Mesenchymal stromal cells derived from exfoliated deciduous teeth express neuronal markers before differentiation induction

Abstract Objective: This study aimed to evaluate neuronal markers in stromal cells from human exfoliated deciduous teeth (SHED) and standardize the isolation and characterization of those cells. Methodology: Healthy primary teeth were collected from children. The cells were isolated by enzymatic digestion with collagenase. By following the International Society for Cell and Gene Therapy (ISCT) guidelines, SHED were characterized by flow cytometry and differentiated into osteogenic, adipogenic, and chondrogenic lineages. Colony-forming unit-fibroblasts (CFU-F) were performed to assess these cells’ potential and efficiency. To clarify the neuronal potential of SHED, the expression of nestin and βIII-tubulin were examined by immunofluorescence and SOX1, SOX2, GFAP, and doublecortin (DCX), nestin, CD56, and CD146 by flow cytometry. Results: SHED showed mesenchymal stromal cells characteristics, such as adhesion to plastic, positive immunophenotypic profile for CD29, CD44, CD73, CD90, CD105, and CD166 markers, reduced expression for CD14, CD19, CD34, CD45, HLA-DR, and differentiation in three lineages confirmed by staining and gene expression for adipogenic differentiation. The average efficiency of colony formation was 16.69%. SHED expressed the neuronal markers nestin and βIII-tubulin; the fluorescent signal intensity was significantly higher in βIII-tubulin (p<0.0001) compared to nestin. Moreover, SHED expressed DCX, GFAP, nestin, SOX1, SOX2, CD56, CD146, and CD271. Therefore, SHED had a potential for neuronal lineage even without induction with culture medium and specific factors. Conclusion: SHEDs may be a new therapeutic strategy for regenerating and repairing neuronal cells and tissues.

Storing MSC, especially those derived from dental tissues, in cell banks under good manufacturing practices conditions allows the use of these samples for future benefit in an autologous or even allogeneic application. 11 Different stromal cell banks specialize in different stromal cell sources, including MSC derived from the bone marrow, umbilical cord, cord blood, adipose tissue, and SHED. 12,13 Collecting and storing SHED can provide advantages compared to cord blood stromal cells: the deciduous teeth is safe to the donor, would usually be discarded, and is more affordable, meaning less than one-third of the cost to store cord blood. As well as MSCs from other sources, SHED can regenerate into solid tissues such as connective, dental, and bone tissue, and demonstrate a remarkable ability to differentiate into neural tissues. 14,15 The possibility of evaluating neuronal markers in SHED before differentiation induction and standardizing the isolation and characterization of those cells would help to develop future strategies for regenerating and repairing neuronal cells/tissues in neurodegenerative diseases.

Methodology
Isolation and cultivation of SHED
The medium was changed on the fifth day of culture.

Stainings
The SHED induced to osteogenic differentiation was fixed to evaluate the calcium crystals, and the Alizarin Red S (Sigma-Aldrich, USA) staining was performed.
The SHED-induced adipogenic differentiation was

Statistical analyses
The data were processed, and statistical significance was estimated using GraphPad Prism software version  Figure 1B). However, the gene expression analyses show that the SHED expressed different adipogenic genes before and after the adipogenic differentiation ( Figure 1D).  Neuronal markers are expressed in the SHED without neuronal induction To clarify the neuronal potential of SHED, we examined the expression of nestin and βIII-tubulin.

The intensity of both markers was measured and
quantified. Regarding neuronal markers, the βIIItubulin, which is specific neuron tubulin, we observed a higher expression when comparing nestin with a significant difference (p<0.0001) ( Figure 3A and Figure 3B). SHED also had a positive expression (>90%) of SOX1, GFAP, nestin, and expression of CD56, CD146, CD271, SOX2, and DCX (<90%) ( Figure   3C). Therefore, SHED had a potential for the neuronal lineage even without induction with culture medium and specific factors.

SHED isolation efficiency was 100% and we
observed no contamination during cell cultivation. Alansary,et al. 19 (2020)  variables, more studies on CD105 must be carried out to understand better which variable acts in the expression. Other studies that used stromal cells from deciduous tooth pulp also found the positive expression of the MSC markers CD29, CD44, and CD166, confirming the similarity to MSC derived from other tissues. 22,23 In this study, all negative markers showed low expression setting up by Dominici, et al. 2 (2006). On the other hand, the other studies cited did not follow the ISCT guidelines (less than 2%). Alansary,et al. 19 (2020)  The adipogenic potential of MSC derived from ectoderm origin has some controversies, encouraging us to find how SHED responds to adipogenic stimulus. Consequently, the expression of CD56 in SHED indicates a great potential to induce these cells to neuronal differentiation, since dental pulp shares a common origin, the neural crest, with neuronal cells.
The CD146 membrane marker is a cell adhesion molecule and its expression is related to the activation of intracellular signaling pathways in the brain. 35 The expression of CD146 in SHED could be related to the neural crest cell migration during embryonic development. Ma, et al. 33 (2021) suggest that CD146 may be related to the quality of SHED. According to these authors, the higher expression of CD146 is Other markers were used to trace the profile of neural precursor cells, such as SOX1, SOX2, GFAP, and DCX. This study shows the expression of these neuronal markers in SHED. The expression of SOX1 in SHED was never described. SHED expressed this marker because SOX1 acts mainly in neurogenesis and is expressed in neural precursor cells. The expression of the SOX2 marker, also known as the pluripotency marker, has already been reported in SHED. 19,28 SHED expressed astrocytes (GFAP) and neuronal intracellular microtubule proteins such as DCX, which may be related to the SHED neural crest's origin, giving a greater differentiation ability in neuronal cells. 43 The DCX expression gives SHED a good advantage compared to bone marrow MSC, which has the mesodermal germ, which does not express DCX. 44

Conclusion
Understanding and elucidating the potential of SHED to express neuronal markers before inducing neuronal differentiation is essential to develop simpler treatment protocols and to transfer them from bench to bedside. Among the advantages of using this source of mesenchymal stromal cells is the accessible collection, which is a non-invasive source with ethical J Appl Oral Sci. 2023;31:e20220489 9/10 criteria that follows the criteria suggested by ISCT.
The storage of different MSCs sources in cell banks is suggested since each source has a different potential. The MSCs are essential in advancing regenerative medicine to treat diseases. Therefore, stromal cell banks can accelerate the safe and efficient use of these cells. SHEDs have enormous potential since they have the same embryonic origin as neurons and express neuronal markers even in the native stage. These characteristics make them an important source for future use in treating neurodegenerative diseases.