Name: isolation, propagation, and prion protein expression during neuronal differentiation of human dental pulp stem cells
Uploaded: 2019-03-18T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells at JoVE.com

This work was supported by &#34;Fondazione Varrone&#34; and Rieti University Hub &#34;Sabina Universitas&#34; to Vincenzo Mattei.
Figure 5 (A, B) reprinted by permission of the publisher Taylor &#38; Francis Ltd from: Role of Prion protein-EGFR multimolecular complex during neuronal differentiation of human dental pulp-derived stem cells. Martellucci, S., Manganelli V., Santacroce C., Santilli F., Piccoli L., Sorice M., Mattei V. Prion. 2018 Mar 4. Taylor &#38; Francis Ltd.

Third molars used in the study were excised from patients (13-19 years old) with no prior history of drug or alcohol consumption, all non-smoking and with appropriate oral hygiene. On the day of explanation, at the Department of Science Dentistry and Maxillofacial of &#34;Sapienza&#34; University of Rome, informed consent was obtained from the patients or the parents. Informed consent was obtained based on ethical considerations and approval of the ethics committee.
1. Tooth and Dental Pulp Extr...

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In this work, we focused on methodology for isolation and neuronal differentiation of hDPSCs; moreover, we evaluated the role of PrPC in this process. There are several methods to isolate and differentiate hDPSCs in neuron-like cells and critical steps during the process. hDPSCs are able to differentiate in several lineages such as chondroblasts, adipocytes, osteoblasts, and neurons. In our paper, we investigated the mechanisms of neuronal differentiation and the presence of PrPC. As discussed above...

Mesenchymal stem cells have been isolated from several tissues, including bone marrow, umbilical cord blood, human dental pulp, adipose tissue, and blood1,2,3,4,5,6. As reported by several authors, hDPSCs show plastic adherence, a typical fibroblast-like morphology. These represent a highly heterogeneous population with distinct clones and differences in proliferative and differentiating capacity7,8. hDPSCs express specific markers for mesenchymal stem cells (i.e. CD44, CD90, CD73, CD105, STRO-1), they are negative for some hematopoietic markers (such as CD14 and CD19) and are capable of in vitro multilineage differentiation9,10,11.
Several authors have shown that these cells are able to differentiate into neuron-like cells using different protocols, that include the addition of NGF, bFGF, EGF in combination&#160;with&#160;the specific media and supplements7,12. Also, many proteins are involved during neuronal differentiation process and, among these, several papers show a relevant role and significant expression of cellular prion protein (PrPC), both in embryonic and adult stem cells13,14. PrPC represents a pleiotropic molecule capable of performing different functions inside cells as copper metabolism, apoptosis, and resistance to oxidative stress15,16,17,18,19,20,21,22.
In our previous paper23, we investigated the role of PrPC in the hDPSCs neuronal differentiation process. In fact, hDPSCs express precociously PrPC and, after neuronal differentiation, it was possible to observe an additional increase. Other authors hypothesized a possible role of PrPC in neuronal differentiation processes of stem cells. Indeed, PrPC drives the differentiation of human embryonic stem cells into neurons, oligodendrocytes, and astrocytes24. The purpose of this study was to emphasize the methodology for obtaining stem cells from dental pulp, its differentiation process and the role of PrPC during neuronal differentiation.

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			<td height="21" style="height:21px;width:269px;">Amphotericin B solution</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2942</td>
			<td style="width:1791px;">It is use to supplement cell culture media, it is a polyene antifungal antibiotic from Streptomyce</td>
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			<td height="21" style="height:21px;">Anti-B3tubulin</td>
			<td>Cell Signaling Technology&#160;</td>
			<td style="width:119px;">#4466</td>
			<td>One of six B-tubulin isoform, it is expressed highly during fetal and postnatal development, remaining high in the peripheral nervous system</td>
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			<td height="21" style="height:21px;width:269px;">Anti-CD105&#160;</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">611314</td>
			<td>Endoglin (CD105), a major glycoprotein of human vascular endothelium, is a type I integral membrane protein with a large extracellular region, a hydrophobic transmembrane region, and a short cytoplasmic tail</td>
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			<td height="21" style="height:21px;width:269px;">Anti-CD44</td>
			<td style="width:161px;">Millipore</td>
			<td style="width:119px;">CBL154-20ul</td>
			<td>Positive cell markers antibodies directed against mesenchymal stem cells</td>
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			<td height="21" style="height:21px;">Anti-CD73&#160;</td>
			<td>Cell Signaling Technology&#160;</td>
			<td style="width:119px;">13160</td>
			<td>CD73 is a 70 kDa glycosyl phosphatidylinositol-anchored, membrane-bound glycoprotein that catalyzes the hydrolysis of extracellular nucleoside monophosphates into bioactive nucleosides</td>
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			<td>Millipore</td>
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			<td>Positive cell markers antibodies directed against mesenchymal stem cells</td>
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			<td height="21" style="height:21px;">Anti-GAP43&#160;</td>
			<td>Cell Signaling Technology&#160;</td>
			<td>#8945</td>
			<td>Is a nervous system specific, growth-associated protein in growth cones and areas of high plasticity</td>
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			<td height="21" style="height:21px;">Anti-mouse PE&#160;</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab7003</td>
			<td>Is an antibody used in in flow cytometry or FACS analysis</td>
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			<td height="21" style="height:21px;">Anti-NFH&#160;</td>
			<td>Cell Signaling Technology&#160;</td>
			<td style="width:119px;">#2836</td>
			<td>Is an antibody that detects endogenous levels of total Neurofilament-H protein</td>
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			<td height="21" style="height:21px;">Anti-PrP mAb EP1802Y&#160;</td>
			<td>Abcam</td>
			<td style="width:119px;">ab52604</td>
			<td>Rabbit monoclonal [EP 1802Y] to Prion protein PrP</td>
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			<td height="21" style="height:21px;">Anti-rabbit CY5&#160;</td>
			<td>Abcam</td>
			<td style="width:119px;">ab6564</td>
			<td>Is an antibody used in in flow cytometry or FACS analysis</td>
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			<td height="21" style="height:21px;">Anti-STRO 1</td>
			<td style="width:161px;">Millipore</td>
			<td style="width:119px;">MAB4315-20ul</td>
			<td>Positive cell markers antibodies directed against mesenchymal stem cells</td>
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			<td height="42" style="height:42px;width:269px;">B27 Supp XF CTS</td>
			<td style="width:161px;">Gibco by life technologies</td>
			<td style="width:119px;">A14867-01</td>
			<td>B-27&#160; can be used to support induction of human neural stem cells (hNSCs) from pluripotent stem cells (PSCs), expansion of hNSCs, differentiation of hNSCs, and maintenance of mature differentiated neurons in culture</td>
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			<td height="21" style="height:21px;">BD Accuri C6 flow cytometer&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:119px;">AC6531180187</td>
			<td>Flow cytometer equipped with a blue laser (488 nm) and a red laser (640 nm)</td>
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			<td height="21" style="height:21px;">BD Accuri C6 Software&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:119px;"></td>
			<td>Controls the BD Accuri C6 flow cytometer system in order to acquire data, generate statistics, and analyze results</td>
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			<td height="21" style="height:21px;width:269px;">bFGF</td>
			<td style="width:161px;">PeproThec, DBA</td>
			<td style="width:119px;">100-18B</td>
			<td>basic Fibroblast Growth Factor&#160;</td>
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			<td height="21" style="height:21px;width:269px;">Centrifuge CL30R</td>
			<td style="width:161px;">Termo fisher Scientific</td>
			<td style="width:119px;">11210908</td>
			<td>it is a device that is used for the separation of fluids,gas or liquid, based on density</td>
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			<td height="21" style="height:21px;width:269px;">CO2 Incubator 3541</td>
			<td style="width:161px;">Termo fisher Scientific</td>
			<td style="width:119px;">317527-185</td>
			<td>it ensures optimal and reproducible growth conditions for cell cultures</td>
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			<td height="21" style="height:21px;">Collagenase, type IV&#160;</td>
			<td>Life Technologies</td>
			<td style="width:119px;">17104019</td>
			<td>Collagenase is a protease that cleaves the bond between a neutral amino acid (X) and glycine in the sequence Pro-X-Glyc-Pro, which is found with high frequency in collagen</td>
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			<td height="21" style="height:21px;width:269px;">Disposable scalpel&#160;</td>
			<td style="width:161px;">Swann-Morton</td>
			<td style="width:119px;">501</td>
			<td>It is use to cut tissues</td>
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			<td height="21" style="height:21px;width:269px;">DMEM-L</td>
			<td>Euroclone</td>
			<td style="width:119px;">ECM0060L</td>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium Low Glucose with L-Glutamine with Sodium Pyruvate</td>
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			<td height="21" style="height:21px;width:269px;">EGF</td>
			<td style="width:161px;">PeproThec, DBA</td>
			<td style="width:119px;">AF-100-15</td>
			<td>Epidermal Growth Factor&#160;</td>
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			<td height="42" style="height:42px;width:269px;">Fetal Bovine Serum</td>
			<td style="width:161px;">Gibco by life technologies</td>
			<td style="width:119px;">10270-106</td>
			<td>FBS is a popular media supplement because it provides a wide array of functions in cell culture. FBS delivers nutrients, growth and attachment factors and protects cells from oxidative damage and apoptosis by mechanisms that are difficult to reproduce in serum-free media (SFM) systems</td>
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			<td height="42" style="height:42px;width:269px;">Filtropur BT50 0.2,500ml Bottle top filter</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">831,823,101</td>
			<td>it is a device that is used for filtration of solutions</td>
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			<td height="21" style="height:21px;width:269px;">Flexitube GeneSolution for PRNP</td>
			<td style="width:161px;">Qiagen</td>
			<td style="width:119px;">GS5621</td>
			<td>4 siRNAs for Entrez gene 5621. Target sequence N.1 TAGAGATTTCATAGCTATTTA&#160; N.2 CAGCAAATAACCATTGGTTAA&#160; N.3. CTGAATCGTTTCATGTAAGAA&#160; N.4&#160; CAGTGACTATGAGGACCGTTA</td>
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			<td height="42" style="height:42px;width:269px;">Hank&#39;s solution 1x</td>
			<td style="width:161px;">Gibco by life technologies</td>
			<td style="width:119px;">240200083</td>
			<td>The essential function of Hanks&#8242; Balanced Salt solution is to maintain pH as well as osmotic balance. It also provides water and essential inorganic ions to cells</td>
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			<td height="21" style="height:21px;width:269px;">HiPerFect Transfection Reagent&#160;</td>
			<td style="width:161px;">Qiagen</td>
			<td style="width:119px;">301705</td>
			<td>HiPerFect Transfection Reagent is a unique blend of cationic and neutral lipids that enables effective siRNA uptake and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations</td>
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			<td height="42" style="height:42px;width:269px;">Neurobasal A&#160;</td>
			<td style="width:161px;">Gibco by life technologies</td>
			<td style="width:119px;">10888022</td>
			<td>Neurobasal-A Medium is a basal medium designed for long-term maintenance and maturation of pure post-natal and adult brain neurons&#160;</td>
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			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">30525-89-4</td>
			<td>Paraformaldehyde has been used for fixing of cells and tissue sections during staining procedures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">penicillin/streptomycin&#160;</td>
			<td style="width:161px;">Euroclone</td>
			<td style="width:119px;">ECB3001D&#160;</td>
			<td>It is use to supplement cell culture media to control bacterial contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline&#160; (PBS)</td>
			<td style="width:161px;">Euroclone</td>
			<td style="width:119px;">ECB4004LX10&#160;</td>
			<td>PBS is a balanced salt solution used for the handling and culturing of mammalian cells. PBS is used to to irrigate, wash, and dilute mammalian cells. Phosphate buffering maintains the pH in the physiological range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">TC-Platte 6 well, Cell+,F</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">833,920,300</td>
			<td>It is a growth surface for adherent cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Tissue culture flask T-25,Cell+,Vented Cap</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">833,910,302</td>
			<td>Tissue culture flask T-25, polystyrene, Cell+ growth surface for sensitive adherent cells, e.g. primary cells, canted neck, ventilation cap, yellow, sterile, Pyrogen-free, non-cytotoxic, 10 pcs./bag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td>Widely used non-ionic surfactant for recovery of membrane components under mild non-denaturing conditions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Trypsin-EDTA&#160;</td>
			<td style="width:161px;">Euroclone</td>
			<td>ECB3052D&#160;</td>
			<td>Trypsin will cleave peptides on the C-terminal side of lysine and arginine amino acid residues. Trypsin is used to remove adherent cells from a culture surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tube</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">62,554,502</td>
			<td>Tube 15ml, 120x17mm, PP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">VBH 36 C2 Compact</td>
			<td style="width:161px;">Steril</td>
			<td>ST-003009000</td>
			<td>Offers totally protection for the enviroment and worker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZEISS Axio Vert.A1 &#8211; Inverted&#160;Microscope</td>
			<td>Zeiss</td>
			<td style="width:119px;">3849000962</td>
			<td>ZEISS Axio Vert.A1 provides a unique entry level price and can provide all contrasting techniques, including brightfield, phase contrast, PlasDIC, VAREL, improved Hoffman Modulation Contrast (iHMC), DIC and fluorescence. Incorporate LED illumination for gentle imaging for fluorescently-labeled cells. Axio Vert.A1 is ergonomically designed for routine work and compact enough to sit inside tissue culture hoods.</td>
		</tr>
	</tbody>
</table>

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.

The isolation and separation procedures of hDPSCs from dental pulp, obtained from the third molar, are complex processes in which small changes can lead a ruinous result. In this paper, we use the protocol of Arthur et al.12 with several improvements. A representative scheme of procedures is shown in Figure 1.
hDPSCs represents a heterogeneous population of cells wit...

Watch this Scientific Journal Video about Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells at JoVE.com

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

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 ...

The standard way to obtain a stem cell from dental pulp is poorly efficient, and this depends on several variable. Our protocol allowed to obtain a successful isolation of stem cell from treated teeth in 90%of the cases. Obtaining a high number of cells in a few weeks is the main advantage.That allow us to use the cell in regenerative medicine and to create a biobank providing other scientists with this useful tool. The procedure for opening the tooth will be performed by Dr.Constantino Santacroce. Subsequently, the protocol will be shown by Stefano Martellucci, the PhD student from my lab, who is the first author of the article.To begin, extract the third molar from the patient, quickly rinse it with PBS, put in a 15-milliliter test tube with the medium, and transfer it to the laboratory in less than two hours. Under a biohazard hood, use a cutter to open the tooth by a coronal cutting pass, parallel and tangent, through the roof of the pulp chamber. With a small excavator, gently remove the pulp, and place it in a test tube.Then, wash with PBS three times, and centrifuge at 2, 500 times g at room temperature for 10 minutes. After the centrifugation, remove the supernatant. Resuspend the pellet in Hank's solution, and transfer the sample to a Petri dish.Remove the Hank's solution by centrifugation at 2, 500 times g at room temperature for 10 minutes. Then, with a disposable scalpel, divide the pulp into approximately one-millimeter slices, and add one milliliter of type IV collagenase. Next, centrifuge the sample at 2, 500 times g at room temperature for 10 minutes.Remove the supernatant, and resuspend the pellet in the medium. Culture it in a T25 flask specific for stem cells at 37 degrees Celsius in 5%carbon dioxide. During the incubation period, change the culture medium every three days.Once the adherent cells reach confluence, add one milliliter of trypsin-EDTA solution to the cells to detach them, and incubate the cells at 37 degrees Celsius for three minutes. Next, add four milliliters of the culture medium in a one-to-five ratio to stop the trypsin action, and centrifuge the cell suspension at 259 times g for six minutes. Remove the supernatant.Then, place the cells in a T25 flask to propagate. Then, detach the cells with one milliliter of trypsin-EDTA at 37 degrees Celsius for three minutes. Centrifuge the cell suspension at 259 times g for six minutes.Remove the supernatant, and proceed to test the cells for cytofluorimetric analysis. To perform the transient prion protein silencing step, first culture the human dental pulp stem cells in six-well plates with two milliliters of the culture medium for 24 hours. Then, add 400 microliters of the small interfering RNA PrP medium to each sample, and incubate them at 37 degrees Celsius for six hours.Without discarding siRNA PrP medium, add 1.6 milliliters of the culture medium in a one-to-five ratio, and leave the cells at 37 degrees Celsius for 72 hours. After the incubation period, remove the supernatant, and wash the cells with two milliliters of PBS three times at room temperature. Then, add two milliliters of the neuronal culture medium, and keep the cells for seven to 14 days in an incubator at 37 degrees Celsius.After the incubation period, wash the cells with two milliliters of PBS three times at room temperature, and proceed to Western blot analysis to test for neuronal surface antigens. To perform the neuronal induction process, culture 2 million human dental pulp stem cells in six-well plates, up to 28 days from the pulp separation. Every three days, discard the supernatant.Wash three times with two milliliters of PBS at room temperature, and replace two milliliters of the neuronal culture medium. After seven to 14 days, detach the cells with one milliliter of trypsin-EDTA at 37 degrees Celsius for three minutes. Then, add four milliliters of the culture medium in a one-to-five ratio to stop the trypsin action.To characterize the human dental pulp stem cells by flow cytometry, culture 2 million per milliliter of the cells in six-well plates with two milliliters of the culture medium. At 28 days post-dental pulp separation or after an additional 14 days with neuronal culture medium, add one milliliter of the trypsin-EDTA at 37 degrees Celsius for three minutes to detach the cells. Next, add four milliliters of the culture medium in a one-to-five ratio to stop the trypsin action, and centrifugate at 259 times g at room temperature for six minutes.Fix the untreated or treated cells with 300 microliters of 4%paraformaldehyde in PBS at four degrees Celsius for 10 minutes. Next, centrifuge, discard the supernatant, and permeabilize the cells with 1%non-ionic surfactant in PBS at room temperature for an additional 10 minutes. Then, centrifuge again, discard the supernatant, and perform the blocking with 5%nonfat dried milk in one milliliters of PBS at room temperature for one hour.Wash three times with one milliliter of PBS, and incubate the cells with anti-CD105, anti-CD44, anti-STRO-1, anti-CD90, anti-CD73, anti-beta-three-tubulin, anti-NFH, and anti-GAP43 monoclonal antibodies at room temperature for one hour. Next, centrifuge the cells again three consecutive times with one milliliter of PBS at six-minute intervals, and then incubate with anti-mouse PE or anti-rabbit CY5 monoclonal antibodies at room temperature for one additional hour. Use the secondary antibodies for gating the immune-positive cells, and analyze all samples with a flow cytometer.Culture 2 million cells per milliliter of the human dental pulp stem cells in six-well plates with two milliliters of the culture medium. At 21 and 28 days post-dental pulp separation or after an additional seven to 14 days with the neuronal culture medium, add one milliliter of the trypsin-EDTA at 37 degrees Celsius for three minutes to detach the cells. Then, centrifuge, discard the supernatant, and then fix the specimens with 4%paraformaldehyde in PBS at four degrees Celsius for 10 minutes.Next, centrifuge, discard the supernatant, and then permeabilize with 1%non-ionic surfactant in PBS at room temperature for 10 minutes. Remove the supernatant, and stain the cells with rabbit anti-PrP monoclonal antibody at room temperature for one hour. Finally, centrifuge, discard the supernatant, and then incubate with anti-rabbit CY5 monoclonal antibody at room temperature for an additional hour.Analyze all samples with a flow cytometer. After pulp separation, clusters of human dental pulp stem cells were expanded on the periphery. A comparison between the growth of these cells before and after the neuronal differentiation induced by EGF/bFGF showed significant changes in the cell morphology and neurites outgrowth.Untreated human dental pulp stem cells expressed multipotent mesenchymal stromal specific surface antigens, such as CD44, CD90, CD105, CD73, and STRO-1. After appropriate neuronal induction stimuli, these cells expressed specific neuronal markers, such as beta-three-tubulin, NFH, and GAP43. Untreated or treated cells did not express hematopoietic markers, such as CD14 and CD19.After 28 days, prion protein was precociously expressed in human dental pulp stem cells compared with the corresponding negative control, and its expression was increased after the neuronal differentiation process induced by EGF/bFGF. Western blot analysis showed that silencing the prion protein by sRNA before EGF/bFGF stimuli could affect the neuronal differentiation process of human dental pulp stem cells by preventing the expression of neuronal markers beta-three-tubulin and NHF. The most important thing is the choice of the enzyme to separate the cells from the pulp.In fact, if the enzyme is too aggressive, the cells could be damaged and their growth and differentiation capacity could be compromised. The dental pulp stem cells are an excellent experimental model which can be employed in vivo and in vitro studies in the regenerative medicine field. Since PrP plays a key role in neuronal differentiation process of dental pulp stem cells, this marker may be an excellent candidate as effective target in neurodegenerative diseases.The most important aspect to advise is the health condition of the patients, such as the absence of infectious diseases.

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Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...