This study was supported by Yeditepe University. All data and figures used in this article were previously published34.

Written informed consent of the patients was obtained after the approval from the Institutional Ethics Committee.
1. DPSC Isolation and Culture
<ol>
	<li>Transfer wisdom teeth obtained from young adults aged between 17 and 20 to 15 mL tubes containing complete Dulbecco&#39;s Modified Eagle Medium (DMEM) [low glucose DMEM media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin (PSA) solution], within 8 h after resection. Keep the tissue material cold (4 &#176;C) during transfer to avoid potential cell death.</li>
	<li>Remove the pulp tissue by sterile extraction forceps from the center of the tooth carefully, place the pulp tissue in the cold complete DMEM medium in 10 cm tissue culture dishes, and mince them into small pieces (2-3 mm) by scalpel. 
	NOTE: All experimental procedures should be carried out under sterile conditions in laminar flow hood. This non-enzymatic technique has been previously used21,22,23.</li>
	<li>Place small pulp tissues inside the tissue culture treated 6-well plates and add 200 &#181;L of complete DMEM media to cover each small pulp tissue pieces.</li>
	<li>Incubate the tissue culture wells at 37 &#176;C in a humidified air atmosphere (80% humidity) with 5% CO2 for 2 h to provide tissue attachment. 
	NOTE: This step could be prolonged to 3-4 h by controlling the evaporation of media.</li>
	<li>Add appropriate volume (2-2.5 mL) of complete DMEM medium to the wells and incubate at 37 &#176;C in a humidified air atmosphere with 5% CO2 for cells to spread from the tissue. 
	NOTE: Cells become visible after approximately 4 days and reach confluency after 8-9 days.</li>
	<li>When cells reach 80% confluency, remove media from 6-well plates, wash with 2 mL of phosphate buffered saline (PBS), and add 2 mL of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Then add 2 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
	<li>Passage cells to the flasks in complete DMEM media and store for further experiments. Add 15 mL of complete DMEM media to the T-75 flasks and transfer cells from two wells of the 6-well plate to one T-75 flasks and incubate at 37 &#176;C with humidified air atmosphere and 5% CO2.</li>
</ol>
2. Characterization of DPSCs
<ol>
	<li>Perform morphological analyses.
	<ol>
		<li>Seed cells (step 1.6) in tissue culture coated flasks (or 6-well plates) in complete DMEM medium for at least 8 passages to observe cell morphology.</li>
		<li>Visualize cells by light microscope and define fibroblast-like cell morphology. Cells should attach to the culture dishes and have spindle-like cell morphology. 
		NOTE: Alternatively, cells can be cultured as single cells for up to 14 days in well-plates to observe colony formation capacity that is a specific characteristic of fibroblasts and MSCs.</li>
	</ol>
	</li>
	<li>Perform surface marker analyses.
	<ol>
		<li>Trypsinize the cells from step 1.7. Remove media from the T-75 tissue culture flask, wash with 2 mL of PBS, and add 2 mL of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Then add 2 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
		<li>Fix the cells with 4% paraformaldehyde for 20 min at room temperature in 1.5 mL tubes and then wash them with 500 &#181;L of PBS 3 times to remove paraformaldehyde.</li>
		<li>Incubate fixed cells with the antibodies against CD29, CD34, CD14, CD45, CD90, CD105, CD166, and CD73 for 1 h at 4 &#176;C in 100 &#181;L of PBS. 
		NOTE: The concentration of antibody used is 0.5 &#181;g/mL. CD34, CD14, and CD45 are used as negative markers, while CD29, CD90, CD105, CD166, and CD73 are used as positive cell surface markers for MSCs.</li>
		<li>Wash cells 3 times with PBS and use respective secondary antibodies such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), etc. for labelling. Incubate cells with 1:500 diluted secondary antibodies in 100 &#181;L of PBS for 30 min at 4 &#176;C and wash 3 times with PBS.</li>
		<li>Keep samples in the dark for flow cytometry analysis and detect positive and negative staining by flow cytometry. 
		NOTE: Use unstained control cells to arrange forward and side scatter. Arrange gating of positively stained populations by excluding dead cells, debris, and un-stained population. Use 100 &#181;m nozzle with 45 psi sheath pressure and collect 10,000 events to determine positive DPSCs by arranging channels.</li>
	</ol>
	</li>
	<li>Perform differentiation of DPSCs.
	<ol>
		<li>Seed 1 &#215; 104 cells onto 24-well plates in complete DMEM media and incubate for 24 h at 37 &#176;C in a humidified air atmosphere with 5% CO2.</li>
		<li>Formulate differentiation media using compete DMEM medium as base media. Prepare osteogenic media by mixing 100 nM dexamethasone,10 mM &#946;-glycerophosphate, and 0.2 mM ascorbic acid. Prepare chondrogenic media by mixing 1&#215; insulin-transferrin-selenium (ITS-G), 100 nM dexamethasone, 100 ng/mL transforming growth factor beta (TGF-&#946;), 14 &#956;g/mL ascorbic acid, and 1 mg/mL bovine serum albumin (BSA). Prepare adipogenic media by mixing 100 nM dexamethasone, 5 &#956;g/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 60 &#956;M indomethacin. 
		NOTE: Differentiation media can be kept at 4 &#176;C for at least one week.</li>
		<li>Change growth media to osteo-, chondro-, or adipo-genic differentiation media and refresh media twice a week for two weeks.</li>
		<li>Confirm differentiation by von Kossa and Alcian blue staining, enzyme activity (alkaline phosphatase activity), immunocytochemistry, and quantitative polymerase chain reaction (qPCR) analyses according to the protocols previously described21.
		<ol>
			<li>Perform von Kossa and Alcian blue stainings on cells that are fixed with 4% paraformaldehyde at room temperature for 10 min. Wash the fixed cells with PBS and stain them with the vonKossa kit according to the manufacturer&#8217;s recommendations to observe calcium deposits.</li>
			<li>Prepare the Alcian blue staining solution by dissolving 1.00 g of Alcian blue dye in 100 mL of 3% (v/v) acetic acid for further staining. Wash the fixed cells with PBS and stain cells for 30 min with Alcian blue solution. Visualize the stained samples by a light microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Preparation of Condition Medium (CM)
<ol>
	<li>Replace media of cells from step 1.7 with fresh complete DMEM 24 h before CM collection. 
	NOTE: Passage 2-4 is recommended.</li>
	<li>Collect condition medium (CM) from cultured DPSCs when cells reach 80% confluency. Centrifuge collected media at 300 x g for 5 min to remove waste tissue material and cell debris. 
	NOTE: Alternatively, use 0.2 &#181;m syringe filters to remove debris from the condition medium.</li>
	<li>Collect supernatant and store at -20 &#176;C for further experiments. 
	NOTE: Keep the supernatant at -80 &#176;C for long-term storage.</li>
</ol>
4. Treatment of Cancer Cells with CM
<ol>
	<li>Perform cell viability analyses.
	<ol>
		<li>Seed PC-3 cells (human prostate cancer cells) onto 96-well plates at a cell density of 5 &#215; 103 cells/well in complete DMEM and incubate in a humidified chamber at 37 &#176;C and 5% CO2 for 24 h.</li>
		<li>Treat cells with 10, 20, 30, 40, and 50% of CM (v/v) mixed with complete DMEM for 24 h.</li>
		<li>Measure cell viability by using 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)-assay as described previously24.</li>
	</ol>
	</li>
	<li>Perform terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses.
	<ol>
		<li>Seed PC-3 cells onto 6-well cell culture plates at a cell density of 2 &#215; 105 cells/well and incubate in a humidified chamber at 37 &#176;C and 5% CO2 overnight.</li>
		<li>Mix 20% CM (v/v) with complete DMEM medium and apply to the cells for 24 h.</li>
		<li>Trypsinize cells and suspend in 50 &#956;L of TUNEL reaction mixture (labeling solution + enzyme solution, supplied with the kit) and incubate at 37 &#176;C for 60 min in a humidified and 5% CO2 atmosphere. 
		NOTE: For trypsinization, remove media from 6-well cell culture plates, wash with 1 mL of PBS and add 500 &#181;L of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Add 1 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
		<li>Rinse with PBS and analyze cells in PBS by using flow cytometry.</li>
	</ol>
	</li>
	<li>Perform qPCR analyses.
	<ol>
		<li>Seed PC-3 cells onto 6-well cell culture plates at a cell density of 2 &#215; 105 cells/well and incubate in a humidified incubator at 37 &#176;C and 5% CO2 overnight.</li>
		<li>Mix 20% CM (v/v) with complete DMEM medium and apply to the cells for 24 h.</li>
		<li>Trypsinize cells and collect cell pellet for RNA isolation and cDNA synthesis. 
		NOTE: Remove media from 6-well cell culture plates, wash with 1 mL of PBS, and add 500 &#181;L of trypsin. Incubate 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Add 1 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
		<li>Perform qPCR experiments according to the previously described protocol25.</li>
	</ol>
	</li>
	<li>Perform cell migration of cancer cells.
	<ol>
		<li>Seed 1 &#215; 105 PC-3 cells onto 12-well plates and incubate in a humidified incubator overnight at 37 &#176;C and 5% CO2.</li>
		<li>Scratch cells with a sterile 200 &#956;L tip and change medium immediately with fresh medium containing various concentrations of CM [e.g., 10, 20, 30, 40, and 50% of CM (v/v) mixed with complete DMEM].</li>
		<li>Observe scratches under an inverted microscope and take pictures at different time intervals (0 and 24 h).</li>
		<li>Measure the scratch closure by using Image J software using the formula: 
		<img alt="Equation" src="/files/ftp_upload/58825/58825eq1.jpg" /> 
		NOTE: Open the scratch image with Image J software. Draw a line that has the same magnitude as the scale bar that already exists in the image. Click analyze, set scale, and observe distance in pixels as the magnification of the drawn line. Write the size of the scale bar to the known distance part, arrange unit (pixels, cm, etc.), and click ok. Go to the analyze section again and click on measurements. This will first give the size of the scale bar as the selected unit. Click on one edge of scratch and drag until reaching the other end of the scratch. Note the value for each time point (0 h and 24 h). Plug these values into the formula above and calculate the scratch closure.</li>
	</ol>
	</li>
</ol>
5. Cell Migration by Indirect Contact of Cancer Cells and DPSCs
<ol>
	<li>Seed 3 &#215; 104 DPSCs onto 24-well plate inserts with 0.4 &#956;m pore and incubate in a humidified incubator overnight at 37 &#176;C.</li>
	<li>Seed PC-3 cells onto 24-well plates at a cell density of 5 &#215; 104 and incubate in a humidified incubator overnight at 37 &#176;C and 5% CO2.</li>
	<li>Scratch PC-3 cells with a sterile 200 &#956;L tip, change medium with fresh medium, and place inserts carrying DPSCs onto PC-3 cells.</li>
	<li>Observe cells under an inverted microscope and take pictures at different time intervals (0 and 24 h) to analyze cell migration.</li>
</ol>
6. Co-culture Assay and Flow Cytometry Analysis
<ol>
	<li>Label PC-3 cells and DPSCs by using PKH67 (green) and PKH26 (red) fluorescent cell linker dyes, respectively26.</li>
	<li>Trypsinize PC-3 and DPSCs cells, respectively. Remove media from T-75 tissue culture flask, wash with 2 mL of PBS, and add 2 mL of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Add 2 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin.</li>
	<li>Centrifuge cells at 300 x g for 5 min, discard supernatant, and resuspend cell pellets in the dye solution prepared in diluent-C buffer supplied by the kit (see Table of Materials).</li>
	<li>Incubate cells in the dye solution for 10 min and terminate the staining reaction by adding 100 &#181;L of FBS. Centrifuge cells at 300 x g for 5 min, discard the supernatant, and wash the cells with complete growth medium before co-culturing.</li>
	<li>Plate labeled cells (5 &#215; 104/well) onto 6-well plates at 1:1 ratio (DPSCs:PC3). Maintain co-cultured cells in complete DMEM.</li>
	<li>Collect cells after 24 h or 48 h incubation periods by centrifugation of cells at 300 x g for 5 min and washing with PBS.</li>
	<li>Resuspend cells in 300 &#181;L of fluorescence-activated cell sorting (FACS) buffer in 5 mL round bottom flow cytometry tubes. Vortex to disperse cell aggregates right before the sample analysis.</li>
	<li>Use a 100 &#181;m nozzle with 45 psi sheath pressure. 
	NOTE: Extremely high flow rate might decrease the sensitivity of fluorescence detection.</li>
	<li>Use unstained control cells and single colored cells to adjust appropriate forward and side scatter laser voltage for cell types and compensation as mentioned previously27. 
	NOTE: Use gating for live cells to exclude cell debris, dead cells, or aggregates.</li>
	<li>Collect 10,000 events (100,000 is preferable) to determine percent positive green DPSCs and red PC-3 cells by arranging FL-1 (green) and FL-2 (red) channels.</li>
</ol>

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E., Y&#305;lmaz, A., Rizvanov, A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+F68+on+cryopreservation+of+mesenchymal+stem+cells+derived+from+human+tooth+germ.">Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ.</a> Applied biochemistry and biotechnology. 171 (7), 1819-1831 (2013).</li><li>Rizvanov, A. 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=Interaction+and+self-organization+of+human+mesenchymal+stem+cells+and+neuro-blastoma+SH-SY5Y+cells+under+co-culture+conditions:+A+novel+system+for+modeling+cancer+cell+micro-environment.">Interaction and self-organization of human mesenchymal stem cells and neuro-blastoma SH-SY5Y cells under co-culture conditions: A novel system for modeling cancer cell micro-environment.</a> European Journal of Pharmaceutics and Biopharmaceutics. 76 (2), 253-259 (2010).</li><li>Troiano, L., 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=Multiparametric+analysis+of+cells+with+different+mitochondrial+membrane+potential+during+apoptosis+by+polychromatic+flow+cytometry.">Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.</a> Nature protocols. 2 (11), 2719 (2007).</li><li>Melzer, C., von der Ohe, J., Lehnert, H., Ungefroren, H., Hass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+niche+models+and+contribution+by+mesenchymal+stroma/stem+cells.">Cancer stem cell niche models and contribution by mesenchymal stroma/stem cells.</a> Molecular cancer. 16 (1), 28 (2017).</li><li>Aguirre, A., Planell, J., Engel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+bone+marrow-derived+endothelial+progenitor+cell/mesenchymal+stem+cell+interaction+in+co-culture+and+its+implications+in+angiogenesis.">Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis.</a> Biochemical and biophysical research communications. 400 (2), 284-291 (2010).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomimetics and Stem Cells. , 29-36 (2013).</li><li>Plotnikov, E., 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=Cell-to-cell+cross-talk+between+mesenchymal+stem+cells+and+cardiomyocytes+in+co-culture.">Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.</a> Journal of cellular and molecular. 12 (5a), 1622-1631 (2008).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cell-cell+communication+in+co-culture.">Studying cell-cell communication in co-culture.</a> Biotechnology journal. 8 (4), 395-396 (2013).</li><li>Brunetti, G., 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=High+expression+of+TRAIL+by+osteoblastic+differentiated+dental+pulp+stem+cells+affects+myeloma+cell+viability.">High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability.</a> Oncology reports. 39 (4), 2031-2039 (2018).</li><li>Do&#287;an, A., Demirci, S., Apdik, H., Apdik, E. A., &#350;ahin, F. <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+cells+(DPSCs)+increase+prostate+cancer+cell+proliferation+and+migration+under+in+vitro+conditions.">Dental pulp stem cells (DPSCs) increase prostate cancer cell proliferation and migration under in vitro conditions.</a> Tissue and Cell. 49 (6), 711-718 (2017).</li></ol>

The authors have nothing to disclose.

Contribution of MSCs to tumor environment is regulated by several interactions including hybrid cell generation via cell fusions, entosis or cytokine and chemokine activities between stem cells and cancer cells28. Structural organization, cell-cell interactions, and secreted factors determine cancer cell behavior in terms of tumor promotion, progression, and metastasis to surrounding tissue. Proper ex vivo model systems to investigate the mechanisms behind the interactions of res...

Mesenchymal stem cells (MSCs), with&#160;the ability of differentiation and contribution to regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose, have been isolated from almost all tissues in the adult body1,2. Other than providing tissue homeostasis by producing resident cells in case of chronic inflammation or an injury, they produce vital cytokines and growth factors to orchestrate angiogenesis, immune system, and tissue remodeling3. The interaction of MSCs with cancer tissue is not well-understood, but accumulating evidence suggests that MSCs might promote tumor initiation, progression, and metastasis4.
The homing ability of MSCs to the injured or chronically inflamed area makes them a valuable candidate for stem cell-based therapies. However, cancer tissues, &#34;never healing wounds&#34;, also release inflammatory cytokines, pro-angiogenic molecules, and vital growth factors, which attract MSCs to the cancerogenous area5. While there are limited reports showing inhibitory effects of MSCs on cancer growth6,7, their cancer progression and metastasis promoting effects have been extensively reported8. MSCs directly or indirectly affect carcinogenesis in different ways including suppressing immune cells, secreting growth factors/cytokines that support cancer cell proliferation and migration, enhancing angiogenic activity, and regulating epithelial-mesenchymal transition (EMT)9,10. Tumor environment consists of several cell types including cancer-associated fibroblasts (CAFs) and/or myofibroblasts, endothelial cells, adipocytes, and immune cells11. Of those, CAFs are the most abundant cell type in the tumor area that secrete various chemokines promoting cancer growth and metastasis8. It has been shown that bone marrow-derived MSCs can differentiate into CAFs in the tumor stroma12.
Dental pulp stem cells (DPSCs), characterized as the first dental tissue-derived MSCs by Gronthos et al.13 in 2000 and then widely investigated by others14,15, express pluripotency markers such as Oct4, Sox2, and Nanog16 and can differentiate into various cell linages17. Gene and protein expression analysis proved that DPSCs produce comparable levels of growth factors/cytokines with other MSCs such as vascular endothelial growth factor (VEGF), angiogenin, fibroblast growth factor 2 (FGF2), interleukin-4 (IL-4), IL-6, IL-10, and stem cell factor (SCF), as well as fms-like tyrosine kinase-3 ligand (Flt-3L) that might promote angiogenesis, modulate immune cells, and support cancer cell proliferation and migration18,19,20. While the interactions of MSCs with cancer environment have been well-documented in the literature, the relationship between DPSCs and cancer cells has not been evaluated yet. In the present study, we established co-culture and condition medium treatment strategies for a highly metastatic prostate cancer cell line, PC-3, and DPSCs to propose potential action of mechanism of dental MSCs in cancer progression and metastasis.

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>11885084</td><td>For cell culture</td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000044</td><td>For cell culture</td></tr><tr><td>PSA</td><td>Lonza</td><td>17-745E</td><td>For cell culture</td></tr><tr><td>Trypsin</td><td>Invitrogen</td><td>25200056</td><td>For cell dissociation</td></tr><tr><td>PBS</td><td>Invitrogen</td><td>10010023</td><td>For washes</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>Component of differentiation media</td></tr><tr><td>&amp;beta;-Glycerophosphate</td><td>Sigma</td><td>G9422</td><td>Component of osteogenic differentiation medium</td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>A4544</td><td>Component of osteo- and chondro-genic differentiation medium</td></tr><tr><td>Insulin-Transferrin-Selenium (ITS &amp;minus;G)</td><td>Invitrogen</td><td>41400045</td><td>Component of chondrogenic differentiation medium</td></tr><tr><td>TGF-&amp;beta;</td><td>Sigma</td><td>SRP3171</td><td>Component of chondrogenic differentiation medium</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I6634</td><td>Component of adipogenic differentiation medium</td></tr><tr><td>Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma</td><td>I7018</td><td>Component of adipogenic differentiation medium</td></tr><tr><td>Indomethacin</td><td>Sigma</td><td>I7378</td><td>Component of adipogenic differentiation medium</td></tr><tr><td>MTS Reagent</td><td>Promega</td><td>G3582</td><td>Cell viability analyses</td></tr><tr><td>TUNEL Assay</td><td>Sigma</td><td>11684795910</td><td>Apoptotic analyses</td></tr><tr><td>24-well plate inserts</td><td>Corning</td><td>3396</td><td>For trans-well migration assay</td></tr><tr><td>PKH67</td><td>Sigma</td><td>PKH67GL</td><td>For co-culture cell staining</td></tr><tr><td>PKH26</td><td>Sigma</td><td>PKH26GL</td><td>For co-culture cell staining</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>For cell fixation</td></tr><tr><td>von Kossa Kit</td><td>BioOptica</td><td>04-170801.A</td><td>For cell staining (differentiation)</td></tr><tr><td>Alcian blue</td><td>Sigma</td><td>A2899</td><td>For cell staining (differentiation)</td></tr></tbody></table>

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.

Figure 1 depicts the general MSC characteristics of DPSCs under culture conditions. DPSCs exert fibroblast-like cell morphology after plating (Figure 1B). MSC surface antigens (CD29, CD73, CD90, CD105, and CD166) are highly expressed while hematopoietic markers (CD34, CD45, and CD14) are negative (Figure 1C). Changes at the morphological and molecular level related to osteo-, chondro-, and adipo-geni...

Watch this Scientific Journal Video about Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions at JoVE.com

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

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 ...

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Research

JoVE Journal

Cancer Research

10.3K Views.  Yeditepe University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het stamcelveld over de rol van volwassen stamcellen in de regulatie van kankercelgedrag. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt de evaluatie van kankercel-en stamcel interacties in vitro zonder het gebruik van levende organismen. Deze techniek heeft potentieel voor kankermetastase als het modellen van de bevorderende rol van volwassen stamcellen in kanker metastase aan harde weefsels zoals ...

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

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Developmental Biology

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

Bioengineering

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

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 ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

Establishing the Primary Culture of Dental Pulp Stem Cells

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

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 ...

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