Multiple sclerosis (MS) is the most prevalent and progressive autoimmune disease that affects the central nervous system, and currently, no drug is available for the treatment. Stem cell therapy has received substantial attention in MS treatment. Recently, we demonstrated the immunosuppressive effects of mesenchymal stem cells derived from neural crest-originated human periodontal ligament tissue (hPDLSCs) in an
Multiple sclerosis (MS) is a chronic debilitating neuroinflammatory disease, which resulted from the activation of immune response against self-antigens residing in the central nervous system (CNS). Activated immune cell infiltration in the brain and spinal cord, degenerated myelin sheath, and severe axonal damage are the typical pathological signatures of MS, which eventually cause severe neurological disabilities [
In recent years, neural crest-originated nonhematopoietic MSCs derived from human dental tissues have attained substantial attention in the field of regenerative medicine for dental and nondental diseases [
In the present study, we investigated whether human PDLSCs (hPDLSCs) could serve as an effective autologous tool for RR-MS patients. To this end, we studied the stemness characteristics of hPDLSCs derived from RR-MS patients in comparison to those of healthy subjects. Cell surface antigen expression, cell proliferation rate, and differentiation capacity were examined. In addition, we investigated the putative modulation of stem cell properties after prolonged
To perform this study, the authors obtained an approval statement from the Ethics Committee at the Medical School, Università degli Studi G. d’Annunzio Chieti-Pescara, Italy (n°266/17.04.14). Informed consent was signed by all patients before sample collection.
Human PDLSCs were isolated from periodontal tissues of healthy donors (
Fluorescein isothiocyanate- (FITC-) conjugated CD14, phycoerythrin- (PE-) conjugated CD29, CD31-FITC, peridinin chlorophyll protein- (PerCP-) cyanine (Cy)5.5-conjugated CD34, CD44-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, CD166-FITC, CD326-PerCP-Cy5.5, Alexa488-conjugated human leukocyte antigen- (HLA-) ABC, and HLA DR-PE were used (BD Biosciences, Franklin Lakes, NJ;
Samples were stained for surface or intracellular antigens, as previously described by Rajan et al. [
Quality control included regular check-up with Rainbow Calibration Particles (6 peaks) and CaliBRITE beads (both from Becton Dickinson). Debris was excluded from the analysis by gating on morphological parameters; 20,000 nondebris events in the morphological gate were recorded for each sample. All antibodies were titrated under assay conditions, and optimal photomultiplier (PMT) gains were established for each channel.
MTT assay and trypan blue exclusion test were deployed to evaluate cell proliferation and viability at different time points (24 h, 48 h, 72 h, and 1 week) as previously described by Rajan et al. [
Human PDLSCs and MS-hPDLSCs at P2 and P15 were subjected to mesengenic differentiation as reported by Rajan et al. [
For neurogenic differentiation, hPDLSCs and MS-hPDLSCs were plated in 24-well plates and were induced with Neurobasal-A Medium (Gibco®) containing B27 (2%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin B (5 mg/ml) (neuroinductive medium) and supplemented with basic fibroblast growth factor (bFGF, 20 ng/ml) (Tema Ricerca, Milan, Italy) for 10 days [
Cells were stained using a senescence detection kit (Ab65351, Abcam, Cambridge, UK) as per the manufacturer’s instructions. The percentage of positively stained cells (blue cells) versus total cells was calculated by randomly choosing 10 microscopic fields under 10x objective magnification at light microscopy (Leica DMIL, Leica Microsystem).
hPDLSCs and MS-hPDLSCs were processed as previously reported by Trubiani et al. [
Total RNA was extracted from hPDLSCs and MS-hPDLSCs at P2 and P15 using the RNeasy Mini Kit (Quiagen, Hilden, Germany). 2
Data were analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Statistical analyses were performed with one-way ANOVA test, followed by a Bonferroni post hoc test for multiple comparisons. A
Immunophenotypic characterization displayed analogous expression of MSC-associated cell surface markers in hPDLSCs obtained from RR-MS patients and healthy subjects. P2 and P15 hPDLSCs of both healthy donors and RR-MS patients revealed similar phenotype results. Expression of surface molecules such as CD29, CD44, CD73, CD90, CD105, CD166, and human leukocyte antigen- (HLA-) ABC was positive in both donor- and RR-MS patient-derived hPDLSCs at P2 and P15 passages (Figure
Flow cytometry phenotype of hPDLSCs and MS-hPDLSCs at P2 and P15. (a) Surface antigen expression profile for CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC. (b) Intracellular positive marker expression profile for NANOG, SOX2, Oct3/4 and SSEA4. Red histograms show the distribution of each antigen expression, and blue histograms represent the distribution of the respective background control.
Cell proliferation and viability abilities were determined using 3-(4,5-dimethyl-thiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay and Trypan blue exclusion test. Proliferation rate of hPDLSCs and MS-hPDLSCs was detected at 24, 48, and 72 h and 1 wk of culture. We noticed that both P2 and P15 cells showed a similar gradual increase in their proliferation rates over a one-week incubation period, regardless of whether they were obtained from healthy donors or RR-MS patients (Figures
Cell proliferation and viability abilities. Cell proliferation and viability of P2 and P15 hPDLSCs at different time points were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay and trypan blue exclusion test. Histograms show MTT assay and trypan blue assay data in P12 and P15 of normal hPDLSCs ((a) and (c), resp.) and MS-hPDLSCs ((b) and (d), resp.).Values are expressed as mean ± S.D.
Then, we have investigated the mesengenic differentiation capacities of hPDLSCs. Alizarin red S staining showed positive staining for osteogenic differentiation under osteogenesis-inducing culture conditions. P2 and P15 hPDLSCs of donors (Figure
Osteogenic differentiation potential of hPDLSCs collected from healthy donors and MS patients. (a) Photomicrographs of hPDLSCs at P2 (A1) and P15 (B1) under standard conditions and hPDLSCs at P2 (A2) and P15 (B2) at the end of osteogenic commitment. (c) MS-hPDLSCs at P2 (C1) and P15 (D1) under standard conditions and MS-hPDLSCs at P2 (C2) and P15 (D2) at the end of osteogenic differentiation. Differentiation potential was assessed by the formation of calcium-rich hydroxyapatite detected with Alizarin Red staining. Mag: 10x. Bar: 10
Oil Red O staining showed positive staining for adipogenic differentiation under adipogenesis-inducing culture conditions. P2 and P15 hPDLSCs of donors (Figure
Adipogenic differentiation potential of hPDLSCs collected from healthy donors and from MS patients. (a) Photomicrographs of hPDLSCs at P2 (A1) and P15 (B1) under standard conditions and hPDLSCs at P2 (A2) and P15 (B2) at the end of adipogenic commitment. (c) MS-hPDLSCs at P2 (C1) and P15 (D1) under standard conditions and MS-hPDLSCs at P2 (C2) and P15 (D2) at the end of adipogenic differentiation. Differentiation potential was evaluated by the formation of cytoplasmic lipid droplets and vacuoles (appear as cherry red spheres) detected with Oil Red O staining. Mag: 10x. Bar: 10
Alcian blue staining displayed positive staining for chondrogenic differentiation under chondrogenesis culture conditions. P2 and P15 hPDLSCs of donors (Figure
Chondrogenic differentiation potential of hPDLSCs collected from healthy donors and from MS patients. (a) Photomicrographs of hPDLSCs at P2 (A1) and P15 (B1) under standard conditions and hPDLSCs at P2 (A2) and P15 (B2) at the end of chondrogenic commitment. (c) MS-hPDLSCs at P2 (C1) and P15 (D1) under standard conditions and MS-hPDLSCs at P2 (C2) and P15 (D2) at the end of chondrogenic differentiation. Differentiation potential was evaluated by the nodule formation and the positivity to alcian blue staining. Mag: 10x. Bar: 10
In addition to the mesengenic differentiation capacity, we have studied the neurogenic differentiation capacity of hPDLSCs. Under neurogenesis induction, hPDLSCs displayed neuron-like morphological changes. Both P2 and P15 hPDLSCs of donors (Figure
Neurogenic differentiation potential of hPDLSCs collected from healthy donors and from MS patients. (a) hPDLSCs at P2 (A1) and P15 (B1) and neurogenically induced hPDLSCs at P2 (A2) and P15 (B2) passages positive to
As prolonged passages result in senescence, we have assessed whether hPDLSCs would undergo senescence after extended passages. Senescence-associated X-gal staining showed a mild positive staining for P2 hPDLSCs of donors (Figure
Cellular senescence assessment. hPDLSCs (a) and MS-hPDLSCs (c) at P2 showed a basal staining for X-gal blue solution, while significant positive staining was observed in hPDLSCs (b) and MS-hPDLSCs (d) at P15. Mag:10x. Bar: 10
Senescence markers p16 and p21 expression. Immunofluorescence staining of p16 in P2 and P15 hPDLSCs ((a) and (b), resp.) and p21 in P2 and P15 hPDLSCs ((d) and (e), resp.). Histograms show relative fold changes of qRT-PCR for p16 (c) and p21 (f) in hPDLSCs. Immunofluorescence staining of p16 in P2 and P15 MS-hPDLSCs ((g) and (h), resp.) and p21 in P2 and P15 MS-hPDLSCs ((j) and (k), resp.). Histograms show relative fold changes of qRT-PCR for p16 (i) and p21 (l) in MS-hPDLSCs. Green, red, and blue fluorescence was applied to stain actin cytoskeleton, p16 or p21, and nuclei, respectively. Mag: 63x. Bar: 20
Lastly, we investigated if hPDLSCs and MS-hPDLSCs could display stemness properties including pluripotency and self-renewal abilities after extended passages. Transcriptional regulatory network analysis revealed differential modulation of expression of genes linked with stemness characteristics between early and late passages in both hPDLSCs and MS-hPDLSCs (Figures
Modulation of genes associated with stemness characteristics at late passage. Histograms show relative expression of mRNA transcripts associated with pluripotency, self-renewal, cell proliferation, and differentiation in hPDLSCs (a) and MS-hPDLSCs (b) at P15. Expression levels of transcripts for P2 and P15 are shown in red and green, respectively. DataAssist software was employed to run a global normalization analysis by using GAPDH, 18s, and HPRT1 as selected internal controls. The reported transcripts evidenced a
IPA network analysis. Peak scoring networks from NANOG, MEIS1, and SIX3, which regulate stemness properties, in P15 hPDLSCs (a) and P15 MS-hPDLSCs (b) are shown. Upregulated genes are shown in red. Downregulated genes are shown in green. Genes not existing in the LRG list but showed interaction with the LRG list are shown as white open nodes. A solid line depicts a direct functional interaction between the gene products. An arrow depicts functional target of the gene product. LRG: locus reference genomic sequence.
Results from clinical trials propose MSC-based treatment as a promising therapeutic tool for RR-MS patients [
In our study, we found that surface antigen markers CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC were positive in healthy donor-derived P2 and P15 hPDLSCs. Interestingly, similar expression of these surface antigens was also found in MS patient-derived P2 and P15 hPDLSCs. These results suggest that MS-linked cellular and biochemical changes did not modulate the stemness properties of hPDLSCs obtained from MS patients at both early and late passages. Moreover, we evaluated cell proliferation and viability abilities. MTT assay and Trypan blue staining assay showed similar cell proliferation and viability capacities in P2 hPDLSCs derived from both MS patients and healthy subjects. Interestingly, we found no defects in the cell viability and cell proliferation abilities of P15 hPDLSCs as well.
Next, we evaluated the differentiation capacity of hPDLSCs towards mesengenic and neurogenic cell lineages. Both MS patients and healthy donor-derived P2 hPDLSCs displayed significant differentiation abilities into osteogenic, adipogenic, chondrogenic, and neurogenic lineages, suggesting stemness characteristics of MS patient-derived hPDLSCs are independent of MS pathology. Previous studies on stemness characteristics of MSCs from patients with inflammatory diseases revealed mixed findings. For example, MSCs obtained from patients with other autoimmune diseases such as rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus showed considerable impairments in proliferation and differentiation properties [
In our study, we studied the senescence property of hPDLSCs. We noticed a moderate expression of senescence-associated
Lastly, we investigated whether prolonged subculture of hPDLSCs and MS-hPDLSCs may affect their stemness properties. We found that genes associated with essential stemness characteristics such as pluripotency, self-renewal, cell proliferation, and differentiation were differentially modulated at late passage. For example, pluripotency and sell-renewal marker MEIS1 was upregulated, while NANOG expression was downregulated at P15 in both normal hPDLSCs and MS-hPDLSCs. Cell proliferation marker STAT3 was upregulated, while another proliferation marker RIF1 was downregulated. SIX3, a neuroectodermal marker, was upregulated during extended passages, suggesting the putative spontaneous differentiation capacity towards neuronal precursor cell lineages. These data suggest that prolonged passages of hPDLSCs may produce modifications in the expression of genes associated with pluripotency, proliferation, and differentiation. Interestingly, we noticed that some gene transcripts were differentially modulated between normal hPDLSCSs and MS-hPDLSCs at late passage. Pluripotency-associated transcripts MYST3, REST, and SKIL were modestly increased in MS-hPDLSCs, while their expression was slightly decreased in normal hPDLSCs in P15. IPA analysis for transcriptional regulatory network suggested that, in addition to the common unidirectionally modulated pathways, some additional but distinct pathways were also activated in normal and in MS-hPDLSCs at extended passages. Given the similar expression of surface antigens and pluripotency markers and no differences in cell proliferation and mesengenic/neurogenic differentiation abilities between normal hPDLSCs and MS-hPDLSCs in both early and late passages, we assume that the differential modulation of gene transcripts that we observed in our study might have resulted from the interaction of heterogeneous pathways which participated in the regulation and maintenance of stemness-related cellular functions.
In summary, our results demonstrated that hPDLSCs derived from RR-MS patients exert typical stemness characteristics similar to that of hPDLSCs derived from healthy subjects. Cell morphology, immunophenotypic, proliferation, and differentiation properties were analogous. hPDLSCs of extended passages showed senescence activation; however, no changes were noticed with respect to the stemness properties. We propose that hPDLSCs could be a simple and efficient alternative autologous source for customized stem cell therapy in MS patients.
The authors declare no conflict of interest.
Francesca Diomede and Thangavelu Soundara Rajan contributed equally to this work.
This work has been supported by ex 60% Funds of Università “G. d’Annunzio”, Chieti-Pescara, Italy and by current research funds 2016 of IRCCS Centro Neurolesi “Bonino-Pulejo” (Messina, Italy).
Modulation of genes associated with stemness characteristics at early and late passages. Histograms show relative expression of mRNA transcripts associated with pluripotency, self-renewal, cell proliferation and differentiation in hPDLSCs and MS-hPDLSCs at P2 (A) and P15 (B). Expression level of transcripts for hPDLSCs and MS-hPDLSCs are shown in green and violet, respectively. DataAssist software was employed to run a global normalization analysis by using GAPDH, 18s and HPRT1 as selected internal controls. The reported transcripts evidenced a