. For example, investigators in 2 studies16,18 reported a lack of expression of GFAP, whereas investigators in 2 other studies17,34 reported expression of GFAP in untreated human MSCs. These conflicting results from various laboratory groups may reflect the lack of a defined set of surface markers for MSCs, which results in inclusion of unidentified subsets of MSCs with slightly differing phenotypic patterns.

In the study reported here, expanded canine MSCs appeared to be morphologically and phenotypically homogeneous, but it was possible that several subsets of MSCs existed in our culture. This was illustrated by discrete properties of expanded cells with regard to their immunoreactivities against β III-tubulin and GFAP; GFAP was expressed in nearly all MSCs, whereas β III-tubulin expression was restricted in approximately three fourths of all MSCs. It was interesting that canine MSCs expressing neuronal-specific proteins also expressed astrocyte-specific proteins (approx three fourths of all MSCs). Therefore, similar to human MSCs,34,37 canine MSCs are considered undifferentiated but can also be characterized as multidifferentiated, which may explain their high plasticity.

Lack of MBP expression on canine MSCs was somewhat predictable because this protein is only expressed in mature oligodendrocytes. Differentiation of MSCs into the oligodendrocyte lineage may require discrete molecular signaling. Markers for earlier stages of oligodendrocytes need to be investigated to further characterize the plasticity of canine MSCs as to their capacity for neural differentiation.

Effects of induction of neural differentiation on cell morphologic characteristics and expression of neuronal or glial markers were evaluated for canine MSCs. The induction protocol, which used dbcAMP and IBMX, can induce a rapid neuronlike morphologic change in human MSCs as a result of increased intra-cellular cAMP concentrations.16 In the study reported here, canine MSCs had neuronlike morphologic characteristics as early as 3 hours after the induction of neuronal differentiation. However, a similar morphologic change was also observed in canine fibroblasts at approximately the same rapidity. Analysis of these observations suggests that the transformation of canine MSCs from fibroblastic to neuronlike morphology was not a specific event associated with the multipotentiality of canine MSCs. Therefore, these dramatic morphologic changes should not be overvalued, and special care must be taken to interpret results of in vitro neural differentiation of MSCs.

As pointed out by several investigators,20–22 a rapid morphologic change of MSCs after neural induction may represent cytotoxic effects of the reagents in the induction medium, which leads to cell shrinkage and subsequent adoption of neuronlike characteristics. Commonly used reagents, alone or in combination, that have effects on MSCs to induce neuronlike characteristics through cytoskeletal shrinkage include butylated hydroxyanisole, dimethyl sulfoxide, and β-mercaptoethanol.

On the basis of immunocytochemical and western blot analyses, we determined that the induction of neural differentiation resulted in increased expression of GFAP on canine MSCs. Therefore, these results may indicate that the neural differentiation method used in our study induces canine MSCs to differentiate preferentially toward the astrocyte lineage. Similar results have been reported for human MSCs34 and murine MSCs treated with dbcAMP and IBMX.19 Although strong expression of β III-tubulin was observed on canine MSCs before and after induction of neural differentiation, expression of this protein was not affected by the induction treatment.

As mentioned previously, canine MSCs became less adherent to culture flasks, and there was a substantial reduction of the cell number during induction of neural differentiation. Consequently, all analyses in the study reported here were performed 5 hours after the onset of induction. However, it would be interesting to further investigate whether a longer induction period would cause emergence of more-mature neuron markers and stable phenotypes. Clearly, additional studies that address functionalities of canine MSC–derived neural cells will be necessary to evaluate the potential of MSCs for use in the treatment of patients with neurologic disorders.

With the advancement of our understanding of regulatory mechanisms that underlie neural differentiation, it may become possible to generate a specific neural cell type from canine MSCs. Such an approach has already been undertaken in an attempt to generate functional neural precursor cells from human embryonic stem cells.38 This approach holds an advantage in that prior induction of embryonic stem cells into neural progenitor cells may reduce the possibility of the development of tumors (eg, teratomas) at the transplantation site. Furthermore, induction of embryonic stem cells into more-specific neural cell types has been proposed. For example, dopaminergic neurons have been generated from human,39,40 monkey,41,42 and murine43 embryonic stem cells and may have potential for treatment of patients with Parkinson's disease. It has also been reported44,45 that purified oligodendrocytes can be generated from human and murine embryonic stem cells. These embryonic stem cell–derived oligodendrocytes have extensive myelinating capacity in vivo; therefore, they hold promise for use in the treatment of humans with demyelinating diseases.

In 1 study,46 investigators reported that rat and human MSCs can be specifically induced by use of gene transfection with Notch intracellular domain to generate functional neurons. In vitro differentiation of rat MSCs into myelinating cells with phenotypic and functional characteristics of Schwann cells has also been described.47,48 In another study,49 investigators determined that transplantation of Schwann cells derived from rat MSCs resulted in enhanced axonal regeneration and functional recovery in rats with a completely transected spinal cord. These studies on MSC differentiation into specific neural cells are particularly promising in that if a stable functional pheno-type can be achieved, MSCs may become the strongest candidate for cellular treatments because autologous transplantations are clearly desirable in a clinical setting. Induction of canine MSCs into specific functional neural cell types may be possible by understanding the transcription factors involved in neurogenesis. Analysis of global gene expression patterns, which has become available through DNA microarrays for the canine genome, would aid in development of such techniques.

A large-scale expansion of transplants is one of the critical prerequisites for clinical applications of cellular transplantation treatments. Canine MSCs can be readily isolated from bone marrow of patients, thus allowing autologous transplantation. These cells can be further expanded in culture while retaining the capacity for multi-lineage differentiation. Canine MSCs have the ability for neural differentiation; therefore, they may have the potential for use in treating dogs with various neurodegenerative diseases and spinal cord injuries. Further investigations on mechanisms of neurogenic abilities of canine MSCs are warranted, which may lead to the development of novel therapeutic strategies to target specific diseases of the CNS. In addition, naturally developing neurologic diseases in dogs may provide unique opportunities for us to evaluate the safety and clinical efficacy of autologous MSC transplantation as a prelude to clinical studies of CNS disorders in humans.

ABBREVIATIONS

MSC

Mesenchymal stem cell

FACS

Fluorescence-assisted cell sorting

FITC

Fluorescein isothiocyanate

MHC

Major histocompatibility complex

IBMX

Methyl-isobutylxanthine

dbcAMP

Dibutyryl cAMP

GFAP

Glial fibrillary acidic protein

MBP

Myelin basic protein

TBS

Tris-buffered saline

a.

Ficoll-Paque, StemCell Technologies, Vancouver, BC, Canada.

b.

R-Phycoerythrin–conjugated mouse anti-canine CD34 monoclonal antibody, BD Biosciences, Franklin Lakes, NJ.

c.

FITC-conjugated rat anti-canine CD45 monoclonal antibody, Serotec, Raleigh, NC.

d.

Mouse anti-canine CD90 monoclonal antibody, VMRD Inc, Pullman, Wash.

e.

Mouse anti-canine MHC-I monoclonal antibody, VMRD Inc, Pullman, Wash.

f.

Mouse anti-canine MHC-II monoclonal antibody, VMRD Inc, Pullman, Wash.

g.

Allophycocyanin-conjugated goat anti-mouse IgM, Caltag Laboratories, Burlingame, Calif.

h.

FITC-conjugated rat anti-mouse IgG2a, BD Biosciences, Franklin Lakes, NJ.

i.

FACS Calibur, Becton Dickinson Immunocytometry Systems, San Jose, Calif.

j.

Cell Quest, BD Biosciences, Franklin Lakes, NJ.

k.

N-2 supplement liquid, Invitrogen Corp, Carlsbad, Calif.

l.

AG07454, Coriell Institute for Medical Research, Camden, NJ.

m.

Mouse anti-β III-tubulin monoclonal antibody, Promega Corp, Madison, Wis.

n.

Mouse anti-GFAP monoclonal antibody, BD Biosciences, Franklin Lakes, NJ.

o.

Mouse anti-MBP monoclonal antibody, HyTest Ltd, Turku, Finland.

p.

Cy2-conjugated goat anti-mouse IgG1, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.

q.

Rhodamine-conjugated goat anti-mouse IgG2b, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.

r.

FITC-conjugated goat anti-mouse IgG2a, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.

s.

VECTASHIELD mounting medium with 4, 6-diamidino-2-phenylindole, Vector Laboratories, Orton Southgate, Peterborough, UK.

t.

Zeiss Axioplan II, Carl Zeiss MicroImaging Inc, Thornwood, NY.

u.

Micro BCA protein assay, Pierce, Rockford, Ill.

v.

Mouse anti-β-actin monoclonal antibody, Abcam Inc, Cambridge, Mass.

w.

Alkaline phosphatase-conjugated goat anti-mouse IgG antibody, Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.

x.

FlourS Multi-imager, Bio-Rad Laboratories, Hercules, Calif.

y.

Quantity One, Bio-Rad Laboratories, Hercules, Calif.

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Contributor Notes

Dr. Deng's present address is Center for Human Genetics, Duke University Medical Center, Durham, NC 27710.

Supported by the University of Florida College of Veterinary Medicine Consolidated Faculty Research Development Award Grant.

Presented in part at the Experimental Biology 2006 meeting, San Francisco, April 2006.

The authors thank Neal Benson and Bhavna Bhardwaj for assistance with flow cyotometric analysis.

Address correspondence to Dr. Kamishina.