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
Spinal cord and peripheral nerve injuries in horses occur after trauma, toxic/metabolic, and infectious diseases. Less frequently, degenerative and hereditary diseases also pose a threat. These events trigger an inflammatory cascade of events that results in poor performance, disability, or death. Additionally, the impact of peripheral nerve injuries in horses is reflected by big financial and emotional investments. Peripheral nerves can be injured by thermal or chemical injuries, compression, crushing, stretching, or transection [
Mesenchymal stromal cells (MSCs) from bone marrow and adipose tissue have been demonstrated to transdifferentiate into cells of other lineages other than mesodermal lineages.
To our knowledge, there are no reports in the literature that describe the ability of equine bone marrow-derived MSCs (eBM-MSCs) to differentiate into cells of neural lineage or demonstrate the clinical benefits after transplantation into horses suffering from neuropathies. We have previously reported that, to improve the clinical outcomes related to stem cell therapies, it is important to assess the biology and function of MSCs prior to their application in clinical cases [
Bone marrow aspirates were obtained from the sternum of 2 young mixed breed (range: 1–4 years old) and 4 adult American Quarter Horse (range: 9–13 years old) mares and 1 young American Quarter Horse gelding as described previously [
These procedures were as described earlier [
Passage 1 of equine MSCs was seeded at a density of 1 × 106 in 100 mm tissue culture dishes (Thermo Scientific, Rochester, NY) and maintained in growth medium for 7 to 10 days until clusters of colonies were observed. The medium was replaced after every 2-3 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.5% of crystal violet (Sigma-Aldrich, Saint Louis, MO). After staining, the dishes were allowed to air-dry and images were acquired with a Fujifilm LAS-4000 imaging system (GE Healthcare Life Sciences).
Low passage (P2–P4) equine MSCs were seeded at a density of 2 × 104 per well in a 24-well tissue culture plate. The medium was replaced every 2-3 days. Cell proliferation was measured at 2, 4, and 7 days after seeding. A CellTiter 96 Aqueous nonradioactive (MTS) assay (Promega, Madison, WI) was used following the manufacturer instructions. Briefly, MTS reagent in a 5 : 1 ratio relative to the media was added to each well and incubated for 3 h at 37°C and 5% CO2. The absorbance at 490 nm was measured and data was obtained using Gen5 data analysis software (Biotek, Winooski, Vermont). Growth medium only with no cells was used as a blank to correct the readings for each of the samples.
Low passage (P2–P4) of equine MSCs was seeded at a cell density of 2 × 105 cells in 60 mm tissue culture dishes (BD Falcon, New Jersey) and maintained at 37°C and 5% CO2 in growth medium. When the cells were roughly 70% confluent, the medium was removed and was replaced with lineage-specific differentiation media as described earlier [
Neural crest-like cell differentiation was conducted in MSCs from the 7 donors aforementioned. Since we need high numbers of eBM-MSCs for neural differentiation, to obtain appropriate protein samples, we selected one young and one middle-aged donor to conduct the western blot and immunofluorescence experiments. The donors were chosen based on their rates of proliferation as demonstrated by the MTS assay described above.
Low (P2) and high (P9) of equine MSCs were seeded at a cell density of 8 to 10 × 106 into either polystyrene or Primaria nitrogen-coated 100 mm tissue culture dishes (Becton Dickinson Labware, Bedford, MA). Cells were maintained in regular growth medium at 37°C and 5% CO2, for at least 48 h to allow attachment. Neural differentiation was induced using a combination of previously described methods [
Nuclear/cytoplasmic fluorescent staining was used to show the neural cell like morphology of MSCs after neural differentiation. Low and high passages of equine MSCs were seeded at a density of 1.25 × 106 on Primaria nitrogen-coated 60 mm tissue culture dishes and maintained in regular growth medium at 37°C and 5% CO2, for at least 48 h to allow attachment. When cells were 80–90% confluent they were chemically induced for neural differentiation as described above. Undifferentiated control MSCs described above were maintained with regular growth medium. For cytoplasmic staining, neurally induced and undifferentiated MSCs at 12 h were stained with 5
Total cell lysates were prepared from undifferentiated and neurally induced equine MSCs from low and high passages 12 h after differentiation using standard protocols. Cells on each dish were gently washed with HBSS buffer and collected via cell scraping. To obtain total proteins in each sample, cells were lysed in 200
Low and high passages of equine MSCs were seeded at a density of 1.25 × 106 on Primaria nitrogen-coated 60 mm tissue culture dishes and maintained in growth medium at 37°C and 5% CO2, for at least 48 h, to allow attachment. When cells were 80–90% confluent, they were chemically induced for neural differentiation as described above. Undifferentiated MSCs used as controls were maintained with regular growth medium. Neurally induced and undifferentiated MSCs were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Sigma) for 10 min, at room temperature, and blocked with 5% normal serum for 30 min, at room temperature. Cells were washed and incubated overnight with 5
A two-tailed Student’s
The stemness of cells from all equine MSC cultures was assessed solely in low passage cells. Once the properties of stem cell were demonstrated, only 1 donor from each group (young and middle-aged) was selected and the population of MSCs was passaged for neural experiments.
Equine MSC cultures expanded from the bone marrow harvest adhered to the polystyrene surface and displayed spindle-shaped fibroblastic morphology. No morphological change was observed in eBM-MSC cultures generated from young and middle-aged donors.
Equine MSCs from young (Figure
Measurement of rate of proliferation of eBM-MSCs. MTS assay was used to compare the rates of proliferation of eBM-MSCs from 4 young horses (numbers 1–4) and 3 middle-aged horses (numbers 5–7) over a period of 7 days. Absorbance is the optical density (OD) measured at 490 nm with this colorimetric assay. Note the rapid rate of self-renewal on all animals at day 7. Mean values of each sample (
Equine MSCs of P1of young and middle-aged horses were capable of growing in clusters over the polystyrene-coated tissue culture dishes at day 10, suggesting that the eBM-MSC cultures established represent the MSCs or the progenitor cells. A representative CFU assay is shown (Figure
Colony forming unit assay of eBM-MSCs. Representative image of CFU formed after 10 days of seeding passage 1 eBM-MSCs from one young (a) and one middle-aged (b) horse. Note the ability of undifferentiated MSCs from both age groups to form CFU.
Low passage equine MSCs from both young and middle-aged donors were capable of differentiation into adipogenic, chondrogenic, and osteogenic cells under lineage-specific chemical induction (Figure
Mesodermal trilineage differentiation assays of eBM-MSCs. Representative images from one young (a) and one middle-aged (b) horse showing oil red-o (A), alcian blue (B), and alizarin red (C) staining of adipogenesis, chondrogenesis, and osteogenesis, respectively, after
All data presented above confirm that equine MSCs generated from each donor are progenitor cells, and they satisfy the criteria to be classified as the adult mesenchymal stromal/progenitor cells.
After ensuring selection of MSCs from low passage cells from the previous experiments, eBM-MSCs were further expanded, passaged, and tested for neural differentiation.
For easy visualization, fluorescence microscopy was used to show an intact nucleus and a “healthy” cytoplasm. TO-PRO-3 stain, the most sensitive probe for nucleic acid detection, along with WGA, specific to the cell membrane, was used to demonstrate the nucleus and the cytoplasmic structure of the neural-like cells. Low and high passage eBM-MSCs from the selected young and middle-aged horses were capable of adopting neural crest-like cell morphology as early as 3 h after chemical induction. These morphological characteristics consisted of elongation of the cell, cell body contraction, and formation of one or multiple cell processes (Figure
Transdifferentiation assay of eBM-MSCs. Representative images from one young (a) and one middle-aged (b) horse showing neural crest-like cells (white arrows) at 12 h after chemical induction. Note the contraction of the cellular body and the appearance of multiple cell processes. Magnification = 40x.
Nuclear/cytoplasmic staining of neural-like eBM-MSCs. Representative confocal images of cytoplasmic (WGA) and nuclear (TO-PRO-3-iodide) fluorescent stains showing the integrity of neural crest-like cells from low passaged eBM-MSCs of one young (a) and one middle-aged (b) horse and from high passaged eBM-MSC of middle-aged (c) horse. Note the typical fibroblast-like morphology of the undifferentiated MSCs (d, e, and f). Scale bar = 25
Finally, as judged by the numbers of cells adhered to the 2 different types of tissue culture plates, the nitrogen-coated plates (Primaria) significantly enhanced cell survival and proliferation roughly, 2-fold higher than the polystyrene-coated plates (
Nuclear/cytoplasmic staining of neural-like eBM-MSCs on different substrates. Representative confocal images of cytoplasmic (WGA) and nuclear (TO-PRO-3-iodide) fluorescent stains showing survival of differentiated cells from one middle-aged horse 12 h after chemical induction, on Primaria, nitrogen-coated (a) and polystyrene-coated (b) tissue culture plates. Note scarce cells on the polystyrene tissue culture plate. Scale bar = 100
The expression of vimentin was confirmed by IF (Figures
Expression of vimentin in undifferentiated and differentiated low passaged eBM-MSCs. Confocal microscopy shows the expression of vimentin (red) in neural crest-like cells 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). Respective undifferentiated controls are in the lower panel. Note the diffuse localization of vimentin, which is expressed in undifferentiated and in neural progenitor cells. Scale bar = 25
Expression of vimentin in undifferentiated and differentiated high passaged eBM-MSCs. Confocal microscopy shows the expression of vimentin (red) in neural crest-like cells from eBM-MSCs of middle-aged horse after 12 h of chemical induction. DAPI was used to stain the nuclei (blue). Undifferentiated control eBM-MSCs are in the right panel. Note the diffuse localization of vimentin, which is expressed in undifferentiated and in neural progenitor cells. Scale bar = 25
Immunoblot analysis was carried out to assess the expression of GFAP and
Expression of neural progenitor proteins by immunoblot analysis. Western blot analysis shows the expression of the neural progenitor proteins,
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
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
Expression of nestin in undifferentiated and differentiated high passaged eBM-MSCs. Confocal microscopy shows the expression of nestin (red) in neural crest-like cells (left panel) and in undifferentiated controls (right panel) from eBM-MSCs of middle-aged horse after 12 h of chemical induction. DAPI was used to stain the nuclei (blue). Note that nestin is inconsistently found perinuclearly or diffusely in the cytoplasm in the differentiated cells. Expression of nestin in the control is perinuclear. Additionally, based on the intensity of the signal, its expression appears less when compared to cells from the low passage. Scale bar = 25
Peripheral nerve injuries are a cause of poor performance in horses. These injuries are difficult to manage and treatment mostly relies on physical therapy and anti-inflammatories; however, the long-term effects are time and personnel consuming. The development of nervous system cells is divided into various stages. After determination of their fate, these cells migrate to specific locations of the nervous system and accomplish different functions [
Adipose-derived and bone marrow-derived MSCs from humans and rats are able to differentiate into neural lineages [
We induced trilineage differentiation (osteogenic, chondrogenic, and adipogenic) on bone marrow-derived MSCs from young and middle-aged horses to characterize their plasticity into mesodermal lineages. We also induced these cells to differentiate into cells of neural lineage to assess their plasticity outside of mesodermal lineages. Cells from all horses were capable of proliferation and underwent differentiation into adipogenic, osteogenic, and chondrogenic lineages. This was consistent with a previous report published from our laboratory [
By the combination of western blot and IF analyses, we found that all MSCs, undifferentiated and differentiated, expressed neural progenitor markers; namely, vimentin, nestin, GFAP, and
Our study mainly relies on morphological changes and neural marker protein expression to describe the events occurring during neural differentiation of eBM-MSCs. Our results agree with previous
The authors declare that they have no competing interests. A research report of a part of this work was presented at the 2014 ACVIM Forum, Nashville, TN.