Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Summary

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Abstract

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 µm pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

Introduction

Mesenchymal stem cells (MSCs), with the ability of differentiation and contribution to regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose, have been isolated from almost all tissues in the adult body^1,2. Other than providing tissue homeostasis by producing resident cells in case of chronic inflammation or an injury, they produce vital cytokines and growth factors to orchestrate angiogenesis, immune system, and tissue remodeling³. The interaction of MSCs with cancer tissue is not well-understood, but accumulating evidence suggests that....

Protocol

Written informed consent of the patients was obtained after the approval from the Institutional Ethics Committee.

1. DPSC Isolation and Culture

1. Transfer wisdom teeth obtained from young adults aged between 17 and 20 to 15 mL tubes containing complete Dulbecco's Modified Eagle Medium (DMEM) [low glucose DMEM media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin (PSA) solution], within 8 h after resection. Keep the tissue material cold (4 °C) during transfer to avoid potential cell death.
2. Remove the pulp tissue by sterile extraction forceps from the center of the tooth carefull....

Results

Figure 1 depicts the general MSC characteristics of DPSCs under culture conditions. DPSCs exert fibroblast-like cell morphology after plating (Figure 1B). MSC surface antigens (CD29, CD73, CD90, CD105, and CD166) are highly expressed while hematopoietic markers (CD34, CD45, and CD14) are negative (Figure 1C). Changes at the morphological and molecular level related to osteo-, chondro-, and adipo-geni.......

Discussion

Contribution of MSCs to tumor environment is regulated by several interactions including hybrid cell generation via cell fusions, entosis or cytokine and chemokine activities between stem cells and cancer cells²⁸. Structural organization, cell-cell interactions, and secreted factors determine cancer cell behavior in terms of tumor promotion, progression, and metastasis to surrounding tissue. Proper ex vivo model systems to investigate the mechanisms behind the interactions of res.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	11885084	For cell culture
FBS	Invitrogen	16000044	For cell culture
PSA	Lonza	17-745E	For cell culture
Trypsin	Invitrogen	25200056	For cell dissociation
PBS	Invitrogen	10010023	For washes
Dexamethasone	Sigma	D4902	Component of differentiation media
β-Glycerophosphate	Sigma	G9422	Component of osteogenic differentiation medium
Ascorbic acid	Sigma	A4544	Component of osteo- and chondro-genic differentiation medium
Insulin-Transferrin-Selenium (ITS −G)	Invitrogen	41400045	Component of chondrogenic differentiation medium
TGF-β	Sigma	SRP3171	Component of chondrogenic differentiation medium
Insulin	Sigma	I6634	Component of adipogenic differentiation medium
Isobutyl-1-methylxanthine (IBMX)	Sigma	I7018	Component of adipogenic differentiation medium
Indomethacin	Sigma	I7378	Component of adipogenic differentiation medium
MTS Reagent	Promega	G3582	Cell viability analyses
TUNEL Assay	Sigma	11684795910	Apoptotic analyses
24-well plate inserts	Corning	3396	For trans-well migration assay
PKH67	Sigma	PKH67GL	For co-culture cell staining
PKH26	Sigma	PKH26GL	For co-culture cell staining
Paraformaldehyde	Sigma	P6148	For cell fixation
von Kossa Kit	BioOptica	04-170801.A	For cell staining (differentiation)
Alcian blue	Sigma	A2899	For cell staining (differentiation)