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Differentiation of equine mesenchymal stromal cells into cells of neural lineage: potential for clinical applications.

Cruz Villagrán C, Amelse L, Neilsen N, Dunlap J, Dhar M - Stem Cells Int (2014)

Bottom Line: Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation.Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype.The effect of cell passage number, however, is inconsistent and further experiments are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative and Experimental Medicine, University of Tennessee, Knoxville, TN 37996, USA ; Department of Large Animal Clinical Sciences, University of Tennessee, Knoxville, TN 37996, USA.

ABSTRACT
Mesenchymal stromal cells (MSCs) are able to differentiate into extramesodermal lineages, including neurons. Positive outcomes were obtained after transplantation of neurally induced MSCs in laboratory animals after nerve injury, but this is unknown in horses. Our objectives were to test the ability of equine MSCs to differentiate into cells of neural lineage in vitro, to assess differences in morphology and lineage-specific protein expression, and to investigate if horse age and cell passage number affected the ability to achieve differentiation. Bone marrow-derived MSCs were obtained from young and adult horses. Following demonstration of stemness, MSCs were neurally induced and microscopically assessed at different time points. Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation. Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype. Expression of neural progenitor proteins, was assessed via western blot or immunofluorescence. In our study, MSCs generated from young and middle-aged horses did not show differences in their ability to undergo differentiation. The effect of cell passage number, however, is inconsistent and further experiments are needed. Ongoing work is aimed at transdifferentiating these cells into Schwann cells for transplantation into a peripheral nerve injury model in horses.

No MeSH data available.


Related in: MedlinePlus

Expression of nestin in undifferentiated and differentiated low passaged eBM-MSCs. Confocal microscopy shows the expression of nestin (red) in neural crest-like cells (upper panels) and in undifferentiated controls (lower panels) from eBM-MSCs of young and middle-aged horses after 3 h and 12 h of chemical induction. DAPI was used to stain the nuclei (blue). Note the predominant perinuclear localization of nestin. Scale bar = 25 μm.
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fig10: Expression of nestin in undifferentiated and differentiated low passaged eBM-MSCs. Confocal microscopy shows the expression of nestin (red) in neural crest-like cells (upper panels) and in undifferentiated controls (lower panels) from eBM-MSCs of young and middle-aged horses after 3 h and 12 h of chemical induction. DAPI was used to stain the nuclei (blue). Note the predominant perinuclear localization of nestin. Scale bar = 25 μm.

Mentions: We were not able to detect nestin expression by western blot analysis, even when 3 different primary antibodies with various dilutions (1 : 1000, 1 : 2000) were used. Interestingly, nestin expression was evident in undifferentiated and neurally differentiated MSCs of low and high passages for both age groups by IF (Figure 10). Nestin expression on cells of low passage for both age groups was observed with no significant differences (P = 0.7325). Additionally, cell passage number did not affect significantly the expression of nestin on the middle-aged horse (P = 0.3467), and no difference on nestin expression was noted between undifferentiated MSCs and neurally induced MSCs for young (P = 0.9616) or middle-aged (P = 0.0830) horses. A perinuclear location of nestin was evident on undifferentiated and neurally differentiated MSCs of low passage and on undifferentiated MSCs of high passage of both age groups (Figure 10). The location of nestin in neurally differentiated cells of high passage, however, was inconsistent, with some cells displaying a perinuclear location and others showing a more diffuse, cytoplasmic expression (Figure 11). It is possible that cells of high passages undergo changes that alter the structure of some filamentous proteins (i.e., nestin).


Differentiation of equine mesenchymal stromal cells into cells of neural lineage: potential for clinical applications.

Cruz Villagrán C, Amelse L, Neilsen N, Dunlap J, Dhar M - Stem Cells Int (2014)

Expression of nestin in undifferentiated and differentiated low passaged eBM-MSCs. Confocal microscopy shows the expression of nestin (red) in neural crest-like cells (upper panels) and in undifferentiated controls (lower panels) from eBM-MSCs of young and middle-aged horses after 3 h and 12 h of chemical induction. DAPI was used to stain the nuclei (blue). Note the predominant perinuclear localization of nestin. Scale bar = 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4260374&req=5

fig10: Expression of nestin in undifferentiated and differentiated low passaged eBM-MSCs. Confocal microscopy shows the expression of nestin (red) in neural crest-like cells (upper panels) and in undifferentiated controls (lower panels) from eBM-MSCs of young and middle-aged horses after 3 h and 12 h of chemical induction. DAPI was used to stain the nuclei (blue). Note the predominant perinuclear localization of nestin. Scale bar = 25 μm.
Mentions: We were not able to detect nestin expression by western blot analysis, even when 3 different primary antibodies with various dilutions (1 : 1000, 1 : 2000) were used. Interestingly, nestin expression was evident in undifferentiated and neurally differentiated MSCs of low and high passages for both age groups by IF (Figure 10). Nestin expression on cells of low passage for both age groups was observed with no significant differences (P = 0.7325). Additionally, cell passage number did not affect significantly the expression of nestin on the middle-aged horse (P = 0.3467), and no difference on nestin expression was noted between undifferentiated MSCs and neurally induced MSCs for young (P = 0.9616) or middle-aged (P = 0.0830) horses. A perinuclear location of nestin was evident on undifferentiated and neurally differentiated MSCs of low passage and on undifferentiated MSCs of high passage of both age groups (Figure 10). The location of nestin in neurally differentiated cells of high passage, however, was inconsistent, with some cells displaying a perinuclear location and others showing a more diffuse, cytoplasmic expression (Figure 11). It is possible that cells of high passages undergo changes that alter the structure of some filamentous proteins (i.e., nestin).

Bottom Line: Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation.Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype.The effect of cell passage number, however, is inconsistent and further experiments are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative and Experimental Medicine, University of Tennessee, Knoxville, TN 37996, USA ; Department of Large Animal Clinical Sciences, University of Tennessee, Knoxville, TN 37996, USA.

ABSTRACT
Mesenchymal stromal cells (MSCs) are able to differentiate into extramesodermal lineages, including neurons. Positive outcomes were obtained after transplantation of neurally induced MSCs in laboratory animals after nerve injury, but this is unknown in horses. Our objectives were to test the ability of equine MSCs to differentiate into cells of neural lineage in vitro, to assess differences in morphology and lineage-specific protein expression, and to investigate if horse age and cell passage number affected the ability to achieve differentiation. Bone marrow-derived MSCs were obtained from young and adult horses. Following demonstration of stemness, MSCs were neurally induced and microscopically assessed at different time points. Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation. Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype. Expression of neural progenitor proteins, was assessed via western blot or immunofluorescence. In our study, MSCs generated from young and middle-aged horses did not show differences in their ability to undergo differentiation. The effect of cell passage number, however, is inconsistent and further experiments are needed. Ongoing work is aimed at transdifferentiating these cells into Schwann cells for transplantation into a peripheral nerve injury model in horses.

No MeSH data available.


Related in: MedlinePlus