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 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 effects^4,5. 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 IPCs^8,9.

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 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.

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 Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% Antibiotic-Antimycotic.
   2. Generate PDX1-lentivirus vectors by co-transfection of 10 µg each of the packaging and envelope plasmids and 20 µg of the targeted gene plasmid into HEK293FT cells using calcium phosphate transfection system, e.g., psPAX2, pMD2.G, and human pWPT-PDX1¹¹. Ensure that the cells are 80%-90% confluent during the time of infection.
   3. Collect the medium containing lentiviral particles at 48 and 72 h after transfection.
   4. Filter the collected medium through a 0.45 µm filter; concentrate the viruses with a centrifugal filter at 100 kDa nominal molecular weight cut-off (NMWCO).
2. PDX1 overexpression
   1. Use passage 3-5 hDPSC that are 70-80% confluent. Trypsinize and count the cells using a cell counter. Seed 1 x 10⁶ hDPSCs onto a 60 mm tissue culture-treated dish and incubate overnight.
   2. 24 h later, add fresh virus particles obtained from step 1.1.4 to transduce polybrene pre-treated cells (4 µg/mL polybrene, 30 min under 37 °C and 5% CO₂) at the desired multiplicity of infection (MOI). For example, in this case, MOI used is 20. Maintain the cells in the cell culture incubator for 24 h at 37 °C with 5% CO₂.
   3. After the 24 h infection period, discard the medium containing viral particles. Add fresh culture media and continue growing the cells for 48 h. All cultures are maintained at 37 °C and 5% CO₂.
   4. Check the aggregated morphology of the transfected cells before proceeding to the induction step. PDX1 transduction is confirmed by gene expression analysis (Supplemental Figure 1).
      NOTE: Check the cell morphology under a microscope before proceeding to the next steps.
   5. Harvest and proceed to a three-step induction protocol (step 1.3) as a microenvironmental induction approach.
3. Three-step induction protocol
   NOTE: The three-step induction protocol resulted in the series of pancreatic differentiation of hDPSCs to definitive endoderm, pancreatic endoderm/endocrine, and pancreatic beta-cells/IPCs using serum-free medium (SFM)-A, SFM-B, and SFM-C, respectively.
   1. Discard the culture medium, and then wash the transduced-hDPSCs with 1x phosphate-buffered solution (PBS).
   2. Add 0.25% trypsin-EDTA solution to the cells and incubate for 1 min at 37 °C, 5% CO₂.
   3. Add the culture medium to stop the trypsin activity and gently flush the cells to get the single cell suspension. Count the cells and aliquot 1 x 10⁶ cells into each collecting tube.
   4. Centrifuge the cell suspension at 468 x g (Relative Centrifugal Force: RCF), 4 °C, for 5 min. Discard the supernatant and save the cell pellet.
   5. Resuspend the pellet in 3 mL of first pancreatic induction medium, i.e., serum-free medium (SFM)-A. Seed the cells on a low attachment culture plate (60 mm). Maintain the cells at 37 °C and 5% CO₂ for 3 days.
      NOTE: Observe cells morphology under an inverted microscope before proceeding to the next step.
   6. Remove SFM-A and add 3 mL of the second induction medium, i.e., SFM-B. Maintain the cells for the next 2 days at 37 °C, 5% CO₂.
      NOTE: Observe cells morphology under an inverted microscope before proceeding to the next step.
   7. Remove SFM-B and add 3 mL of the third induction medium, i.e., SFM-C. Maintain the cells for the next 5 days in a cell culture incubator maintained at 37 °C with 5% CO₂. Change the medium every 48 h. Observe their morphology after 5 days.
      NOTE: Each induction medium is supplemented with different reagents as described; SFM-A: 1% bovine serum albumin (BSA), 1x insulin-transferrin-selenium (ITS), 4 nM of activin A, 1 nM of sodium butyrate, and 50 µM of beta-mercaptoethanol; SFM-B: 1% BSA, 1x ITS, and 0.3 mM taurine; and SFM C: 1.5% BSA, 1x ITS, 3 mM taurine, 100 nM of glucagon-like peptide (GLP)-1, 1 mM nicotinamide, and 1x non-essential amino acids (NEAAs).
   8. Make sure to check the cell morphology under an inverted microscope after each step. The cells will become more aggregated and float as colonies.
   9. Collect cell colonies for further analysis. Check for colony morphology and size. Perform pancreatic gene marker expression analysis and functional analysis (Glucose stimulated C-peptide secretion assay).

2. Non-integrative induction protocol

NOTE: The non-integrative protocol is the backbone protocol for delivering the IPCs with the three-step induction process as a microenvironmental induction approach^8,9.

1. Use passage 3-5 hDPSCs at 70%-80% confluency.
2. Discard the culture medium and wash hDPSCs with 1x PBS.
3. Add 0.25% trypsin-EDTA solution onto the cells and incubate for 1 min at 37 °C, 5% CO₂.
4. Add the culture medium to stop the trypsin activity and gently pipette the cells to obtain the single cell suspension. Count cells and aliquot 1 x 10⁶ cells into each collection tube.
   1. Centrifuge the cell suspension at 468 x g (RCF), 4 °C, 5 min. Discard the supernatant.
   2. Resuspend the pellet in SFM-A and seed onto a low attachment culture plate (60 mm). Culture the cells at 37 °C and 5% CO₂ for 3 days. Check the cell morphology under an inverted microscope.
   3. Remove SFM-A, add SFM-B (Day 3) and then maintain the cells for the next 2 days under 37 °C, 5% CO₂.
   4. Remove SFM-B, add SFM-C (Day 5) and then maintain the cells for the next 5 days under 37 °C, 5% CO₂. SFM-C is changed every 48 h.
      NOTE: Each induction medium is supplemented with different reagents as described; SFM-A: 1% BSA, 1x ITS, 4 nM of activin A, 1 nM of sodium butyrate, and 50 µM of beta-mercaptoethanol; SFM-B: 1% BSA, 1x ITS, and 0.3 mM taurine; and SFM C: 1.5% BSA, 1x ITS, 3 mM taurine, 100 nM of GLP-1, 1 mM nicotinamide, and 1x NEAAs.
   5. Make sure to check the cell morphology under the inverted microscope after each step and at Day 10.
      NOTE: The cells will become more aggregated and will float as colonies.
   6. Collect cell colonies for further analysis. Check for colony morphology and size. Perform pancreatic gene marker expression analysis and functional analysis (Glucose stimulated C-peptide secretion assay).

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 throughout ...

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

List of materials used in this article

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