Name: in vitro induction of human dental pulp stem cells toward pancreatic lineages
Uploaded: 2021-09-25T14:30:00
Description: Watch this Scientific Journal Video about In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages at JoVE.com

SK, WR, and QDL were supported by the Veterinary Stem Cell and Bioengineering Research Unit, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University. TO and PP were supported by Chulalongkorn Academic Advancement into Its 2nd Century Project. CS was supported by a research supporting grant of the Faculty of Veterinary Science, Chulalongkorn Academic Advancement into Its 2nd Century Project, Veterinary Stem Cell and Bioengineering Research Unit, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University, and Government Research Fund.

This work was performed in accordance with the Declaration of Helsinki and approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University. Human DPSCs (hDPSCs) were isolated from human dental pulp tissues extracted from both premolars and molars due to wisdom teeth issues. Informed consent was obtained from the patients under an approved protocol (HREC-DCU 2018/054).
1. Integrative induction protocol
<ol>
	<li>Preparation of lentiviral vector carrying PDX...

<ol>
	<li>Cho, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IDF+diabetes+atlas:+Global+estimates+of+diabetes+prevalence+for+2017+and+projections+for+2045.">IDF diabetes atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045.</a> Diabetes Research and Clinical Practice. 138, 271-281 (2018).</li><li>Danaei, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National,+regional,+and+global+trends+in+fasting+plasma+glucose+and+diabetes+prevalence+since+1980:+systematic+analysis+of+health+examination+surveys+and+epidemiological+studies+with+370+country-years+and+2.7+million+participants.">National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants.</a> Lancet. 378 (9785), 31-40 (2011).</li><li>Diabetes Care. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+hypoglycemia+in+diabetes.">Minimizing hypoglycemia in diabetes.</a> Diabetes Care. 38 (8), 1583 (2015).</li><li>Health Quality Ontario. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation:+an+evidence-based+analysis.">Islet transplantation: an evidence-based analysis.</a> Ontario Health Technology Assessment Series. 3 (4), 1-45 (2003).</li><li>Brennan, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+follow-up+of+the+Edmonton+protocol+of+islet+transplantation+in+the+United+States.">Long-term follow-up of the Edmonton protocol of islet transplantation in the United States.</a> American Journal of Transplantation. 16 (2), 509-517 (2016).</li><li>Korsgren, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+encapsulation:+Physiological+possibilities+and+limitations.">Islet encapsulation: Physiological possibilities and limitations.</a> Diabetes. 66 (7), 1748-1754 (2017).</li><li>Kuncorojakti, S., Srisuwatanasagul, S., Kradangnga, K., Sawangmake, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-Producing+Cell+Transplantation+Platform+for+Veterinary+Practice.">Insulin-Producing Cell Transplantation Platform for Veterinary Practice.</a> Frontiers in Veterinary Science. 7, 4 (2020).</li><li>Sawangmake, C., Nowwarote, N., Pavasant, P., Chansiripornchai, P., Osathanon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+feasibility+study+of+an+in+vitro+differentiation+potential+toward+insulin-producing+cells+by+dental+tissue-derived+mesenchymal+stem+cells.">A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells.</a> Biochemical and Biophysical Research Communications. 452 (3), 581-587 (2014).</li><li>Sawangmake, C., Rodprasert, W., Osathanon, T., Pavasant, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+protocols+for+an+in+vitro+generation+of+pancreatic+progenitors+from+human+dental+pulp+stem+cells.">Integrative protocols for an in vitro generation of pancreatic progenitors from human dental pulp stem cells.</a> Biochemical and Biophysical Research Communications. 530 (1), 222-229 (2020).</li><li>Kuncorojakti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate/Pluronic+F127-based+encapsulation+supports+viability+and+functionality+of+human+dental+pulp+stem+cell-derived+insulin-producing+cells.">Alginate/Pluronic F127-based encapsulation supports viability and functionality of human dental pulp stem cell-derived insulin-producing cells.</a> Journal of Biological Engineering. 14, 23 (2020).</li><li>Ritz-Laser, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+the+beta-cell+specific+transcription+factor+Pdx1+inhibits+glucagon+gene+transcription.">Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcription.</a> Diabetologia. 46 (6), 810-821 (2003).</li><li>Pampusch, M. S., Skinner, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+and+expansion+of+primary+T+cells+in+nine+days+with+maintenance+of+central+memory+phenotype.">Transduction and expansion of primary T cells in nine days with maintenance of central memory phenotype.</a> Journal of Visualized Experiments: JoVE. (157), (2020).</li><li>Fraga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+transfection+efficiency+of+pIDUA/nanoemulsion+complexes+in+a+mucopolysaccharidosis+type+I+murine+model.">Factors influencing transfection efficiency of pIDUA/nanoemulsion complexes in a mucopolysaccharidosis type I murine model.</a> International Journal of Nanomedicine. 12, 2061-2067 (2017).</li><li>Balak, J. R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+ex+vivo+lentiviral+transduction+of+primary+human+pancreatic+exocrine+cells.">Highly efficient ex vivo lentiviral transduction of primary human pancreatic exocrine cells.</a> Scientific Reports. 9 (1), 15870 (2019).</li><li>Balaji, S., Zhou, Y., Opara, E. C., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinations+of+Activin+A+or+nicotinamide+with+the+pancreatic+transcription+factor+PDX1+support+differentiation+of+human+amnion+epithelial+cells+toward+a+pancreatic+lineage.">Combinations of Activin A or nicotinamide with the pancreatic transcription factor PDX1 support differentiation of human amnion epithelial cells toward a pancreatic lineage.</a> Cellular Reprogramming. 19 (4), 255-262 (2017).</li><li>Spaeth, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+novel+role+for+the+Pdx1+transcription+factor+in+islet+&#946;-Cell+maturation+and+proliferation+during+weaning.">Defining a novel role for the Pdx1 transcription factor in islet &#946;-Cell maturation and proliferation during weaning.</a> Diabetes. 66 (11), 2830-2839 (2017).</li><li>Bastidas-Ponce, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxa2+and+Pdx1+cooperatively+regulate+postnatal+maturation+of+pancreatic+&#946;-cells.">Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic &#946;-cells.</a> Molecular Metabolism. 6 (6), 524-534 (2017).</li><li>Zhu, Y., Liu, Q., Zhou, Z., Ikeda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDX1,+Neurogenin-3,+and+MAFA:+critical+transcription+regulators+for+beta+cell+development+and+regeneration.">PDX1, Neurogenin-3, and MAFA: critical transcription regulators for beta cell development and regeneration.</a> Stem Cell Research &amp; Therapy. 8 (1), 240 (2017).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+transcriptome+profiling+of+progenitor-like+colonies+derived+from+adult+mouse+pancreas.">Culturing and transcriptome profiling of progenitor-like colonies derived from adult mouse pancreas.</a> Stem Cell Research &amp; Therapy. 8 (1), 172 (2017).</li><li>Tiedemann, H. B., Schneltzer, E., Beckers, J., Przemeck, G. K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hrabe+de+Angelis,+M.+Modeling+coexistence+of+oscillation+and+Delta/Notch-mediated+lateral+inhibition+in+pancreas+development+and+neurogenesis.">Hrabe de Angelis, M. Modeling coexistence of oscillation and Delta/Notch-mediated lateral inhibition in pancreas development and neurogenesis.</a> Journal of Theoretical Biology. 430, 32-44 (2017).</li><li>Xu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+promotes+the+differentiation+of+human+dental+pulp+mesenchymal+stem+cells+into+insulin-producing+cells+for+improving+the+diabetes+therapy.">Three-dimensional culture promotes the differentiation of human dental pulp mesenchymal stem cells into insulin-producing cells for improving the diabetes therapy.</a> Frontiers in Pharmacology. 10, 1576 (2019).</li><li>Grimm, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+under+microgravity+conditions-use+of+stem+cells+and+specialized+cells.">Tissue engineering under microgravity conditions-use of stem cells and specialized cells.</a> Stem Cells and Development. 27 (12), 787-804 (2018).</li><li>Tran, R., Moraes, C., Hoesli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+clustering+enhances+PDX1+and+NKX6.1+expression+in+pancreatic+endoderm+cells+derived+from+pluripotent+stem+cells.">Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells.</a> Scientific Reports. 10 (1), 1190 (2020).</li><li>Li, X. Y., Zhai, W. J., Teng, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+pancreatic+development.">Notch signaling in pancreatic development.</a> International Journal of Molecular Sciences. 17 (1), 48 (2015).</li><li>Motoyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+specific+soluble+factors+promotes+the+functional+maturation+of+transcription+factor-mediated,+pancreatic+transdifferentiated+cells.">Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells.</a> PLoS One. 13 (5), 0197175 (2018).</li><li>Baldan, J., Houbracken, I., Rooman, I., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+pancreatic+acinar+cells+dedifferentiate+into+an+embryonic+progenitor-like+state+in+3D+suspension+culture.">Adult human pancreatic acinar cells dedifferentiate into an embryonic progenitor-like state in 3D suspension culture.</a> Scientific Reports. 9 (1), 4040 (2019).</li><li>Wedeken, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+murine+pancreatic+progenitors+require+epidermal+growth+factor+and+nicotinamide+for+self-renewal+and+differentiation+in+a+serum-+and+conditioned+medium-free+culture.">Adult murine pancreatic progenitors require epidermal growth factor and nicotinamide for self-renewal and differentiation in a serum- and conditioned medium-free culture.</a> Stem Cells and Development. 26 (8), 599-607 (2017).</li><li>Trott, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+self-renewing+pancreatic+progenitors+derived+from+human+pluripotent+stem+cells.">Long-term culture of self-renewing pancreatic progenitors derived from human pluripotent stem cells.</a> Stem Cell Reports. 8 (6), 1675-1688 (2017).</li><li>Kim, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+EMSC-islet+co-localizing+composites+for+xenogeneic+porcine+islet+transplantation.">Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.</a> Biochemical and Biophysical Research Communications. 497 (2), 506-512 (2018).</li><li>Gauthaman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extra-embryonic+human+Wharton's+jelly+stem+cells+do+not+induce+tumorigenesis,+unlike+human+embryonic+stem+cells.">Extra-embryonic human Wharton's jelly stem cells do not induce tumorigenesis, unlike human embryonic stem cells.</a> Reproductive BioMedicine Online. 24 (2), 235-246 (2012).</li><li>Schiesser, J. V., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+beta+cells+from+human+pluripotent+stem+cells:+are+we+there+yet.">Generation of beta cells from human pluripotent stem cells: are we there yet.</a> Annals of the New York Academy of Sciences. 1311, 124-137 (2014).</li><li>Chmielowiec, J., Borowiak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+and+expansion+of+human+pluripotent+stem+cell-derived+pancreatic+progenitors.">In vitro differentiation and expansion of human pluripotent stem cell-derived pancreatic progenitors.</a> The Review of Diabetic Studies. 11 (1), 19-34 (2014).</li></ol>

Achieving higher IPCs production from MSCs plays an essential role in diabetes therapy. The critical steps of the integrative protocol rely on the quality of cells to be used for the transduction and the quality of transduced cells. Some cell requirements that should be checked for successful transduction are ensuring cell healthiness, cell banking management, and cells are in a mitotically active state. Further, monitoring the viability of transduced cells also plays an important role. Less successful transduction is ca...

Diabetes mellitus is an ongoing global concern. An International Diabetes Federation (IDF) report estimated that the global prevalence of diabetes would increase from 151 million in 2000 to 415 million in 20151,2. The latest epidemiology-based study has predicted that the estimated worldwide diabetes prevalence will increase from 451 million in 2017 to 693 million in 20451. The success of pancreatic islet transplantation using the Edmonton protocol was first demonstrated in 2000, when it was shown to maintain endogenous insulin production and stabilize the normoglycemic condition in type I diabetic patients3. However, the application of the Edmonton protocol still faces a bottleneck problem. The limited number of cadaveric pancreas donors is the main issue since each patient with type I diabetes requires at least 2-4 islet donors. Furthermore, the long-term use of immunosuppressive agents may cause life-threatening side effects4,5. To address this, the development of a potential therapy for diabetes in the past decade has mainly focused on the generation of effective insulin-producing cells (IPCs) from various sources of stem cells6.
Stem cells became an alternative treatment in many diseases, including diabetes type I, which is caused by the loss of beta-cells. Transplantation of IPCs is the new promising method for controlling blood glucose in these patients7. Two approaches for generating IPCs, integrative and non-integrative induction protocols, are presented in this article. The induction protocol mimicked the natural pancreatic developmental process to get the matured and functional IPCs8,9.
For this study, hDPSCs were characterized by flow cytometry for MSC surface marker detection, multilineage differentiation potential, and RT-qPCR to determine the expression of stemness property and proliferative gene markers (data not shown)8,9,10. hDPSCs were induced toward definitive endoderm, pancreatic endoderm, pancreatic endocrine, and pancreatic beta-cells or IPCs (Figure 1), respectively7. To induce the cells, a three-step induction approach was used as a backbone protocol. This protocol was called a non-integrative protocol. In the case of integrative protocol, the essential pancreatic transcription factor, PDX1, was overexpressed in hDPSCs followed by the induction of&#160;overexpressed PDX1 in hDPSCs using a three-step differentiation protocol. The difference between non-integrative and integrative protocol is the overexpression of PDX1 in integrative protocol and not in the non-integrative protocol. The pancreatic differentiation was compared between the integrative and non-integrative protocols in this study.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific Corporation, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#174; 60 mm TC-treated Culture Dish</td>
			<td>Corning&#174;</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>12800017</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#8482;</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) powder, pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>P3813-10PAK</td>
			<td>One pack is used for preparing 1 L of PBS solution with sterile DDI</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral Vector Carrying PDX1 Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon&#174; Ultra-15 Centrifugal Filter</td>
			<td>Merck Millipore, USA</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>Human pWPT-PDX1 plasmid</td>
			<td>Addgene</td>
			<td>12256</td>
			<td>Gift from Didier Trono; http://n2t.net/addgene:12256; RRID: Addgene_12256</td>
		</tr>
		<tr>
			<td>Millex-HV Syringe Filter Unit, 0.45&#160;&#181;m</td>
			<td>Merck Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>Gift from Didier Trono; http://n2t.net/addgene:12259; RRID: Addgene_12259</td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Merck Millipore</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2 plasmid</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Gift from Didier Trono; http://n2t.net/addgene:12260; RRID: Addgene_12260</td>
		</tr>
		<tr>
			<td>Three-step Induction Protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A Recombinant Human Protein</td>
			<td>Merck Millipore</td>
			<td>GF300</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA, Cohn fraction V, fatty acid free)</td>
			<td>Sigma-Aldrich</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucagon-like peptide (GLP)-1</td>
			<td>Sigma-Aldrich</td>
			<td>G3265</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids (NEAAs)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-treated cell culture dish, 60mm</td>
			<td>Eppendorf</td>
			<td>30701011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 this article, the outcomes of both the induction protocols were compared. The diagrams of both induction protocols are illustrated in Figure 2A,C. In both the protocols, the evaluation was performed under a light microscope, and images were analyzed with ImageJ. hDPSCs were able to form colony-like structures from the first day of induction in both induction protocols. The colony&#39;s morphology was round and dense, and all colonies floated in the culture vessels through...

Watch this Scientific Journal Video about In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages at JoVE.com

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).

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. ...

This video is showing the in vitro induction of human dental pulp stem cells toward pancreatic lineages. Stem cells give an alternative treatment in many diseases including diabetes type I which cause biolaging of beta cells. Transplantation of insulin producing cells will be the new promising method for causing lifelong.In this video two approaches for generating insulin producing cell, including the integrative and non integrative induction protocol are described. And in this part the integration protocol for inducing insulin producing cells from human dental pulp stem cell is explained. For the induction protocol is the for mixing the the natural pancreatic developmental process.To get the natural and the functional insulin producing cells. For generating insulin producing cells from mesenchymal stem cells like dental pulp stem cells, multi step induction protocol is employed. Mesenchymal stem cells are used in the word definitive endoderm, pancreatic endoderm, pancreatic endocrine and then pancreatic beta-cell or insulin producing cells respectively.To induce the cells, three step induction approach is used as a backbone protocol. The integrative protocol employed the over expiration of the essential pancreatic transcription factor, PDX-1, by human dental pulp stem cells. Then the PDX-1 over expressed stem cells will be further induced using three step differentiation protocol.The pancreatic differentiation potential, by the integrative protocol, will be compared with the non integrative protocol as illustrated in the animation. This idea was performed under the approved protocol by the Human Research Ethic Committee, Faculty of Dentistry, Chulalongkorn University with patients'informed consent. This protocol started with over expiration of PDX-1.Human dental pulp stem cells in parts of 2 to 5, with 70-80%confluence, are used for the transduction. Cells are trypsinized and counted. Then one million of cells are seated onto the transfection vessel and incubated overnight.24 hours later freshly prepared antivirus carrying PDX-1 gene is added to the cultured vessel according to the desired multiplicity of infection. The transfected cells are then kept in the incubator for 24 hours. Then fresh cultured medium is changed and maintained for another 48 period.Morphology of transfected cells is checked before using for further induction step. The human dental pulp stem cells with PDX-1 over expiration are then Tryptimized and suspended in the first pancreatic induction medium, so called medium A.In this step, low attachment cultured vessel is used. Morphology of PDX-1 over expressed human dental pulp stem cells on day 3 after Medium A induction is observed.Cells with good morphology will be then used for further inductions step. Next step, the second induction medium, so called medium B is added to the cultured vessel. The morphology of PDX-1 over expressed human dental pulp stem cells on day two after medium induction is observed.Cells with good morphology will be then used for further induction step. Next step, the third induction media, so called medium C is added to the cultured vessel. 5 days later cell morphology is observed.Cell colonies are collected for further analysis. Including colony morphology and size, pancreatic gene marker expression and functional property regarding Glucose-stimulated C-peptide secretion or GSCS assay. For the non-integrative induction approach, three step induction protocol mentioned previously is employed.One million of cells are re-suspended in the medium A and seated on to low attachment culture vessel. Then cultured for three days. After morphology are checking, series of induction medium, including medium B and C are subsequently added to the cultured vessel on day three and five, respectively.At day 10, cell morphology is observed. Cell colonies are collected for further analysis, including colony morphology and size, pancreatic gene marker expression, and functional property regarding Glucose-stimulated C-peptide secretion or GSCS assay. The outcomes of both induction protocols are compared in both protocols.Human dental pulp stem cells are able to form colony like structure since the very first day of the induction. The colonies appearance is round and dense. All of the colonies are floating in the culture vessels throughout the induction period.The total colony count of the integrative induction protocol is a bit higher from that of non-integrative induction. The evolution under colony sized percentage shows a beneficial trend, from small to medium sized colony formation upon integrative induction, which is important to reduce the necrotic core inside the colony. Pancreatic gene marker analysis revealed that the integrative inductive protocol yields the colonies with high expiration of pancreatic markers, including MAP-A, GLUT-2, insulin, and GLP-1R, suggesting the differentiation toward the measured insulin producing cells.To the function glucose C-peptide secretion is important. The results show that both induction protocol yields the colonies with good response to glucose challenge. From this study, both of pancreatic induction protocols are able to generate the insulin producing cells in vitro in term of pancreatic marker expiration and functional property, the protocols shows a better quality of insulin producing cell formation.Suggesting the trends of human mesenchymal stem cells like human dental pulp stem cell application in stem cell based diabetes treatment in the near future.

Research

JoVE Journal

Bioengineering

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 ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

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

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 ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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 ...

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, ...