The spontaneous expression of neural markers by mesenchymal stem cells (MSCs) has been considered to be a demonstration of MSCs’ predisposition to differentiate towards neural lineages. In view of their application in cell therapy for neurodegenerative diseases, it is very important to deepen the knowledge about this distinctive biological property of MSCs. In this study, we evaluated the expression of neuronal and glial markers in undifferentiated rat MSCs (rMSCs) at different culture passages (from early to late). rMSCs spontaneously expressed neural markers depending on culture passage, and they were coexpressed or not with the neural progenitor marker nestin. In contrast, the number of rMSCs expressing mesengenic differentiation markers was very low or even completely absent. Moreover, rMSCs at late culture passages were not senescent cells and maintained the MSC immunophenotype. However, their differentiation capabilities were altered. In conclusion, our results support the concept of MSCs as multidifferentiated cells and suggest the existence of immature and mature neurally fated rMSC subpopulations. A possible correlation between specific MSC subpopulations and specific neural lineages could optimize the use of MSCs in cell transplantation therapy for the treatment of neurological diseases.
Cellular therapies using mesenchymal stem cells (MSCs) represent a promising approach in regenerative medicine, tissue-engineering, and autoimmune disease treatment. Clinical studies have confirmed the therapeutic potential of MSCs [
The growing interest in rMSCs has led to a number of studies in which their biochemical, genomic, immunophenotypic, and differentiation properties have been examined [
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rMSCs were collected, after sacrifice (according to the European Directive 86/609/EEC) from femurs and tibias of 10-week-old female Sprague Dawley (
rMSC cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. After 48 h, the nonadherent cells were removed, and the cells attaching to the culture flasks were cultured in
The rMSCs isolated from rat bone marrow were characterized for their immunophenotype by flow cytometry analysis and for their capacity to differentiate towards mesengenic lineages using specific protocols, as described in a previous work [
rMSCs were characterized for genomic instability at several passages [
rMSCs (P0, P1, P2, P4, P8, P16, P24, P40, and P80) from 5 rats, respectively, were seeded at approximately 104 cells/dish on noncoated glass slides into 35 mm diameter dishes using a culture medium composed of
Cells were fixed with 4% (w/v) paraformaldehyde for 10 min, washed with PBS, and treated for 10 min with 0.1 M glycine to quench autofluorescence. Then cells were incubated for 1 hour at room temperature with blocking solution (5% (w/v) BSA, 0.6% (v/v) Triton X-100 in PBS) and, subsequently, for 30 min at 37°C with 1 mg/mL RNAse in blocking-solution. Incubation with the following primary antibodies (diluted in blocking solution) was performed overnight at 4°C: anti-Nestin (1 : 50;
rMSCs were washed twice with ice-cold PBS, and total cellular extracts were prepared as previously described [
Differences in the number (%) of cells expressing a specific differentiation marker among passages were analyzed by using one-way analysis of variance (ANOVA). For each marker, an average value of positive cells, after 14 days of culture, was calculated from at least 3 experiments. Data were expressed as mean ± SD. Comparisons of mean values among the passages were analyzed using a Tukey’s multiple comparison test. A five percent probability (
The present study is an offshoot of a previous work by our group [
The rMSCs, isolated from rat bone marrow and used in our experiments, according to the international criteria proposed for the definition of MSCs [
In our culture conditions, rMSCs at P0 and P1 were morphologically heterogeneous (Figure
Cultures of undifferentiated rMSCs at different passages. At P1 rMSC cultures were morphologically heterogeneous (a). At P2 rMSC cultures consisted of a more homogeneous population of cells, most of which had a large flattened morphology (arrow). Relatively elongated cells were also present (arrow head) (b). At P50 rMSCs lost their morphology appearing more rounded (c). Bars:100
rMSCs, at P40 and P80, maintained the immunophenotype observed at P2 [
rMSC characterization. (a–d) rMSCs, at P80, were characterized by flow cytometry analysis for the expression of the following markers (pink histograms): CD29 (a), CD90 (b), CD34 (c), and CD45 (d). Isotype-matching IgGs were used to determine nonspecific signals (white histograms). (e and f) rMSC differentiation, at P80, after treatment with specific induction media: osteogenic differentiation was evaluated by alizarin red staining that visualizes calcium deposits (e), adipogenic differentiation was evaluated by oil red O staining that labels lipid droplets, not observed in adipogenic treated rMSC cultures at P80 (f). Bar: 100
In all the culture passages examined rMSCs maintained their capacity to actively divide (data not shown), and rMSCs at P140 are already in culture for expansion. Moreover, as demonstrated by
rMSCs senescence.
At different passages and at different times (see Section
The specificity of the antibodies used against neural antigens was confirmed by control cultures represented by DRG primary cultures, in which neurons and glial cells were present (Figure
Control cultures represented by DRG primary cultures, in which neurons and glial cells were present. Phalloidin-staining labeled actin filaments in blue (c). Only neurons were positive for
The data reported in Table
Undifferentiated rMSC expression of differentiation markers at several passages after 14 days of culture. The number of positive cells for each marker is expressed as %
Marker | Passage | Significant differences (ANOVA) | ||||||||
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P0 | P1 | P2 | P4 | P8 | P16 | P24 | P40 | |||
Nestin | 55 ± 21,2 | 52,5 ± 24,8 | 26,25 ± 1,8 | 56 ± 5,5 | 61,3 ± 11,1 | 68,3 ± 10,4 | 28,8 ± 1,8 | 3,5 ± 0,7 |
|
P16 versus P40. |
|
P0 versus P40; | |||||||||
P1 versus P40; | ||||||||||
P4 versus P40; | ||||||||||
P8 versus P40. | ||||||||||
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P2 versus P16; | |||||||||
P16 versus P24. | ||||||||||
| ||||||||||
|
36,5 ± 2,1 | 72,5 ± 3,5 | 64,2 ± 3,8 | 56,7 ± 7,6 | 30 ± 8,2 | 11,7 ± 3,8 | 12,5 ± 3,5 | 0,9 ± 0,14 |
|
P0 versus P1, P2, P16, P24, P40; |
P1 versus P8, P16, P24, P40; | ||||||||||
P2 versus P8, P16, P40; | ||||||||||
P4 versus P8, P16, P40; | ||||||||||
P8 versus P40. | ||||||||||
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P0 versus P4; | |||||||||
P8 versus P16, P24. | ||||||||||
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P1 versus P4. | |||||||||
| ||||||||||
NeuN | 36 ± 1,4 | 18,8 ± 1,8 | 47,5 ± 17,7 | 50 ± 8,7 | 20 ± 7 | 7 ± 4,2 | 0,9 ± 0,14 | — |
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P0 versus P24, P40; |
P2 versus P16, P24, P40; | ||||||||||
P4 versus P16, P24, P40. | ||||||||||
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P0 versus P16; | |||||||||
P1 versus P2, P4; | ||||||||||
P2 versus P8; | ||||||||||
P4 versus P8. | ||||||||||
| ||||||||||
NF | — | — | 0,9 ± 0,11 | 0,8 ± 0,14 | 0,85 ± 0,10 | 0,88 ± 0,16 | 0,85 ± 0,21 | — |
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P0 versus all except P1 and P40; |
P1 versus all except P0 and P40; | ||||||||||
P2 versus P40; | ||||||||||
P4 versus P40; | ||||||||||
P8 versus P40; | ||||||||||
P16 versus P40; | ||||||||||
P24 versus P40. | ||||||||||
| ||||||||||
GFAP | 55 ± 7,1 | 19 ± 7,1 | 11 ± 2,8 | 7,2 ± 3 | 11 ± 4,2 | 5,8 ± 2,5 | 3 ± 1,4 | — |
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P0 versus all; |
P1 versus P40. | ||||||||||
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P1 versus P24. | |||||||||
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P1 versus P16. | |||||||||
| ||||||||||
S100 | 10,3 ± 0,4 | 3,8 ± 1,8 | 0,95 ± 0,07 | 0,93 ± 0,1 | 0,98 ± 0,04 | 0,9 ± 0,04 | 0,93 ± 0,04 | — |
|
P0 versus all; |
P1 versus P40. | ||||||||||
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P1 versus P2, P4, P8, P16, P24. | |||||||||
| ||||||||||
PPAR |
3,9 ± 0,14 | 4,25 ± 0,35 | 2,45 ± 0,07 | 0,78 ± 0,04 | 0,68 ± 0,11 | 0,8 ± 0,14 | 0,85 ± 0,21 | 0,83 ± 0,11 |
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P0 versus all except P1; |
P1 versus all except P0; | ||||||||||
P2 versus all. | ||||||||||
| ||||||||||
OPN | 12,2 ± 0,4 | 11 ± 5,7 | 25 ± 14,1 | 10 ± 2,8 | 9,75 ± 3,9 | 5,25 ± 3,2 | 0,9 ± 0,1 | 0,83 ± 0,2 |
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P2 versus P24, P40. |
|
P2 versus P16. |
The neuroprogenitor marker nestin was expressed at P0 and P1 by about 50% of rMSCs (Table
Spontaneous expression of neural and mesengenic markers by undifferentiated rMSCs (P4) after 14 days of culture. Phalloidin staining labeled actin filaments in red (a, d, g, j, m, p, and s), and neural and mesengenic markers were labelled in green (b, e, h, k, n, q, t, and w). Propidium iodide labeled nuclei in red (v), used instead of phalloidin for a better labelling visualization after superimposition, being OPN expressed as dots. Most of cells were, respectively, positive for nestin (b) and
At P0 about 30% of rMSCs were positive for the early neuronal marker
The late neuronal marker NeuN (Figure
The late neuronal marker neurofilament (NF) was not expressed by rMSCs at any culture time examined at P0 and P1. At P2, P4, P8, P16, and P24, we observed no more than 1% of cells that were NF positive (Figure
The glial marker GFAP was mainly expressed by rMSCs at P0, where about 50% of cells were GFAP positive. From P1 to the subsequent passages GFAP expression decreased progressively, and at P40 and P80 no rMSCs expressed this marker (Table
Spontaneous coexpression of neural markers by undifferentiated rMSCs (P4) after 14 days of culture. Nestin was labelled in green (a, d, and g), and the other neural markers in red:
The expression of the glial marker S100 was evident at P0 and P1 and was extended, respectively, to about 10% and less than 5% of cells. At P2, P4, P8, P16, and P24 the percentage of positive rMSCs decreased to less than 1% (Figure
From the earliest passages (P0, P1, and P2), the adipogenic marker PPAR
At P0 and P1 the osteogenic marker osteopontin (OPN) was expressed by about 10–15% of cells that were frequently clustered. At P2 we observed more clustered OPN-positive cells. From P4 to subsequent passages (Figure
At different passages and at different times (see Section
Nestin,
rMSC expression of neural markers by immunoblotting. Protein extracts (15
In this study we have demonstrated that rMSCs, in the absence of any differentiative agent, are able to spontaneously express neural markers. This finding is not new, but, compared to the literature data, our study is more extensive and thorough in terms of the panel of markers and number of passages. Our results show that the percentage of rMSCs expressing neural markers depends on the culture passage. In fact, at late passages, in which cells are not senescent and maintained the MSC immunophenotype, the expression of neural markers decreases or is absent in comparison with early passages. On the other hand, the spontaneous expression of mesengenic differentiation markers, in rMSCs, is very low or absent at all passages examined.
The ability of undifferentiated rMSCs to express neural proteins confirms that MSCs are multidifferentiated cells [
The biological origin of MSCs may account for the expression of neural markers by undifferentiated rMSCs. It has been demonstrated that, during embryonic development, MSCs are generated from the neural crest and that neural crest-derived MSCs may persist in adult bone marrow [
The expression of nestin is considered to be one of the initial steps in the MSC progression to neural lineage. We have found that, in the presence of serum, nestin is expressed by a high percentage of undifferentiated rMSCs even at very early culture passages. In contrast, previous publications have reported that the presence of serum inhibited nestin expression and that, only after serum starvation, was an enrichment in nestin-expressing cells observed [
In the literature the existence of MSC subpopulations has been proposed [
In conclusion, we have demonstrated the spontaneous expression of neural markers by undifferentiated rMSCs, thereby supporting the concept of MSCs as multidifferentiated cells. Moreover, the presence of distinct rMSC subpopulations suggests that the controversial results regarding MSC neuronal differentiation could derive from the use of the whole population of MSCs, whereas the use of a neutrally fated subpopulation of MSCs could optimize their differentiation. The possibility of linking a specific MSC subpopulation with a specific neural lineage provides a framework for optimizing future transplantation studies aimed at treating neurodegenerative diseases.
The authors are grateful to Dr. E. Genton for language assistance.