The spontaneous expression of neural markers, already demonstrated in bone marrow (BM) mesenchymal stem cells (MSCs), has been considered as evidence of the MSCs’ predisposition to differentiate toward neural lineages, supporting their use in stem cell-based therapy for neural repair. In this study we have evaluated, by immunocytochemistry, immunoblotting, and flow cytometry experiments, the expression of neural markers in undifferentiated MSCs from different sources: human adipose stem cells (hASCs), human skin-derived mesenchymal stem cells (hS-MSCs), human periodontal ligament stem cells (hPDLSCs,) and human dental pulp stem cells (hDPSCs). Our results demonstrate that the neuronal markers
Mesenchymal stem cells (MSCs) from bone marrow (BM-MSCs) are the adult stem cells that have been best characterized but other similar populations have been isolated and described from several tissues such as adipose tissue, umbilical cord blood, skin, skeletal muscle, and teeth. When MSCs are isolated from various tissues, a heterogeneous pool of cells may be obtained, so it is crucial to distinguish MSCs from other cells that may exhibit a similar phenotype in culture [
We and other researchers have shown that undifferentiated BM-MSCs express neuronal and glial differentiation markers [
hASC isolation and characterization were performed as previously described [
hS-MSC isolation and characterization were performed as previously described [
hS-MSCs expressed MSC-related cell surface antigens [
Healthy human third molars extracted during orthodontic treatment were collected from patients, after obtaining written informed consent, at the Department of Dentistry, San Gerardo Hospital, Monza. All interventions were performed under anesthesia due to total mucosal or bone impaction of dental elements. After local anesthesia, performed in a plessic way in association with mepivacaine 2% with a 1 : 100000 adrenalin ratio, a full thickness flap was completed. Moreover, through osteotomy, impacted third molars were enucleated and then preserved for a maximum of 1 hour in a sterile saline solution (PBS). The following procedures were done under sterile conditions.
To obtain hPDLSCs, periodontal ligament tissue was gently scraped from the surface of the middle third of the root. The tissue was placed in a 75 cm2 culture flask with complete DMEM and incubated at 37°C in 5% CO2. To obtain hDPSCs, the teeth were sectioned longitudinally in a mesiodistal direction with a piezoelectric ultrasonic device (OT7 insert) under abundant irrigation with sterile salt solution (0.9% NaCl) to expose the pulp chamber. The pulp tissue was separated by means of an MOD.31W hand excavator; it was then placed in a 75 cm2 culture flask with complete DMEM plus 10% FBS and incubated at 37°C in 5% CO2.
hPDLSCs and hDPSCs expressed MSC-related cell surface antigens and were able to differentiate toward mesodermal lineages (paper in preparation).
For the experiments performed in this study, hPDLSCs and hDPSCs were cultured in complete DMEM plus 10% defined FBS in a 5% CO2 humidified incubator at 37°C.
The expression of differentiation markers was determined in hASCs (P3, P6, and P16), hS-MSCs (P4, P8, and P16), hPDLSCs (P2, P4, and P8), and hDPSCs (P2, P4, and P8) by immunofluorescence experiments after 14 days from plating on glass slides in 35 mm diameter dishes (104 cells/dish) in the culture medium used for cell expansion without adding any differentiative agent.
Cells were fixed with 4% paraformaldehyde for 10 min and then treated with 0.1 M glycine (Sigma-Aldrich, St. Louis, MO) for 10 min. Cells were incubated for 30 min at room temperature with a blocking solution (5% BSA, 0.6% Triton X-100 in PBS) and for 30 min at
Total cellular extracts from hASCs, hS-MSCs, hPDLSCs, and hDPSCs were prepared as previously described [
hASCs, hS-MSCs, hPDLSCs, and hDPSCs were trypsinized and centrifuged at 500 g for 5 min. Then, after washes with PBS, cells were fixed in 2% paraformaldehyde, permeabilized with 0.5% saponin for 15 min (for
Differences in the number (%) of cells expressing a specific differentiation marker in different 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. Data were expressed as means ± SD. Comparisons of mean values for the passages were analyzed using Tukey’s multiple comparison test. A five percent probability
At different culture passages and for each analysed passage at day 14 from plating we evaluated, in undifferentiated hASCs, hS-MSCs, hPDLSCs, and hDPSCs, and cultured, in the presence of serum and in the absence of any differentiative agent, the expression of the following differentiation markers: the neuronal markers of
For each marker, the percentage of hASC positive cells was obtained by averaging the experimental results using cells from 3 healthy donors. The data reported in Table
Undifferentiated hASC expression of differentiation markers at different passages after 14 days of culture. The number of positive cells for each marker is expressed as % ± SD (—: no positive cells).
Marker | Passage | Significant differences ( |
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P3 | P6 | P16 | ||
Nestin |
|
|
— | All ns |
|
|
|
|
All ns |
NeuN |
|
|
|
All ns |
GFAP | — |
|
|
P6 versus P16 |
A small number of undifferentiated hASCs expressed the neuroprogenitor marker nestin (Figure
Spontaneous expression of neural markers by undifferentiated hASCs (P3), after 14 days of culture. Actin filaments were stained in red by phalloidin ((a), (d), and (g)), and neural markers were labelled in green. Few cells were nestin positive (b). Most of cells were
As shown in Figure
Moreover, double immunolabeling studies revealed that undifferentiated hASCs that were positive for NeuN also expressed
Spontaneous coexpression of neural markers by undifferentiated hASCs (P6) after 14 days of culture. NeuN-positive cells ((a): red) were always
For each marker, the percentage of hS-MSC positive cells was obtained by averaging the experimental results using cells from 3 healthy donors. The data reported in Table
Undifferentiated hS-MSC expression of differentiation markers at different passages after 14 days of culture. The number of positive cells for each marker is expressed as % ± SD (—: no positive cells). When the values are very heterogeneous we report the % of positive cells
Marker | Passage | Significant differences ( |
||
---|---|---|---|---|
P4 | P8 | P16 | ||
Nestin |
|
@ |
|
All ns |
|
|
|
|
All ns |
NeuN |
|
|
∞ | All ns |
GFAP | — | # | * | All ns |
#Donor 1 and donor 2: negative; donor 3: 30%.
*Donor 1: negative; donor 2 and donor 3: 70%.
The expression of the neuroprogenitor marker nestin was extended to about 10–15% of cells at P4 (Figure
Spontaneous expression of neural markers by undifferentiated hS-MSCs (P4) after 14 days of culture. Actin filaments were stained in red by phalloidin ((a), (d), and (g)), and neural markers were labelled in green. Some cells were nestin positive (b). Most of the cells were
Undifferentiated hS-MSCs expressed the early neuronal marker
The glial marker GFAP was not expressed at P4, while at P8 its expression was observed only in hS-MSCs deriving from one of the donors examined where about 30% of cells were GFAP positive. At P16, hS-MSCs obtained from two of the donors examined were GFAP positive (about 70%), while cells from the third donor were GFAP negative. Often GFAP expression was not equally distributed in the cytoplasm, and zones with different labelling intensities were observed.
Undifferentiated hS-MSCs did not express S100 (glial marker), PPAR
Moreover, double immunolabeling studies revealed that undifferentiated hS-MSCs that were positive for NeuN also expressed
Spontaneous coexpression of neural markers by undifferentiated hS-MSCs (P8) after 14 days of culture. Most of cells were
For each marker, the percentage of hPDLSC positive cells was obtained by averaging experimental results using cells from 3 healthy donors. The data reported in Table
Undifferentiated hPDLSC expression of differentiation markers at different passages after 14 days of culture. The number of positive cells for each marker is expressed as % ± SD.
Marker | Passage | Significant differences ( |
||
---|---|---|---|---|
P2 | P4 | P8 | ||
Nestin |
|
|
|
All ns |
|
|
|
|
All ns |
NeuN |
|
|
|
All ns |
At all the examined passages about 30% of undifferentiated hPDLSCs expressed the neuroprogenitor marker nestin (Figure
Spontaneous expression of neural markers by undifferentiated hPDLSCs (P4), after 14 days of culture. Actin filaments were stained in red ((a), (d), and (g)), and neural markers were labelled in green. About one-third of the cells in culture were nestin positive (b). Most of cells were
The expression of the late neuronal marker NF, the glial markers GFAP and S100, and the mesengenic markers PPAR
Moreover, undifferentiated hPDLSCs that were positive for NeuN also expressed
Spontaneous coexpression of neural markers by undifferentiated hPDLSCs (P8) after 14 days of culture. Most of cells were
For each marker, the percentage of hDPSC positive cells was obtained by averaging experimental results using cells from 3 healthy donors. The data reported in Table
Undifferentiated hDPSC expression of differentiation markers at different passages after 14 days of culture. The number of positive cells for each marker is expressed as % ± SD.
Marker | Passage | Significant differences ( |
||
---|---|---|---|---|
P2 | P4 | P8 | ||
Nestin |
|
|
|
All ns |
|
|
|
|
All ns |
NeuN |
|
|
|
All ns |
At all the examined passages about 30% of undifferentiated hDPSCs expressed the neuroprogenitor marker nestin (Figure
Spontaneous expression of neural markers by undifferentiated hDPSCs (P4), after 14 days of culture. Actin filaments were labelled in red ((a), (d), and (g)), and neural markers were labelled in green. About one-third of the cells in culture were nestin positive (b). Most of cells were
The expression of the late neuronal marker NF and the glial markers GFAP and S100 was not observed at any culture passage examined. Undifferentiated hDPSCs did not express PPAR
Moreover, double immunolabeling studies revealed that undifferentiated hDPSCs that were positive for NeuN also expressed
Spontaneous coexpression of neural markers by undifferentiated hDPSCs (P8) after 14 days of culture. Most of cells were
Among the neural markers evaluated, only
Overall means of the percentage of cells expressing
Marker | MSC type | Significant differences ( |
|||
---|---|---|---|---|---|
hASCs | hS-MSCs | hPDLSCs | hDPSCs | ||
|
|
|
|
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All ns |
NeuN |
|
|
|
|
All ns |
Stem cells expression of
The undifferentiated stem cell expression of
In this study, we have demonstrated that the early neuronal marker
Until now the expressions of
In our work a very high percentage of hASCs, hS-MSCs, hPDLSCs, and hDPSCs are
NeuN is a neuron-specific nuclear protein and its expression is found only in postmitotic neurons [
In our study, the percentage of cells expressing
An interesting aspect of our study is that none of the markers for mesodermal differentiation was spontaneously expressed by the various types of stem cells we looked at. This finding supports the hypothesis that the expression of the neuronal markers, observed at very high levels, is not aspecific but is probably related to the role played by these proteins in undifferentiated stem cells.
In a previous study, we showed
In conclusion, in this study we have demonstrated that, like hBM-MSCs, undifferentiated stem cells from various sources such as hASCs, hS-MSCs, hDPSCs, and hPDLSCs spontaneously express the neuronal markers
Last but not least, our results confirm that the evaluation of marker expression can never be used as the only evidence to prove the neuronal differentiation of MSC-like cells; morphological changes and functional properties should also be evaluated.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Anna T. Brini and Monia Orciani contributed equally to this work and are listed in alphabetical order.
The authors are grateful to Dr. E. Genton for her language assistance. They would like to thank Dr. A. Scuteri for doing the DRG total protein extracts and Dr. R. Rigolio for flow cytometry analysis assistance.