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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-isobuty-l-methyl-xanthine&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>AS001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue&#160;</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 (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</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&#160;</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 &#181;m )</td>
			<td>HiMedia Laboratories&#160; 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&#160; Ltd.Mumbai,India</td>
			<td>1101 | 1102</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 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&#160; 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&#160;</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&#178;)</td>
			<td>Corning Inc.NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCG4/TCG6</td>
			<td>T25/T75</td>
		</tr>
		<tr>
			<td>Culture Plates</td>
			<td>HiMedia Laboratories&#160; 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&#160;</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&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160; 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&#160;</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&#160; 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&#160; Ltd.Mumbai,India</td>
			<td>TCP017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;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&#160;</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&#160;</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 &#181;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)&#160;</td>
			<td>STEMCELL Technologies Inc.Vancouver, BC, V5Z 1B3,Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</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++ and Mg++</td>
			<td>HiMedia Laboratories&#160; 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&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade Size 15&#160;</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&#160; 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-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</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.
<st...

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

1.1K Views.  Hamad Medical Corporation. 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.

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 ...

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 ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

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 ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...