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Characterization of Dental-Pulp Derived Islet-like Cell Aggregates and the Role of GABA

  • Author(s): Egli, Jennifer Lynn
  • Advisor(s): Pae, Eung-Kwon
  • Kang, Mo
  • et al.
Abstract

Diabetes Mellitus (DM) is a growing health concern with 8.3% of Americans suffering from the effects of the disease. The use of multipotent mesenchymal progenitor cells, known as stem cells from human exfoliated deciduous teeth (SHED), to generate insulin-releasing islet-like cell aggregates (ICAs) is considered to be a potential treatment mode for Type-I DM. ɤ-Aminobutyric acid (GABA) has recently been shown to be a strong secretogogue of insulin and increases β-cell mass, reversing DM in rats. This investigation focused on the use of insulin-producing ICAs to examine the roles of GABA in an autocrine excitatory secretory mechanism in pancreatic β-cells. The aim of this study was to investigate the role of GABA on insulin secretion utilizing the in vitro system, insulin-secreting ICAs derived from SHED.

SHED was propagated using a 10-day differentiation protocol to generate ICAs. Day-10 ICAs were stained with dithizone (DTZ) to confirm zinc-positive β-cell phenotype. Real-time quantitative reverse transcriptase PCR (qRT-PCR) on Day-5 and Day-10 ICAs was used to confirm the presence of pancreatic markers PDX-1, NeuroD, Nkx6.1, Isl-1, Glut-2, insulin, and GABAA receptors, and to demonstrate temporal changes in expression levels. Day-10 ICAs were challenged with glucose and GABA to demonstrate dose-dependent insulin secretion using enzyme-linked immunosorbent assay (ELISA).

Results demonstrate that SHED ICAs were able to differentiate toward the pancreatic lineage. Day-10 ICAs demonstrated a weak positive DTZ staining. A statistically significant temporal increase of the β-cell markers was shown from Day-5 to Day-10 ICAs (p<0.05) using qRT-PCR; albeit, Day 10 ICAs did not reach pancreatic marker transcript levels previously reported. Day-10 ICAs did secrete insulin in response to glucose, however, not in a dose-dependent manner, possibly due to insufficient storage of insulin because of immaturity of the aggregates or a lack of intracellular zinc concentration. Day-10 ICAs demonstrated a statistically significant increase in the transcript levels of GABA receptors (i.e. GABAα-1, GABAα2, GABAα3, and GABAα6), indicating that the aggregates may respond to GABA during challenge (p<0.05). Preliminary results utilizing GABA in addition to glucose challenge indicates that GABA may increase insulin secretion by the ICAs; however, further studies need to be done to confirm this finding. We conclude that SHED is capable of differentiating toward the pancreatic lineage and producing insulin; therefore, these cells may be a possible source for β-cell replacement therapy in Type-I DM patients.

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