Diabetes Research

Type 1 diabetes stem cell treatment advances utilizing autologous dental stem cells

by Doctor Dharmini Pathmanathan, DMD, PhD

Diabetes is a chronic degenerative disease of the beta-cells of the pancreas leading to a lifelong dependency on insulin treatments.  While the cure for diabetes would be transplantation of cadaveric pancreatic islet cells to the diabetic individual, the scarcity of transplantable organs and side effects arising from a lifelong immunosuppressive regimen limits its therapeutic potential and is thus far from an ideal option. Similarly, the use of embryonic stem cells (ES), a favored option due to enormous differentiation potential of ES, is limited in its application in regenerative medicine because of persistent ethical and legal concerns. Thus, mesenchymal stem cells (MSCs) have been extensively studied for their ability to differentiate into insulin-producing cells. However, limited sources and the invasive procedures necessary to procure these cells restrict their application.  Given the limitations of the above procedures, locating a source of MSCs that is readily available and accessible and without any significant donor morbidity would be an ideal solution. Recent article in the Journal of Dental Research presented a benchmark study in which dental pulp stem cells (DPSCs) were differentiated into functional pancreatic cells. DPSCs were isolated from deciduous teeth that were already planned for serial extraction as part of orthodontic management. In short, the DPSCs were differentiated to islet-like cell aggregates (ICAs) using various growth factors and their identity confirmed using expression studies. In vitro functional studies showing that the DPSCs differentiated into ICAs could indeed respond to a glucose challenge with insulin production provided further confirmation for their potential use in islet transplantation programs. These findings add to the body of evidence regarding DPSCs ability to be differentiated into pancreatic cell lineage and their potential as a source of human tissue that could be used for an autologous stem cell therapy for diabetes without fear of rejection. Since DPSCs do not have the same restrictions as ES, are readily accessible, can be harvested from the individual himself, and virtually eliminate the possibility of rejection and therefore the use of immunosuppressants, DPSCs are considered to be an ideal source for MSCs for emerging diabetes stem cell treatments. Beyond diabetes, these findings also validate and encourage the banking of dental pulp stem cells for use in autologous stem cell therapy. 





Mo Chen, Chang H. Lee, Ang Li, Michael Huang, Tracy Shen, Rujing Yang, Shantanu Lal, Jeremy J. Mao
Tissue Engineering and Regenerative Medicine Laboratory, Columbia University Medical Center, New York, NY
Department of Biochemistry, Xian Jiaotong University, Xi’an, China

 Introduction

Insulin-producing cells (IPCs) have been derived from embryonic stem cells and postnatal stem cells isolated from anatomic structures such as amniotic fluid, bone marrow, and adipose tissue (1,2). However, a common challenge for this task is insulin yield. Tooth pulp is neural crest-derived mesenchymal tissue, and its genesis relies on epithelial- mesenchymal interactions.  Dental-pulp stem/progenitor cells (DSCs) express the embryonic stem cell markers, Nanog and Oct4, suggesting their primitive status. In this study, we first isolated DSCs from human deciduous and adult teeth, and demonstrated their multi-lineage differentiation capacity into osteoblasts/odontoblasts, chondrocytes, and myocytes. We then studied the differentiation of polyclonal and monoclonal DSCs towards insulin-producing cells (IPCs) and found that both polyclonal and cloned DSCs differentiated into IPCs. Finally, we differentiated DSC clones into endoderm pancreatic cells and characterized critical markers associated with IPC differentiation.  Objective: To derive insulin-producing cells from dental-pulp stem/progenitor cells.

Methods and Materials - Subjects and Cell Culture

Exfoliating deciduous incisors and permanent third molars of multiple donors were collected with IRB approval. The dental pulps were isolated and enzyme-digested. Mononucleated and adherent cells were cultured in DMEM-LG medium containing 10% FBS and 1% antibiotics in 10 cm cell culture dishes.  Single cells in suspension were then isolated from heterogeneous DSCs and cultured under the same conditions for 2 weeks. Following this, the monoclonal cells were transferred to 6-well culture plates.

Differentiation of Insulin Producing Cells (IPCs)

DSC clones were expanded and subjected to insulin-producing cell differentiation conditions. Briefly, 2.5×105  DSCs were suspended in DMEM-LG medium containing 10% FBS and
centrifuged for 5 min. Then, the cells were transferred into 1:1
DMEM/F-12 medium containing glucose, Insulin-Transferin- Selenium-A, IBMX, Wnt3a, and 5 µg/mL fibronectin, and
subsequently cultured for 2 days. The cells were then
switched to DMEM/F-12 medium containing glucose, nicotinamide, N2 supplement, B27 supplement noggin, and
fibronectin for 4 days. After suspension culture, the cell pellets
were washed with PBS, fixed with 4% paraformaldehyde, and sectioned.  The expression of proinsulin, insulin, and Pdx-1 were detected by immunofluorescence and ELISA.

Results

Insulin-producing cell differentiation
Overall, heterogeneous DSCs had a low yield of IPC differentiation.  Differentiated heterogeneous DSCs expressed significantly more insulin, Pdx-1, and C-peptide, compared to the undifferentiated DSCs. 20 clones isolated from 3 permanent teeth and 6 clones isolated from 2 deciduous teeth were used for IPC differentiation. Immunostaining demonstrated that 2 of 20 permanent teeth clones and 5 of 6 deciduous teeth clones were Stro-1 positive. Upon IPC differentiation, 2 permanent and 2 deciduous teeth clones demonstrated strong proinsulin and Pdx-1 staining (Fig.1). Insulin production by heterogeneous IPCs was further validated by ELISA. Polyclonal DSCs produced twice the amount of insulin in comparison with bone marrow-derived MSCs (Fig. 2). At this time, the efficiency of cloned DSCs regarding proinsulin and Pdx-1 expression is markedly higher than that of polyclonal DSCs.

 



Fig.1. Immunostaining of proinsulin and Pdx-1 of dental-pulp stem/progenitor cells.





Fig.2. Insulin secretion of MSC- and TSC- derived IPCs.


Cell Marker Analysis

Of the 4 DSC clones that were differentiated into IPCs, one permanent and one deciduous clone were Stro-1 positive, whereas the other two clones were Stro-1 negative. Whether Stro-1 is an accurate surrogate marker for IPCs warrants additional investigation.  Interestingly, the strongest insulin- producing IPC clone was positive for both Nanog and Oct4, whereas the other 3 clones were positive for either Nanog or Oct4.  These findings suggest that Nanog and/or Oct4, two hallmarks of embryonic stem cells, expressed by fractions of dental-pulp stem/progenitor cells, are indicative, but not obligatory, markers for IPCs differentiation.

Discussion

We demonstrate, for the first time, that insulin-producing cells can be derived from dental-pulp stem/progenitor cells, both polyclonal and monoclonal populations.  Nanog and Oct4, two hallmarks expressed by embryonic stem cells, appear to be indicative, but not obligatory, markers for IPC differentiation. The insulin yield of polyclonal DSCs was approximately two fold higher than that of bone marrow-derived MSCs subjected to the same IPC differentiation protocol. We anticipate that clonal DSCs to have greater insulin yield than polyclonal DSCs, because cloned DSCs have higher differentiation efficiency towards IPCs than polyclonal DSCs. These discoveries offer a potential for utilizing dental-pulp stem/progenitor cells towards the derivation of insulin-
producing cells. Advantages of IPC differentiation from dental- pulp stem/progenitor cells include: 1) DSCs are readily
accessible from exfoliating/extracted teeth that are otherwise
discarded as medical waste, 2) DSCs as postnatal stem cells are not subjected to ethical controversy, and 3) rapid proliferation of DSCs provide a potential for expansion.

Reference

1.  D'Amour KA et al. (2006) Production of pancreatic hormone- expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24:1392-1401.
2.  Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R (2001) Differentiation of embryonic stem cells to insulin-secreting
structures similar to pancreatic islets. Science 292:1389-1394.

Disclosure

The work is sponsored by NIH R01EB005256 to J.J.M.




Differentiation of dental pulp stem cells into islet-like aggregates

Govindasamy V, Ronald VS, Abdullah AN, Nathan KR, Ab Aziz ZA, Abdullah M, Musa S, Kasim NH, Bhonde RR.

Source - Stempeutics Research Malaysia Sdn Bhd, (773817-K), Lot G-E-2A, Enterprise 4, Technology Park Malaysia, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

Abstract

The post-natal dental pulp tissue contains a population of multipotent mesenchymal progenitor cells known as dental pulp stromal/stem cells (DPSCs), with high proliferative potential for self-renewal. In this investigation, we explored the potential of DPSCs to differentiate into pancreatic cell lineage resembling islet-like cell aggregates (ICAs). We isolated, propagated, and characterized DPSCs and demonstrated that these could be differentiated into adipogenic, chondrogenic, and osteogenic lineage upon exposure to an appropriate cocktail of differentiating agents. Using a three-step protocol reported previously by our group, we succeeded in obtaining ICAs from DPSCs. The identity of ICAs was confirmed as islets by dithiozone-positive staining, as well as by expression of C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. There were several-fold up-regulations of these transcription factors proportional to days of differentiation as compared with undifferentiated DPSCs. Day 10 ICAs released insulin and C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Our results demonstrated for the first time that DPSCs could be differentiated into pancreatic cell lineage and offer an unconventional and non-controversial source of human tissue that could be used for autologous stem cell therapy in diabetes.

PMID:21335539

[PubMed - indexed for MEDLINE]