The authors are grateful to Dr. Mathew Mazhavancheril, Director and Head of the Pushpagiri Research Centre in Thiruvalla, for his support in documenting the procedures at the Research Centre.

The protocol outlined herein conforms to the guidelines of the institutional human research ethics committee (IRB, Pushpagiri Research Center, Kerala). The use of extracted teeth was conducted following ethical standards to ensure the integrity, dignity, and rights of the participants. The participants selected for this study were healthy individuals under 30 years of age who required tooth extraction for orthodontic treatment. Those with extensive dental caries or severe periodontitis were excluded from the study. Deciduous teeth were collected from children who required the extraction of retained teeth. Informed written consent was also obtained from the subjects involved in this study.
1. Extraction and transport of teeth
<ol>
	<li>Collection of deciduous and permanent teeth
	<ol>
		<li>Explain to the participants the procedure&#39;s purpose and the potential use of the extracted teeth for research purposes.</li>
		<li>Perform teeth extraction under local anesthesia by administering an anesthetic agent in the vicinity of the tooth to be extracted. Specifically, 1.75 mL of lidocaine mixed with epinephrine (see Table of Materials) at a 1:200,000 ratio was injected for this study. Selecting teeth that do not exhibit severe dental caries or periodontitis is preferable20,21.</li>
	</ol>
	</li>
	<li>Transportation of the extracted teeth 
	NOTE: Working under aseptic conditions is recommended to avoid contamination. This includes wearing personal protective equipment, such as gloves and a lab coat, and working in a sterile environment, such as a biohazard laminar flow hood.
	<ol>
		<li>After the tooth is extracted, rinse it with sterile phosphate-buffered saline (PBS) solution to remove the blood and other debris. Using sterile forceps and scalpel, carefully remove any attached soft tissue remnants from the tooth&#39;s surface (Figure 2A).</li>
		<li>Immerse the tooth in a disinfectant solution, such as 70% ethanol, for 10 s. After the disinfection, rinse the tooth with sterile PBS to wash away any residual disinfectant.</li>
		<li>Place the tooth in a sterile container containing the transport medium. 
		NOTE: The transport medium consisted of Alpha-MEM, a commercial culture medium supplemented with antibiotics: 100 U/mL penicillin/streptomycin (see Table of Materials). This medium supplied essential nutrients to the cells within the extracted tooth, ensuring their survival until laboratory processing. The antibiotics served to inhibit bacterial growth.</li>
		<li>Maintain a temperature of 4 &#176;C during transport to slow cellular metabolic activities and delay cell death. Transport the tooth to the laboratory for the next steps, including processing and isolating the dental pulp stem cells.</li>
	</ol>
	</li>
</ol>
2. Collection of pulp tissue
<ol>
	<li>Secure the tooth using dental forceps to ensure it stays in place during the procedure. Use a diamond disc to cut the tooth in a dental handpiece (see Table of Materials) connected to a water coolant (Figure 2B). 
	NOTE: The goal is to expose the pulp chamber without causing unnecessary damage to the pulp tissue within.</li>
	<li>Employ a dental excavator (see Table of Materials) to remove the pulp tissue. This instrument enables the extraction of pulp without inflicting damage, thereby preserving the viability of the dental pulp stem cells (Figure 2C).</li>
	<li>Place the obtained tissue on a glass Petri dish. Wash the pulp tissue with phosphate-buffered saline.</li>
</ol>
3. Digestion of pulp tissue and cell isolation
<ol>
	<li>Tissue mincing and digestion
	<ol>
		<li>With a sterile surgical blade, carefully mince the pulp tissue into small fragments (Figure 2D) and place it in a mini tissue grinder with PBS to obtain a finely homogenized mixture. This process increases the surface area of the tissue, facilitating more efficient enzyme action during digestion.</li>
		<li>Transfer the minced homogenous tissue fragments into a 15 mL tube and enzymatically digest utilizing a mixture of 3 mg/mL collagenase type I and 4 mg/mL dispase (see Table of Materials). These enzymes were dissolved in Hank&#39;s Balanced Salt Solution (HBSS) to achieve the desired concentrations.</li>
		<li>Incubate the pulp tissue in the enzyme mixture (step 3.1.2) for about 2 h at 37 &#176;C. After incubation, neutralize the enzymes, and collect the cells by centrifugation (step 3.2.1) for further culture.</li>
	</ol>
	</li>
	<li>Centrifugation and resuspension
	<ol>
		<li>After incubation, transfer the digested tissue to a 15 mL centrifuge tube and spin at 300 x g for 5 min at room temperature to pellet the cells. This step separates the cells from the remaining undigested tissue fragments and enzymes. Carefully discard the supernatant medium, ensuring not to disturb the cell pellet at the bottom of the tube.</li>
		<li>Resuspend the cell pellet in a fresh culture medium. This medium provides the necessary nutrients and environment for the isolated cells to survive and proliferate.</li>
	</ol>
	</li>
</ol>
4. Cell culture
<ol>
	<li>Plating and incubation of cells
	<ol>
		<li>Carefully transfer the resuspended cells to a 25 cm&#178; culture flask. 
		NOTE: All material from a single pulp is transferred into a single 25 cm2 cell culture flask. A media volume of 5-7 mL ensures sufficient coverage and nutrient availability for the cells. Ensure that the culture medium for DPSCs is Dulbecco&#39;s Modified Eagle Medium (DMEM), supplemented with 10%-20% fetal bovine serum (FBS) (see Table of Materials) to provide necessary growth factors. Further, supplement the medium with 1% penicillin/streptomycin to prevent bacterial contamination. Ensure the cells are evenly distributed throughout the flask to promote homogeneous growth.</li>
		<li>Incubate the flask at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	</ol>
	</li>
	<li>Medium change and monitoring confluency
	<ol>
		<li>Change the culture medium every 2-3 days to provide fresh nutrients and remove metabolic waste. To do this, carefully aspirate the old medium using a sterile serological pipette and replace it with a fresh medium.</li>
		<li>Regularly monitor the cell culture under a microscope to track the cells&#39; growth and check for any signs of contamination.</li>
		<li>Continue this process until the cells reach 80%-90% confluency, the point at which the cells cover most of the flask surface area but are not overly crowded.</li>
	</ol>
	</li>
	<li>Cell trypsinization and passaging or freezing
	<ol>
		<li>Once the cells reach 80%-90% confluency, remove the culture medium and wash the cells with phosphate-buffered saline (PBS) to remove the residual medium and non-adherent cells.</li>
		<li>Add a solution of 0.25% trypsin-EDTA to the flask to detach the cells from the flask&#39;s surface. Incubate for 2-5 min at 37 &#176;C until the cells lift off.</li>
		<li>Neutralize the trypsin by adding an equal volume of culture medium, then gently pipette the cell suspension up and down to ensure complete cell detachment.</li>
		<li>Centrifuge the cell suspension at 300 x g for 5 min at room temperature to pellet the cells.</li>
		<li>Resuspend the cells in a fresh culture medium and either replate them (passage) for further growth or freeze them for future use. When freezing, use a freezing medium that contains a cryoprotectant, like DMSO, to protect the cells from damage during freezing.</li>
	</ol>
	</li>
</ol>
5. Characterization of DPSCs
<ol>
	<li>Flow cytometry analysis
	<ol>
		<li>To confirm the mesenchymal stem cell nature of the isolated cells, perform flow cytometry analysis22,23.Initially, trypsinize and collect cells as described in step 4.3.</li>
		<li>Determine the cell viability by the trypan dye exclusion technique. Perform the analysis using a cell viability analyzer (see Table of Materials) during the 2nd and 8th passages. Perform phenotypic analysis via a flow cytometer during the 3rd and 7th passages.</li>
		<li>After incubation, wash the cells in a flow cytometry buffer to remove excess antibodies and analyze the cells using a flow cytometer. 
		NOTE: The flow cytometry buffer is composed of phosphate-buffered saline (1x PBS), 1%-2% fetal bovine serum (FBS), and 0.1% sodium azide (NaN3). The FBS or BSA blocks the non-specific binding of antibodies, while the sodium azide acts as a preservative. A high percentage of cells should express the mesenchymal stem cell markers and lack the hematopoietic markers, confirming their identity as DPSCs24.</li>
		<li>Detach the DPSCs and stain them with immunofluorescence antibodies for flow cytometry analysis. 
		NOTE: The selection of markers for flow cytometry analysis of mesenchymal stem cells (MSCs) is based on the consensus established by the International Society for Cellular Therapy (ISCT), which states that MSCs should express CD105, CD73, and CD90, and lack the expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA class II25.&#160;These markers are commonly used in studies investigating dental pulp stem cells26.The concentrations of antibodies for staining can vary depending on the specific antibody used, and it is recommended to follow the manufacturer&#39;s instructions (see Table of Materials) for antibody dilutions and incubation conditions25.</li>
		<li>Establish the classification criteria27,28 for the expression of CD markers as mentioned: less than 10%, no expression; between 11% and 40%, low expression; a range of 41% to 70%, moderate expression; above 71%, high expression.</li>
		<li>Additionally, confirm the absence of hematopoietic markers by incubating a separate cell aliquot with antibodies against CD34 and CD45.</li>
	</ol>
	</li>
</ol>
6. Multilineage differentiation
NOTE: The following steps outline protocols for osteogenic, adipogenic, and chondrogenic differentiation of dental stem cells. Begin by seeding cultures at a density of 1 x 105 cells per well in a fibronectin-coated tissue culture plate with complete medium (CM). Monitor cell growth until 80%-90% confluency is achieved before initiating the desired differentiation protocol. To evaluate the DPSCs&#39; multilineage differentiation potential, initiate the differentiation process towards osteoblasts, adipocytes, and chondrocytes by seeding cells into 24-well plates and culturing them in appropriate differentiation media.
<ol>
	<li>Osteogenic differentiation
	<ol>
		<li>Initiate by culturing DPSCs in a regular growth medium under standard conditions until they reach 70%-80% confluence.</li>
		<li>Remove the growth medium from the cells, then wash the cells once with 1x PBS. Add the Osteogenic Differentiation Medium (ODM) to the cells. 
		NOTE: The ODM consists of Alpha Minimum Essential Medium (&#945;-MEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 &#181;g/mL streptomycin, 0.25 &#181;g/mL amphotericin B, 50 &#181;g/mL ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone (see Table of Materials).</li>
		<li>Incubate the cells in the osteogenic medium at 37 &#176;C with 5% CO2. Change the osteogenic medium every 2-3 days. 
		NOTE: After 14 to 21 days, the DPSCs should have differentiated into osteoblast-like cells. This can be confirmed by checking for an increase in the expression of osteoblast-specific markers, such as alkaline phosphatase, or by using staining techniques to detect mineralized matrix, such as Alizarin Red S staining.</li>
		<li>Fix the cells by incubating them with 70% ice-cold ethanol for 1 h at -20 &#176;C.</li>
		<li>Stain the fixed cells with 40 nM Alizarin Red S (pH 4.2) staining solution at room temperature in the dark for 10-15 min. Visualize the staining under a microscope to confirm osteogenic differentiation (Figure 3). Confirm the differentiation by staining the cells with Alizarin Red S, which identifies calcium deposits indicative of mineralization29,30.</li>
	</ol>
	</li>
	<li>Adipogenic differentiation
	<ol>
		<li>As detailed in the earlier section, culture DPSCs under standard conditions in a humidified atmosphere with 5% CO2 at 37 &#176;C. Passage the cells when they reach around 70%-80% confluence.</li>
		<li>Aspirate the medium and wash the cells once with 1x PBS. Replace the washed-out medium with adipogenic differentiation medium. This medium generally consists of DMEM, 10% FBS, 10 &#181;g/mL insulin, 1 &#181;M dexamethasone, 200 &#181;M indomethacin, and 0.5 mM IBMX (see Table of Materials).</li>
		<li>Change the differentiation medium every 3-4 days. After about 3 weeks, wash the adipogenic-differentiated DPSCs with PBS and fix them with 4% paraformaldehyde (PFA) for 20 min at room temperature.</li>
		<li>Stain the fixed cells with Oil Red O (see Table of Materials) to visualize lipid droplet accumulation (Figure 4). To further confirm adipogenic differentiation, perform gene expression analysis of adipogenic markers such as peroxisome proliferator-activated receptor gamma (PPAR&#947;) and adiponectin31,32.</li>
	</ol>
	</li>
	<li>Chondrogenic differentiation
	<ol>
		<li>DPSCs cultures maintained in a humidified atmosphere with 5% CO2 at 37 &#176;C are used for Chondrogenic differentiation. Passage the cells when they reach around 70%-80% confluence.</li>
		<li>Aspirate the medium and wash the cells once with 1x PBS. Replace the washed-out medium with chondrogenic differentiation medium, which typically contains high glucose Dulbecco&#39;s Modified Eagle Medium (DMEM), 1% ITS, 100 nM dexamethasone, 50 &#181;g/mL ascorbate-2-phosphate, 40 &#181;g/mL proline, and 10 ng/mL Transforming Growth Factor-beta 3 (TGF-&#946;3) (see Table of Materials).</li>
		<li>Maintain the cells in the chondrogenic differentiation medium, changing the medium every 2-3 days. After about three weeks, the DPSCs should have differentiated into chondrocyte-like cells.</li>
		<li>Fix the cells by incubating them with 4% paraformaldehyde (PFA) for 20 min at room temperature. After fixation, stain the cells with Alcian Blue (see Table of Materials) and visualize them under a microscope to confirm chondrogenic differentiation (Figure 5).</li>
		<li>Confirm the differentiation of DPSCs into chondrocytes using techniques such as Alcian Blue staining for proteoglycans or gene expression analysis of chondrogenic markers like aggrecan, SOX9, and type II collagen33,34.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The protocol outlines the isolation, culture, and characterization of dental pulp stem cells (DPSCs) from human deciduous and permanent teeth. It includes a description of the storage and proliferation of these cells, as well as the assessment of their in vitro differentiation potential into osteoblasts, adipocytes, and chondrocytes35.
Chen et al.36&#160;demonstrated that Dental Pulp Stem Cells (DPSCs) could be ob...

Stem cell research has flourished in biomedical science due to its promising applications in regenerative medicine and tissue engineering. Dental pulp stem cells (DPSCs), derived from the pulp tissue of both human deciduous and permanent teeth, have attracted significant interest as a source of stem cells due to their ready availability and multipotent capacity1,2. These cells have the potential to differentiate into various cell types, including adipocytes, osteoblasts, and chondrocytes, as confirmed by numerous studies3.
Over the past few decades, research and therapeutic applications of stem cells have surged. The expansive potential of stem cells calls for diversifying the sources from which they are obtained. Several factors influence the efficiency, viability, and stemness of chosen cells. Despite the existence of various known stem cell reservoirs, such as bone marrow and adipose tissues, the invasive procedures, site morbidity, and ethical concerns linked to these sources often limit their exploration4,5. Among the various stem cell sources, dental stem cells have gained attention due to their easy accessibility, high plasticity, and diverse potential applications. Human dental pulp stem cells, in particular, have been extensively researched for their therapeutic prospects6. Teeth, commonly discarded as medical waste, hold a wealth of mesenchymal stem cells7. Safeguarding this valuable stem cell pool requires collective efforts from patients, dentists, and doctors to ensure that these resources are not wasted, making each dental pulp stem cell available for future regenerative requirements.
Dental pulp-derived stem cells, such as human adult dental pulp stem cells (DPSCs) and stem cells from exfoliated human deciduous teeth (SHED), are located in the perivascular niche of the dental pulp. These cells are believed to originate from cranial neural crest cells and exhibit early markers for both mesenchymal stem cells (MSCs) and neuroectodermal stem cells. DPSCs and SHEDs have demonstrated multipotency and the ability to regenerate diverse tissue types8.
Potential sources of dental stem cells encompass healthy deciduous and permanent teeth. Stem cells constitute only about 1% of the total cell population in the pulp, highlighting the importance of effective isolation and expansion techniques9. Consequently, the extraction and expansion of these stem cells are pivotal steps in DPSC isolation10. Extracted or exfoliated teeth need to be stored in a nutrient-rich transport medium, such as phosphate-buffered saline (PBS) or Hanks-buffered saline solution (HBSS).
Obtaining dental pulp can be achieved through various methods, contingent on the tooth type7,11. For deciduous teeth with resorbed roots, extraction can be performed via the root apex. Similarly, sterile barbed broaches can be used to obtain pulp from permanent teeth with an immature open apex. In cases of permanent teeth with fully developed roots, accessing the pulp chamber involves separating the dental crown from the root. This is accomplished by cutting the tooth using a diamond disc at the cementoenamel junction. This incision exposes the pulp chamber, enabling retrieval of the pulp tissue12,13,14.
Dental pulp stem cells (DPSCs) can be isolated through enzymatic digestion (ED) or outgrowth from tissue explants (OG), also known as spontaneous growth. The ED method employs enzymes, primarily collagenase I and dispase, to break down the tissue into single-cell suspensions15,16. The OG method, simpler and quicker, entails chopping the pulp fragments and directly placing them into a culture plate, allowing cells to grow from the tissue explants17. Researchers have utilized and compared both techniques to assess cell proliferation rates, preservation of isolated stem cell properties, differentiation, and surface marker expression18. Establishing and standardizing protocols for acquiring DPSCs with high efficiency and stemness can pave the way for effective and safe therapies19. This protocol encompasses extracting DPSCs using enzymatic digestion, lab processing, preservation, and cell differentiation with colorimetric staining for adipogenesis, osteogenesis, and chondrogenesis.
The protocol outlined in this article presents a step-by-step procedure, beginning with the initial isolation of dental pulp from the tooth, followed by culture and maintenance of DPSCs in the laboratory, and concluding with their characterization using specific stem cell markers (Figure 1). The techniques for inducing these stem cells into different cell lineages, highlighting their multipotency, are also described.

<table><tbody><tr><td>3-isobuty-l-methyl-xanthine&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>I5879</td><td></td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>AS001</td><td></td></tr><tr><td>Alcian Blue&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>RM471</td><td></td></tr><tr><td>Alizarin Red S staining solution</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>GRM894</td><td></td></tr><tr><td>Alkaline phosphatase -Staining kit</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Alpha Minimum Essential Medium (&amp;alpha;-MEM)&amp;nbsp;</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td>Gibco</td></tr><tr><td>Alpha Minimum Essential Medium (&amp;alpha;-MEM)&amp;nbsp;</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td>Gibco</td></tr><tr><td>Alpha-MEM, or Alpha Minimum Essential Medium</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td>Gibco</td></tr><tr><td>Alpha-MEM, or Alpha Minimum Essential Medium</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td>Gibco</td></tr><tr><td>Antibiotic/Antimycotic</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>P4333</td><td></td></tr><tr><td>Ascorbate-2-phosphate</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>012-04802</td><td></td></tr><tr><td>Beta-glycerophosphate&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>G9422-10G</td><td></td></tr><tr><td>Biosafety cabinet-Laminar flow hood</td><td>Labconco Corporation,MO 64132-2696,USA</td><td></td><td></td></tr><tr><td>CD90, CD105, CD73, CD34, CD45, and HLA-DR</td><td>BioLegend, Inc.CA 92121,USA</td><td></td><td></td></tr><tr><td>Cell strainer (70 &amp;micro;m )</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>TCP025</td><td>Cell strainer</td></tr><tr><td>Centrifuge</td><td>REMI Elektrotechnik Limited (REMI)</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>1101 | 1102</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington Biochem. Corp. NJ 08701, USA</td><td></td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington Biochem. Corp. NJ 08701, USA</td><td></td><td></td></tr><tr><td>Complete Growth Medium</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>AT006</td><td>DMEM</td></tr><tr><td>Conical tubes (15 or 50 )</td><td>Thermo Fisher Scientific, MA, USA</td><td>546021P/546041P</td><td>15 mL and 50 mL</td></tr><tr><td>Cryo freezing container</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td>15-350-50</td><td></td></tr><tr><td>Cryolabels</td><td>Label India:</td><td></td><td></td></tr><tr><td>Cryovial storage boxes&amp;nbsp;</td><td>Cryostore Storage Boxes</td><td></td><td></td></tr><tr><td>Cryovials</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Cryovials (1.8 mL)</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td>PW1282</td><td>Self standing</td></tr><tr><td>Culture flask (25 cm&amp;sup2;)</td><td>Corning Inc.NY 14831,USA</td><td></td><td></td></tr><tr><td>Culture flasks</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>TCG4/TCG6</td><td>T25/T75</td></tr><tr><td>Culture Plates</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>TCP129/TCP008</td><td>60 mm/100 mm</td></tr><tr><td>Dental Diamond Discs</td><td>Komet SC 29730, USA</td><td></td><td>Komet&amp;nbsp;</td></tr><tr><td>Dental Spoon Excavator</td><td>Brasseler,GA 31419,USA</td><td>5023591U0</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>D4902-25MG</td><td></td></tr><tr><td>Dexamethosone</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>D4902-25MG</td><td></td></tr><tr><td>Dexamethosone</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>D4902-25MG</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>TC185</td><td></td></tr><tr><td>Dispase</td><td>Roche Diagnostics,Mannheim,Germany.</td><td></td><td></td></tr><tr><td>Dispase</td><td>Roche Diagnostics GmbH, Mannheim,Germany</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific, MA, USA</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Ethanol (70%)</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>MB106</td><td></td></tr><tr><td>Ethanol -70%</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td>Fisher Scientific</td></tr><tr><td>Extraction forceps&amp;nbsp;</td><td>Dentsply Sirona, USA</td><td></td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific Inc.,MA,USA</td><td>F2442-500ML</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific Inc.,MA,USA</td><td>F2442-500ML</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>RM9954</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific Inc.,MA,USA</td><td>F2442-500ML</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific Inc.,MA,USA</td><td>F2442-500ML</td><td></td></tr><tr><td>Fibronectin-coated tissue culture plate</td><td>Corning Inc.Corning, NY 14831,USA</td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences,CA 95131,USA</td><td></td><td></td></tr><tr><td>Flow cytometry buffer</td><td>BD Biosciences,CA 95131,USA</td><td></td><td></td></tr><tr><td>Glass cover slip 22 x 22 mm</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>TCP017</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Lonza Group Ltd,4002 Basel, Switzerland</td><td></td><td></td></tr><tr><td>High-speed dental handpiece&amp;nbsp;</td><td>NSK Ltd,Tokyo 8216, Japan</td><td></td><td>Ti-Max Z series</td></tr><tr><td>Horse Serum</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>IBMX, or 3-isobutyl-1-methylxanthine</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td></td><td></td></tr><tr><td>Indomethacin</td><td>Pfizer Inc. NY 10017,USA</td><td></td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS)</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td>I5523</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS)</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td>I5523</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS) premix</td><td>Corning Incorporated,MA 01876,USA</td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus Corp.,Tokyo 163-0914,Japan</td><td></td><td></td></tr><tr><td>Isopropanol (60% )</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>I9516</td><td></td></tr><tr><td>Isopropyl alcohol&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>MB063</td><td></td></tr><tr><td>Laminar flow hood</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td></td><td></td></tr><tr><td>Lidocaine mixed with epinephrine</td><td>DENTSPLY,NC 28277,USA</td><td></td><td>Citanest</td></tr><tr><td>Liquid Nitrogen</td><td>Air Liquide,75007 Paris,France</td><td></td><td></td></tr><tr><td>Liquid nitrogen storage tank</td><td>Cryo Scientific Systems Pvt. Ltd.</td><td></td><td></td></tr><tr><td>Micropipettes</td><td>Eppendorf AG,22339 Hamburg,Germany</td><td>30020</td><td>Accupipet-2-20 &amp;micro;L</td></tr><tr><td>Mini tissue grinder</td><td>Bio-Rad Lab, Inc. CA 94547,USA</td><td></td><td>ReadyPrep mini grinders</td></tr><tr><td>Minus 80 freezer</td><td>Blue Star Limited</td><td></td><td></td></tr><tr><td>Neubauer counting chamber</td><td>Marienfeld Superior,arktheidenfeld,Germany</td><td></td><td></td></tr><tr><td>Oil red O stain</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>1024190250</td><td></td></tr><tr><td>Osteogenic Differentiation Medium (ODM)&amp;nbsp;</td><td>STEMCELL Technologies Inc.Vancouver, BC, V5Z 1B3,Canada</td><td></td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>TCL119</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td><td></td><td></td></tr><tr><td>Phosphate Buffered Solution (PBS) without Ca&lt;sup&gt;++&lt;/sup&gt; and Mg&lt;sup&gt;++&lt;/sup&gt;</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>TS1101</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td></td><td>Gibco</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo Fisher Scientific,MA, USA</td><td></td><td>Gibco</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo Fisher Scientific, MA, USA</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo Fisher Scientific, MA, USA</td><td>P3813-1PAK</td><td>1x PBS, pH 7.4</td></tr><tr><td>Proline&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td></td><td></td></tr><tr><td>Scalpel Blade Size 15&amp;nbsp;</td><td>Swann-Morton Ltd, Sheffield, S6 2BJ,UK</td><td>BDF-6955C</td><td></td></tr><tr><td>Sodium Hypochlorite</td><td>HiMedia Laboratories&amp;nbsp; Ltd.Mumbai,India</td><td>AS102</td><td>4% w/v solution</td></tr><tr><td>Sterile centrifuge tubes</td><td>Tarsons Products Pvt. Ltd.</td><td></td><td></td></tr><tr><td>Sterile container -20 mL</td><td>3M Center, MN 55144-1000,USA</td><td></td><td>3 M</td></tr><tr><td>Sterile phosphate-buffered saline (PBS)</td><td>Sigma Aldrich, USA</td><td>P3813-1PAK</td><td>1x PBS, pH 7.4</td></tr><tr><td>Sterile pipettes (2, 5, and 10 mL )</td><td>Eppendorf AG,22339 Hamburg,Germany</td><td></td><td></td></tr><tr><td>Sterile pipettes and tips</td><td>Eppendorf India Limited</td><td></td><td></td></tr><tr><td>Surgical Blade Handle</td><td>Becton, Dickinson and Co.,NJ,USA</td><td>371030</td><td>BP Handle 3</td></tr><tr><td>Transforming Growth Factor-beta 3 (TGF-&amp;beta;3)</td><td>R&amp;amp;D Systems, Inc.MN 55413,USA</td><td></td><td></td></tr><tr><td>Transforming Growth Factor-beta 3 (TGF-&amp;beta;3)</td><td>R&amp;amp;D Systems, Inc.MN 55413,USA</td><td></td><td></td></tr><tr><td>Trypan Blue 0.4%&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td></td><td></td></tr><tr><td>Trypan Blue 0.4%&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>TCL046</td><td></td></tr><tr><td>Trypan Blue 0.4%&amp;nbsp;</td><td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td><td>TCL046</td><td></td></tr><tr><td>Trypsin-EDTA&amp;nbsp;</td><td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td><td></td><td></td></tr><tr><td>Trypsin-EDTA 0.25%</td><td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td><td></td><td></td></tr><tr><td>Water bath</td><td>Thermo Fisher Scientific ,MA 02451,USA</td><td>BSW-01D</td><td></td></tr></tbody></table>

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.

The successful execution of the outlined protocol yielded dental pulp stem cells (DPSCs) capable of multilineage differentiation, demonstrating their multipotency.
Viability assays 
The viability of the DPSCs was assessed using a Trypan Blue exclusion assay at various time points. The results show consistently high viability (greater than 95%) throughout the culture period, demonstrating the robustness of our isolation and culture protocol.
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Read this Scientific Article Protocol about Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth at JoVE.com

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

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 ...

isolation-culture-characterization-dental-pulp-stem-cells-from-human

Research

JoVE Journal

Bioengineering

1.3K Views.  Hamad Medical Corporation. Begin met het vastzetten van de getrokken menselijke tand op zijn plaats met een tandtang. Snijd vervolgens de tand af met behulp van een diamantschijf die is verbonden met een tandheelkundig handstuk en een waterkoelmiddel. Verwijder met een tandgraafmachine het pulpaweefsel van de tand.Gebruik vervolgens een steriel chirurgisch mes om het pulpaweefsel voorzichtig in kleine fragmenten te hakken. Plaats deze fragmenten vervolgens in een mini-weefselvermaler met PBS om een homogeen meng...

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetics

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

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

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

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

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

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 ...

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

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