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

Summary

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).

Abstract

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.

Introduction

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 2015^1,2. 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....

Protocol

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 PDX1
   1. Use human embryonic kidney (HEK) 293FT cells for viral packaging. Culture and maintain these cells in Dulbecco's Modified Eagle....

Results

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.......

Discussion

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.......

Materials

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