Name: Author Spotlight: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells
Uploaded: 2023-05-05T13:50:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Tissue Regeneration and Disease Modelling with Dental Pulp Stem Cells at JoVE.com

We acknowledge the funding support to AK from the Department of Health Research (DHR), ICMR, Govt. of India (DHR-NRI Grant # R.12015/01/2022-HR). SR has received funding from ICMR, Govt. of India (Grant # 2020-7593/SCR-BMS) and PS has received fellowship from CSIR, Govt. of India. We are also thankful to Ms. Sandhya Tokhi and Ms. Bhupinder Kaur for assistance in flow cytometry, and central sophisticated instrumentation core (CSIC) and PGIMER, Chandigarh for providing infrastructural support.

All the procedures described in the study have been approved by the Institute Ethics Committee (IEC# 9195/PG-12 ITRG/2571-72) of PGIMER, Chandigarh. All the cell culture related experiments need to be performed in a Class II biological safety cabinet (BSC) following aseptic technique. Dental pulp was obtained from healthy teeth of three (F/14, M/14, and M/20) patients undergoing third molar extractions for orthodontic reasons. Before the sample collection, written informed consent was obtained from the patient/guardian i...

Establishing the Primary Culture of Dental Pulp Stem Cells

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+dental+pulp+stem+cell+transplantation+in+a+rat+model+of+Alzheimer's+disease.">Therapeutic potential of dental pulp stem cell transplantation in a rat model of Alzheimer's disease.</a> Neural Regeneration Research. 16 (5), 893-898 (2021).</li><li>Kabir, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imperative+role+of+dental+pulp+stem+cells+in+regenerative+therapies:+a+systematic+review.">Imperative role of dental pulp stem cells in regenerative therapies: a systematic review.</a> Nigerian Journal of Surgery. 20 (1), 1-8 (2014).</li><li>Kumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cell+secretome+ameliorates+d-galactose+induced+accelerated+aging+in+rat+model.">Dental pulp stem cell secretome ameliorates d-galactose induced accelerated aging in rat model.</a> Cell Biochemistry and Function. 40 (5), 535-545 (2022).</li><li>Hirata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+therapeutic+benefits+of+factors+derived+from+dental+pulp+stem+cells+for+mouse+liver+fibrosis.">Multifaceted therapeutic benefits of factors derived from dental pulp stem cells for mouse liver fibrosis.</a> Stem Cells Translational Medicine. 5 (10), 1416-1424 (2016).</li><li>Fujii, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regeneration+by+human+dental+pulp+stem+cells+using+a+helioxanthin+derivative+and+cell-sheet+technology.">regeneration by human dental pulp stem cells using a helioxanthin derivative and cell-sheet technology.</a> Stem Cell Research &amp; Therapy. 9 (1), 24 (2018).</li><li>Ferrua, C. 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Stem cells have pinned the hopes of curing numerous diseases, owing to their plasticity, robustness, immunomodulatory properties, paracrine mechanisms, and homing efficiencies. Dental pulp tissue is considered the most potent and valuable source of stem cells, with eminent plasticity and a regenerative capability. Here, we demonstrate the isolation of DPSCs, utilizing the widely adopted explant culture method, in which the cells migrate from pieces of pulp tissue or explants to grow into a homogenous cell culture that mo...

Adult stem cells have transpired into a powerful therapeutic tool for cell-directed treatments and therapies due to their plasticity, paracrine mechanisms, and immunomodulatory properties1,2,3. The encouraging data from stem cell-based preclinical studies have inspired researchers to work for the bench to-bedside translation. The type of stem cells used for stem cell therapy plays a significant role in successful outcomes. In preclinical and clinical studies, the most widely reported source for mesenchymal stem cells (MSCs) remain bone marrow4,5. However, major drawbacks to&#160;using bone marrow-derived stem cells (BMSCs) include their rare population, highly invasive procedures for isolation, and their limited ability to expand. Therefore, alternative sources of MSCs are being explored. In this regard, dental tissues, with their ease of accessibility, enormous plasticity, high regenerative potential, and high proliferative ability, have now been deemed as a rich and potential alternative source of stem cells6,7,8,9,10.
Dental pulp stem cells (DPSCs) were the first type of dental stem cells to be isolated and characterized by Gronthos in 200011. DPSCs have grabbed the attention for tissue engineering applications because of their high proliferation rate, significant differentiation potential, ease of accessibility with effortless culturing, and, most importantly, their ability to be obtained from a discarded tooth without any ethical concern12.&#160;The limitations posed by other stem cell sources, such as BMSCs and adipose-derived stem cells (ADSCs), in their isolation and inadequate self-renewal capacities are circumvented by DPSCs13. Human DPSCs can be obtained from human primary teeth, permanent teeth, wisdom teeth, exfoliated deciduous teeth (SHEDs), and apical papillae. Moreover, DPSCs can also be isolated from supernumerary teeth, which are generally discarded14. DPSCs express neural crest-associated markers and have the potential to differentiate into neuronal cells both in vitro and in vivo15. In addition to their neurogenic potential, DPSCs can differentiate into other cell lineages, such as osteogenic, chondrogenic, adipogenic, hepatic, and myogenic, when given specific differentiation conditions13. Thus, these multipotent cells hold great potential for cell-based therapy and can be employed for the regeneration of various tissues. Studies have also reported the potential role of DPSCs in the reconstruction of the cornea16, repair of myocardial infarction17, and their potential therapeutic role in diseases like limb ischemia18, Alzheimer&#39;s 19, Parkinson&#39;s 20, and aging21. Therefore, dental tissue-derived stem cells can be used not only for dental regeneration, but also for the repair and regeneration of non-dental organs like eyes16, hearts17, livers22, bones23 etc.
There are two particular methods for the isolation of an MSC population from pulp tissue -&#160;enzymatic digestion and explant culture24,25. Successful establishment of primary cultures without any significant difference in the quantity and properties of DPSCs have been reported by both these methods26. In this study, we have focused on the isolation of DPSCs by the explant method27, since this method generates DPSCs without contamination of hematopoietic and endothelial cells, as compared to enzymatic digestion which can result in fibroblast contamination28.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>Costar</td>
			<td>3516</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>EZstain chondrocyte staining kit, HiMedia</td>
			<td>CCK029</td>
			<td></td>
		</tr>
		<tr>
			<td>alizarin red S stain</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-008</td>
			<td>Osteogenic stain</td>
		</tr>
		<tr>
			<td>Antibiotic cocktail</td>
			<td>Himedia</td>
			<td>A002-5X50ML</td>
			<td>To prevent culture contamination</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Himedia</td>
			<td>TC094-25G</td>
			<td>Chondrogenic induction</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>bFGF ( basic Fibroblast Growth Factor)</td>
			<td>Gibco</td>
			<td>PHG0024</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>CD 105</td>
			<td>BD-Pharmingen</td>
			<td>560839</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 35</td>
			<td>Biolegend</td>
			<td>343604</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 45</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 73</td>
			<td>Biolegend</td>
			<td>344016</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 90</td>
			<td>Biolegend</td>
			<td>328107</td>
			<td>Characterization</td>
		</tr>
		<tr>
			<td>cetyl pyridinium chloride (CPC)</td>
			<td>Sigma-Aldrich</td>
			<td>1104006</td>
			<td>For Alizarin Red extraction</td>
		</tr>
		<tr>
			<td>Dexamethasone 21-phosphate disodium</td>
			<td>Sigma-Aldrich</td>
			<td>D1159-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Himedia</td>
			<td>TS1006-5L</td>
			<td>For washing purpose</td>
		</tr>
		<tr>
			<td>EGF (Epidermal Growth Factor)</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>For hepatic and neural&#160; induction</td>
		</tr>
		<tr>
			<td>EVOS LED microscope</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For&#160; fluorescence imaging</td>
		</tr>
		<tr>
			<td>EZ stain Chondrocyte staining kit</td>
			<td>Himedia</td>
			<td>CCK029-1KT</td>
			<td>Chondro stain Kit</td>
		</tr>
		<tr>
			<td>FACS Canto flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>For cell characterization</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For primary culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>G5 supplement</td>
			<td>Gibco</td>
			<td>17503012</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>HGF( Hepatocyte Growth Factor)</td>
			<td>Sigma-Aldrich</td>
			<td>H1404</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>Biolegend</td>
			<td>307605</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Human TGF-&#946;3</td>
			<td>Peprotech</td>
			<td>#100-36E-10U</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenous acid premix</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>ITS premix</td>
			<td>Corning</td>
			<td>354350</td>
			<td></td>
		</tr>
		<tr>
			<td>LDL Uptake Assay kit</td>
			<td>Abcam</td>
			<td>ab133127</td>
			<td>For hepatic characterization</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td>For hepatic induction</td>
		</tr>
		<tr>
			<td>MAP2 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>M4403</td>
			<td>For neural characterization</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>Neural Basal Media</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>NFM antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
			<td>For neural characterization</td>
		</tr>
		<tr>
			<td>Nikon Elipse TS100 microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>For&#160; fluorescence imaging</td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>01391-250Ml</td>
			<td>Adipogenic stain</td>
		</tr>
		<tr>
			<td>Oncostatin M</td>
			<td>R&#38;D Systems</td>
			<td>295-OM-010/CF</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Tarson</td>
			<td>460090-90MM</td>
			<td>For tissue cutting</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>15655-100G</td>
			<td>Osteogenic induction</td>
		</tr>
		<tr>
			<td>Propan-2-ol</td>
			<td>Thermo Fisher</td>
			<td>Q13827</td>
			<td>For Oil Red O extraction</td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution</td>
			<td>Sigma life sciences</td>
			<td>S8636-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td>For cell passaging</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>merck</td>
			<td>WHA1001325</td>
			<td>filter paper</td>
		</tr>
		<tr>
			<td>&#945;- Minimum Essential Media (&#945;-MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643-10X 1L</td>
			<td>Media for primary culture</td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422-50G</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of dental pulp stem cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Author Spotlight: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells

Primary Culture of Dental Pulp Stem Cells

We are investigating the potential of DPSCs in cell-based and cell-free applications for disease models, while studying the cellular and molecular mechanisms involved in tissue regeneration. Additionally, we are exploring the role of stem cell-derived secretory factors in controlling stem cell fate and promoting tissue regeneration. Our current focus is on using 3D culture techniques to gain deeper understanding of molecular mechanisms, genetic and epigenetic changes associated with tissue regeneration.We are incorporating advanced techniques such as next-gen sequencing, RNA sequencing, and chip sequencing to unravel regulatory domains governing various cellular processes. Using explant methods to isolate DPSC, a homogeneous stem cell population can be efficiently harvested, free from other cells like endothelial and pericytes. These cell types remain in the cell culture when DPSCs are established using enzymatic procedures.Our research using DPSCs as a cellular model for drug screening and tissue regeneration can develop novel therapies, advanced personalized medicine, and have far-reaching implications in regenerative medicine including craniofacial reconstruction and in vivo bone regeneration after dental extraction. We are currently investigating how DPSCs can be used to address bone defects, such as long bone and calvarial defects. Additionally, we are exploring the potential therapeutic implications of secretory molecules derived from DPSCs in various disease models, including neurodegeneration and glaucoma.

Here, we describe how researchers can establish a pure culture of DPSCs via the explant method6,7,8,9,10 and induce them toward multiple lineages to establish the purity of culture for downstream applications.
We established a primary culture of DPSCs from the small tissue of pulp extracted from the third molar tooth of pat...

Read this Scientific Article Protocol about Author Spotlight: Advancing Tissue Regeneration and Disease Modelling with Dental Pulp Stem Cells at JoVE.com

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...