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Bioengineering

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

Published: September 25th, 2021

DOI:

10.3791/62497

1Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science, Chulalongkorn University, 2Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga, 3Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, 4Center of Excellence in Regenerative Dentistry, Faculty of Dentistry, Chulalongkorn University, 5Veterinary Stem Cell and Bioengineering Research Unit, Faculty of Veterinary Science, Chulalongkorn University, 6Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University
* These authors contributed equally

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

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

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 typ....

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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

  1. Preparation of lentiviral vector carrying PDX.......

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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's morphology was round and dense, and all colonies floated in the culture vessels through.......

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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.......

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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.

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

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