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.

in-vitro-induction-human-dental-pulp-stem-cells-toward-pancreatic

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 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 ...

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 indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

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

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

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. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

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)

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 ...

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 similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic 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

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

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

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

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 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 ...

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 robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

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 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, ...