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.

Establishing the Primary Culture of Dental Pulp Stem Cells

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<li>Hirata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multifaceted+therapeutic+benefits+of+factors+derived+from+dental+pulp+stem+cells+for+mouse+liver+fibrosis%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">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/pubmed?term=((regeneration+by+human+dental+pulp+stem+cells+using+a+helioxanthin+derivative+and+cell-sheet+technology%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">regeneration by human dental pulp stem cells using a helioxanthin derivative and cell-sheet technology.</a> Stem Cell Research & Therapy. 9 (1), 24(2018).</li>
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</ol>

The authors declare no financial or non-financial conflict of interest.

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 morphologically resembles spindle-shaped fibroblast-like cells. The explant method yields more uniform cultures of DPSCs, devoid of other cell types such as endothelial cells and pericytes, which remain during the enzymatic procedure for DPSC culture establishment34. We showed that DPSCs derived by the explant method express ISCT-recommended mesenchymal cell surface CD markers, such as CD90, CD73, and CD105. They are also devoid of CD45, CD34, and HLA-DR marker-expressing cells, which indicate the homogeneity and purity of DPSC culture. The lack of HLA-DR also makes them suitable for transplantation purposes, with minimum rejection possibilities. There are a few critical steps in the protocol to ensure the efficiency and purity of DPSC culture. The explants should be washed at least four times with PBS before being put in the culture plates to avoid any possible contamination. Also, the explants should adhere well to the culture well surface, as detached explants would not yield any cells. Hence, utmost care should be taken while handling explants during media change. The media should be added carefully, dropwise along the corners of the well, to not disturb the explants. Once the cells grow in the plate wells, tissue pieces should be removed carefully using the tip, in order not to damage any surrounding cells. During the retrieval of DPSCs at the first passage from plate wells, there might be very few cells, making it impossible to see any visible cell pellet in the tube after centrifugation. Some cells might also stick to the walls of the centrifuge tube, making it hard to get any cells during the upscaling process. Care should be taken to rinse the culture plate and 15 mL tube well with PBS to retrieve as many cells as possible. All cell-based differentiation experiments should be performed in DPSCs below passage number eight, as higher passages might lead to the accumulation of genetic aberrations and spontaneous differentiation, hence affecting the results. 
There are also a few limitations of the protocol. The explant culture is a slow protocol and yields enough cells after 15-20 days of culture only. In between, there is a risk of contamination, and the explant (dental pulp pieces) can get detached from the plate surface, which would not yield any cells later. Hence, utmost care should be taken while changing the media. Also, repeated PBS washings are important before seeding the explants, as teeth are rich in oral micro flora and there are very high chances of contamination in the culture if proper washing is not performed. Human DPSCs are ectodermal in origin and during tooth development; they migrate from the neural crest and later differentiate into mesenchymal cells. This confers special properties of both mesodermal and ectodermal lineage in them34. Arthur et al. postulated that the neural crest-derived cells are predisposed to neural differentiation35; the spontaneous neural differentiation of DPSCs was also reported by Kim et al.36. Another study has indicated the potential role of the distal C-terminus L-type voltage-gated calcium channel in directing DPSCs to attain a neuronal phenotype37. Previously, our lab demonstrated the cryopreservation efficiency of these DPSCs by preserving them at -80 &#176;C for 5 years10. These cells were able to maintain stemness characteristics, proliferation, and differentiation ability, as well as a stable karyotype without any structural or numerical abnormalities. In addition, in a comparative analysis, our lab also demonstrated the superior tendency of DPSCs toward neurogenic7 and osteogenic8 lineages, as compared to BMSCs. Their superior competence and inherit tendency toward osteogenic and neural lineage make these cells the best-suited candidate for stem cell therapies in bone disorders and neurodegenerative diseases. 
Altogether, we describe here an efficient protocol to obtain a pure population of DPSCs by the explant method, as evident by their stem cell marker expression and multilineage differentiation capability.

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><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&#039;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&amp;nbsp; induction</td></tr><tr><td>EVOS LED microscope</td><td>Invitrogen</td><td></td><td>For&amp;nbsp; 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>&amp;nbsp;Human TGF-&amp;beta;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&amp;nbsp; 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&amp;amp;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>&amp;alpha;- Minimum Essential Media (&amp;alpha;-MEM)</td><td>Sigma-Aldrich</td><td>M0643-10X 1L</td><td>Media for primary culture</td></tr><tr><td>&amp;beta;-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.

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 patients, as shown in Figure 1A. Initially, very few rounded cells were observed in the immediate vicinity of explants or tissue on the fourth and fifth day post-explant seeding. However, by the 12th day, the count of cells that emerged out of the explants increased, and by the end of second week (14th-16th day), most of the explants got surrounded by many adherent cells with spindle-shaped morphology. At this time, the explant could be removed from the plates and the cells were allowed to grow in the presence of complete media. After reaching 70%-75% confluence (third week), the cells were sub-cultured and expanded for further experimental setup. Through the explant method, we obtained a uniform culture of stem cells with positivity of MSC CD markers CD90, CD105, CD73, and an almost negligible cell population of another cell type, endothelial cell marker CD34 and hematopoietic cell marker CD45 (Figure 1B).
We also differentiated DPSCs successfully into cells of osteogenic lineage, as evident by positive alizarin red stain uptake in calcified granules in osteocytes, adipogenic lineage, as demonstrated by the uptake of oil red O staining by lipid droplets in differentiated adipocytes, and chondrogenic lineage, as shown by positive Alcian blue staining (Figure 2). DPSCs also showed efficient differentiation toward hepatic-like lineage, as evident by the typical cuboidal morphology and uptake of LDL-550 by live cell (Figure 3A) staining. The differentiated hepatocyte-like cells were fixed and processed for staining with the LDL receptor antibody. Figure 3B shows the positive expression of the LDL receptor in differentiated hepatocyte- like cells along with LDL-550. DPSCs also demonstrated a marked ability to differentiate into neuronal-like cells, as shown by the positive staining of MAP-2 and NFM, typical neuronal proteins (Figure 4). In a nutshell, our study shows that DPSCs can be isolated and cultured as a pure population, with a &#62;95% purity for stem cell markers, and differentiated into various lineages for further downstream applications like cell therapy, drug testing, and disease modeling.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65223/65223fig01.jpg" /> 
Figure 1: Primary culture of DPSCs. (A). Differential interference contrast (DIC) images showing various stages in development of DPSC primary culture: 1) explant at the initial budding stage with rounded bubble type cells coming out of dental tissue on the second day of seeding; 2) establishment of a mess-like network of cells coming out of tissue on the fourth day; 3) cells with full spindle-shaped morphology at confluence on the 13th day and cells covering the entire surface of the culture dish after the removal of tissue. Scale bar = 100 &#181;m, (B). Stem cell marker characterization by flow cytometry with histograms and a bar graph showing the positive expression of stemness markers and negative expression of non-stem cell markers in DPSCs (n = 3). Each experiment was repeated in triplicate at least three times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65223/65223fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65223/65223fig02.jpg" /> 
Figure 2: Multilineage differentiation of DPSCs. Bright field microscopic images showing alizarin red staining after osteogenic differentiation, oil red O staining post-adipogenic differentiation, and Alcian blue staining after chondrogenic differentiation in DPSCs. Scale bar = 100 &#181;m (n = 3). Each experiment was repeated in triplicate at least three times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65223/65223fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65223/65223fig03.jpg" /> 
Figure 3: Differentiation of DPSCs toward hepatocyte-like cells and uptake of LDL. (A). Light microscopic images for control and hepatic-like cells differentiated from DPSCs; the last image on the extreme right shows LDL uptake in the live differentiated cells. (B). Fluorescent microscopic images showing the expression of the LDL receptor (green) and uptake of LDL-550 (red) (fixed cells) in DPSCs post-hepatic differentiation. DAPI was used as a nuclear stain. Scale bar = 200 &#181;m (n = 3). Each experiment was repeated in triplicate at least three times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65223/65223fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65223/65223fig04.jpg" /> 
Figure 4: Differentiation of DPSCs to neuronal lineage. Fluorescent microscopic images for DPSCs after 41 days of neural differentiation, showing a positive expression of neural lineage-specific antibodies&#39; neurofilament and MAP-2. PI was used as a nuclear stain. Scale bar = 20 &#181;m (n = 3). Each experiment was repeated in triplicate at least three times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65223/65223fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

primary-culture-of-dental-pulp-stem-cells

Nanyang Technological University

Research

JoVE Journal

Biology

2.1K Views.  PGIMER. We onderzoeken het potentieel van DPSC's in celgebaseerde en celvrije toepassingen voor ziektemodellen, terwijl we de cellulaire en moleculaire mechanismen bestuderen die betrokken zijn bij weefselregeneratie. Daarnaast onderzoeken we de rol van van stamcellen afgeleide secretoire factoren bij het beheersen van het lot van stamcellen en het bevorderen van weefselregeneratie. Onze huidige focus ligt op het gebruik van 3D-kweektechnieken om een beter begrip te krijgen van moleculaire mechanisme...

Video: Auteur in de schijnwerpers: Weefselregeneratie en ziektemodellering bevorderen met stamcellen van tandpulp

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Cancer Research

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicine

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very important to obtain adult stem cells from different tissues such as adipose, umbilical cord, bone marrow and blood. Among the possible sources, dental pulp is particularly interesting because it is easy to obtain in respect of bioethical considerations. Indeed, human Dental Pulp Stem Cells (hDPSCs) are a type of adult stem cells able to differentiate in neuronal-like cells and can be obtained from the third molar of healthy patients (13-19 ages). In particular, the dental pulp was removed with an excavator, cut into small slices, treated with collagenase IV and cultured in a flask. To induce the neuronal differentiation, hDPSCs were stimulated with EGF/bFGF for 2 weeks. Previously, we have demonstrated that during the differentiation process the content of cellular prion Protein (PrPC) in hDPSCs increased. The cytofluorimetric analysis showed an early expression of PrPC that increased after neuronal differentiation process. Ablation of PrPC by siRNA PrP prevented neuronal differentiation induced by EGF/bFGF. In this paper, we illustrate that as we enhanced the isolation, separation and in vitro cultivation methods of hDPSCs with several easy procedures, more efficient cell clones were obtained and large-scale expansion of the mesenchymal stem cells (MSCs) was observed. We also show how the hDPSCs, obtained with methods detailed in the protocol, are an excellent experimental model to study the neuronal differentiation process of MSCs and subsequent cellular and molecular processes.

Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation process.

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

Developmental Biology

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

Bioengineering

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Source: Barkley, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60809">A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.</a>&#160;J. Vis. Exp. (2020).
This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of neurons towards the mesenchymal cells establishing a co-culture system.

1. Plate Preparation
NOTE: Be sure the plate lid is on during all incubation and rinsing steps outside of the sterile tissue culture hood to prevent ...

Primary Culture of Dental Pulp Stem Cells

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