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| ORIGINAL ARTICLE |
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| Year : 2020 | Volume
: 4
| Issue : 1 | Page : 7-11 |
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Induction of cardiomyogenesis in stem cells isolated from human exfoliated deciduous teeth
Herman S Cheung
Department of Biomedical Engineering; Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida, USA
| Date of Submission | 12-Oct-2019 |
| Date of Acceptance | 23-Nov-2019 |
| Date of Web Publication | 03-Mar-2020 |
Correspondence Address:
Prof. Herman S Cheung
Department of Biomedical Engineering, University of Miami, 1251 Memorial Dr. Miami, FL 33146
USA
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/hm.hm_68_19
Background: The natural transition from neonatal deciduous teeth to adult permanent teeth is a physiological phenomenon. Miura et al. reported the isolation and characterization of stem cells from the pulp of human exfoliated deciduous teeth (SHED). The great majority of current stem cell therapies use human adult stem cells. Since adult stem cells are multi-potent. SHED have demonstrated to have the capability to differentiate into osteogenic and odontogenic cells, adipocytes, and neural cells. When transplanted in immunocompromised mice, SHED were able to form bone. In addition, SHED have been able to differentiate into functional odontoblast and angiogenic endothelial cells. Aims and Objective: There are two objectives in our study; First, we want to confirm that SHED cells differentiate into osteogenic and neurotic cells. Second, we shall also examine whether SHED stem cells can also differentiate into cardiomyocytes. Material and Method: SHED stem cells are subjected to cardiomyogenic, neurogenic, and osteogenic induction treatment. The treated cells are the subjected to real time PCR and mmuno-histochemical analysis. The presence of calcium deposits with Alizarin Red S staining for SHED cells treated with osteogenic media was used to confirm osteogenic differentiation. Results: Our study confirmed that SHED cells differentiate into osteogenic and neurotic cells. For the first time we showed that SHED cells can also differentiate into cardiomyocytes. Conclusion: Due to their potentials and its neural crest origin, SHED are an ideal stem cell source for tissue regeneration.
Keywords: Cardiomyogenesis, regenerative potential, SHED stem cells
How to cite this article:
Cheung HS. Induction of cardiomyogenesis in stem cells isolated from human exfoliated deciduous teeth. Heart Mind 2020;4:7-11 |
| Introduction | |  |
Stem cells have shown to be a great promise to serve as therapeutic agents in the regeneration of tissues damaged by disease or injury. Stem cells are undifferentiated tissue progenitor cells that can proliferate, self–renew, and form one or more differentiated cell types.[1] In accordance with their developmental potential, stem cells are categorized as embryonic stem cells (pluripotent) and adult stem cells (multipotent and unipotent).[1],[2] Embryonic stem cells are pluripotent cells found in the blastocyte. These cells are capable of differentiating into three germ layers: endoderm, ectoderm, and mesoderm. Multipotent stem cells can be found in cell niches of adult tissue and organs; however, they produce a limited range of differentiated cells. Therefore, their differentiation potential is limited. Unipotent stem cells have a limited proliferative potential and can differentiate into only one specific cell lineage.[1],[2]
The neural crest is a transient structure present during embryonic development. Neural crest cells arise in the neural folds of the forming neural tube. While the neural tube generates the spinal cord, neural crest cells sustain an epithelial-to-mesenchymal transformation, become migratory, and translocate to different regions of the embryo.[3],[4] Overall, neural crest cells contribute to the craniofacial skeleton, the cornea, thyroid and thymus, septal of the heart, the adrenal gland, and pigment cells in the skin, sensory nerves, autonomic nerves, Schwann cells, and teeth.[5],[6],[7],[8]
The natural transition from the baby teeth to the adult permanent teeth is a physiological phenomenon. Since every child has 20 deciduous teeth, collecting them offers a noninvasive and ethically free method to isolate stem cells.[9] SHED in 2003, stem cells were isolated and characterized from the pulp of human exfoliated deciduous teeth.[10]
SHED have demonstrated to have the capability to differentiate into osteogenic and odontogenic cells,[10] adipocytes, and neural cells.[11],[12] When transplanted in immunocompromised mice, SHED were able to form bone.[13],[14] In addition, SHED have been able to differentiate into functional odontoblast,[10] angiogenic endothelial cells,[13] and neuronlike cells.[12] Due to their potentials and its neural crest origin, SHED are an ideal stem cell source for tissue regeneration. The aim of the present study is to demonstrate that SHED can differentiate into cardiomyocytes, osteocytes, and neural cells.
| Method and Procedures | | |
Culture and subculture of cells
SHED cells were a gift provided by the National Institute of Dental and Craniofacial Research (NIDCR, Bethesda, MD, USA) with the institution ethics approval before shipping. The University of Miami Human Use Committee also approved the use of SHED in this study. They were isolated using the technique described by Miura et al.[9] Cells are maintained in culture media that consists of high-glucose Dulbecco's Modified Eagle's medium (HG-DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B at 37°C in 10% CO2(HG-DMEM).
SHED control was cultured in control HG-DMEM. For cardiomyogenic, neurogenic, and osteogenic induction, SHED were seeded at a density of 300 cells/cm2 on 0.1% gelatin-coated six-well plates and 12-well plates and were treated with HG-DMEM pluses the differentiation supplement, as outlined below.
Cardiogenic differentiation
Cardio-induced cells were treated with standard tissue cardiomyogenic media consisting of HG-DMEM and 2%FBS and supplemented with the following growth factors: transforming growth factor β3 (TGFβ3), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor, fibroblast growth factor 4 (FGF4), Dickopff-related protein 1 (DKK1), cardiogenol-C, and oxytocin. Media changes were performed every 2–3 days for a period of 10 days. At the end of the treatment period, cells were fixed and analyzed for their immunohistochemical expression. In addition, RNA was collected from the cultures in the six-well plates using Trizol reagent to analyze cardiomyogenic gene expression using real-time quantitative polymerase chain reaction (qPCR) techniques.[15]
Neurogenic differentiation
Neuro-induced SHED cells were treated with neurogenic media. Neurogenic media was prepared using the following growth factors: insulin-like growth factor 1, activin A, DKK1, dimethyl sulfoxide, β-mercaptoethanol, neurotrophin-3, epidermal growth factor, and basic FGF. Media changes were performed every 2–3 days for a period of 10 days. At the end of the treatment period, cells were fixed and analyzed for their immunohistochemical expression. In addition, RNA was collected from the cells cultured in the six-well plates using Trizol reagent to analyze neurogenic gene expression using real-time qPCR technique.[14]
Osteogenic differentiation
Osteo-induced cells were treated with osteogenic media. Osteogenic media was prepared using HG-DMEM and 2% FBS and supplemented with the following growth factors: BMP2, -TGFβ, β-glycerphosphate, dexamethasone, and ascorbic acid. Media changes were performed every 2–3 days for a period of 10 days. At the end of the treatment period, cells were fixed and analyzed using mineralization assays. In addition, RNA was collected from the cultures in the six-well plates using Trizol reagent to analyze osteogenic gene expression using real-time qPCR techniques.[16]
Immunohistochemistry analysis
For immunohistochemical analysis, cells were first rinsed with phosphate-buffered saline (PBS) two times at room temperature followed by fixation in cold methanol or 10% formaldehyde for 10 min at room temperature. Samples were then washed three times in PBS.
Further incubation in 0.1% Triton X-100 for 10 min was performed for nuclear marker staining. All samples were blocked using blocking buffer (PBS-T, 1% FBS, and 2% BSA) for 1 h at room temperature. After blocking, the samples were incubated in a primary antibody overnight at room temperature. After overnight incubation with the primary antibodies, the samples were washed twice in PBS and were then incubated with the appropriate secondary antibodies at a 1:200 dilution for 2 h.[15]
RNA isolation, reverse transcription, and polymerase chain reactions
RNA isolation and real-time PCR (RT PCR) protocol were done according to the protocol that we published earlier.[15] Briefly, total RNA was isolated by means of Trizol reagents following the manufacturers' recommended protocol and resuspended in RNase- and DNase-free water. The RNA suspension was then frozen at −80°C overnight and quantified using a NanoDrop® spectrophotometer (Nanodrop Products, Wilmington DE). Reverse transcription of RNA to cDNA was performed by means of the High-capacity cDNA Reverse Transcription Kit (Applied Biosystems) as per the manufacturers' recommended protocol.
RT PCR was carried out using the TaqMan® Fast universal PCR Master Mix (Applied Biosystems) and TaqMan® probe predesigned primers (electronic supplementary material) for all the human genes explored along with 20 ng of cDNA for each sample. The reactions were performed using the Applied Biosystems StepOne plus RT PCR system. Each reaction (n = 4) was run in triplicate for each assay, and gene expression quantification was carried out by means of the comparative Ct method. Data were normalized to GAPDH, and the significance over the static controls is presented in the analysis.[15]
Statistical analysis
All numerical values presented in this study reflect the mean–standard deviation. Statistical analyses were performed by means of two-tailed student t-tests, and statistical significance was determined by any statistical test returning at P < 0.05.
| Results | | |
[Figure 1] illustrates where SHED cells derived from the dental pulp. [Figure 2] represents the microscopic examination of differentiation of SHED cells into osteocytes/chondrocytes, neural cells, and cardiomyocytes. | Figure 2: Differentiation of stem cells from the pulp of human exfoliated deciduous teeth cells into osteocytes/chondrocytes, neural and cardiomyocytes osteocytes/chondrocytes neural cells cardiomyocytes
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Immuno-histochemical analysis of cardiomyogenic gene markers in SHED cells treated with cardiomyogenic media. [Figure 3]a illustrates the expression of MEF (green) and nucleic acids (blue), [Figure 3]b illustrates the expression of GATA4 (red) and nucleic acids (blue) [Figure 3]c illustrates the expression of Nkx 2-5 (green) and nucleic acids (blue), and [Figure 3]d illustrates the expression of cardiac troponin (green) and nucleic acids (blue). | Figure 3:(a-d) Immunohistochemical analysis of cardiomyogenic gene markers in stem cells from the pulp of human exfoliated deciduous teeth cells treated with cardiomyogenic media. (a) Expression of MEF (green) and nucleic acids (blue) (b) Expression of GATA4 (red) and nucleic acids (blue). (c) Expression of Nk × 2-5 (green) and nucleic acids (blue). (d) Expression of cardiac troponin (green) and nucleic acids (blue)
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Results from immunohistochemistry for glial fibrillary acidic protein and TUBB3 analysis. [Figure 4]a illustrates the expression of GFAP (red) and nucleic acids (blue) in SHED cells treated with neurogenic media and [Figure 4]b illustrates the expression of tubulin3 (green) and nucleic acids (blue) in SHED populations treated with neurogenic media. | Figure 4: Immunohistochemical analysis of neurogenic gene markers. (a) Expression of glial fibrillary acidic protein (red) and nucleic acids (blue) in stem cells from the pulp of human exfoliated deciduous teeth cells treated with neurogenic media. (b) Expression of TUBB3 (green) and nucleic acids (blue) in stem cells from the pulp of human exfoliated deciduous teeth populations treated with neurogenic media
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The initiation of osteogenic differentiation was evident by the presence of calcium deposits with Alizarin Red S staining for SHED cells treated with osteogenic media for a 10-day period [Figure 5]. Moreover, the statistically significant increase of the gene RUNX2 (P < 0.05) confirmed the precommitment into the osteolineage [Figure 6]. | Figure 5: Alizarin red staining for stem cells from the pulp of human exfoliated deciduous teeth cells treated with osteogenic media for a 10-day period
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 | Figure 6: Expression of RUNX2 for stem cells from the pulp of human exfoliated deciduous teeth treated with control and osteogenic media for a 10-day period. Genes were quantified and normalized against GAPDH. Data are represented as the mean + standard error of the mean, n = 3 (significance = P < 0.05)
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| Discussion | | |
Since SHED cells are derived from a neural crest stem cell (NCSC), SHED stem cells' population is heterogeneous. Mesenchymal stem cells make up the majority population with a small population of residual NSCS. Our data confirmed the previous studies that SHED cells can differentiate into neurogenic cells and osteogenic.[10],[12],[13] Our qPCR and immunohistochemical results demonstrate that the neural induction treatment drove the SHED population into a neuronal lineage. From the immunohistochemistry analysis, the presence of BTUB3 is strongly marked. It can be observed neuronal processes that are branching and connecting to other cells. In addition, the gene expression for NEFM (mature neuronal marker)[14] is increased, and the TUBB3 (immature neuronal marker)[12],[13],[14] expression is decreased.
We also demonstrated that SHED population differentiated into the osteogenic lineage. The Alizarin Red S staining confirmed the presence of extracellular calcium deposits, indicating that the treatment drove the stem cells into an osteogenic lineage. Moreover, there is a statistically significant increase in the gene expression of RUNX2 (P < 0.05). The actions and regulation of the Runx 2 transcript factor involve the ability to facilitate the junction of numerous osteogenic signaling pathways.[17] This central control gene was activated at a higher level than in the nonselected population. Our present study confirmed that the heterogeneous population of SHED cells can differentiate into the osteogenic lineage.[10],[11],[12]
We showed for the first time that SHED cells can differentiate into the cardiomyogenic lineage. Immunohistochemistry analysis confirmed the presence of early markers in the cardiomyocyte differentiation. The markers identified include Nkx 2-5, GATA4, and MEF. Nkx 2.5 associates with members of the GATA family transcription factors (GATA4/5/6) turn on cardiac structural genes, such as actin, myosin light chain, myosin heavy chain, troponins, and desmin.[18],[19] Moreover, the MEF transcription factor also plays an important role in the cardiomyocyte differentiation by regulating cardiac muscle structural genes.[18],[19] MEF, NKx 2.5, and GATA4 were localized in the cell nuclei at a higher expression than in the cardio-treated nonselected cells. Furthermore, the cardiac-specific protein cardiac troponin T was expressed in the SHED.
In summary, our data demonstrated that the multipotent potential of SHED cells to differentiate into cells derived from both the ectodermal and mesodermal germ layers.[10],[11],[12] Recent studies have shown the potential of SHED to differentiate into the pancreatic cell lineage,[20],[21] which is derived from the endodermal germ layer. This may be explained by the fact that SHED stem cells also contain a small residual population of NCSC, which is pluripotent stem cells. It would be an interesting future study to isolate these pluripotent NCSC cells using our Connex-43 + marker technology to confirm the finding.[15] Due to their potentials and its neural crest origin, SHED are an ideal stem cell source for tissue regeneration.
Financial support and sponsorship
The U.S. Department of Veteran Affairs Senior Research Career Scientist Award and Merit Review Grant supported our work.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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