Alginate/Pluronic F127-based encapsulation supports viability and functionality of human dental pulp stem cell-derived insulin-producing cells

Abstract

Background: Current approach for diabetes treatment remained several adverse events varied from gastrointestinal to life-threatening symptoms. Regenerative therapy regarding Edmonton protocol has been facing serious limitations involving protocol efficiency and safety. This led to the study for alternative insulin-producing cell (IPC) resource and transplantation platform. In this study, evaluation of encapsulated human dental pulp-derived stem cell (hDPSC)-derived IPCs by alginate (ALG) and pluronic F127-coated alginate (ALGPA) was performed.

Results: The results showed that ALG and ALGPA preserved hDPSC viability and allowed glucose and insulin diffusion in and out. ALG and ALGPA-encapsulated hDPSC-derived IPCs maintained viability for at least 336 h and sustained pancreatic endoderm marker (NGN3), pancreatic islet markers (NKX6.1, MAF-A, ISL-1, GLUT-2 and INSULIN), and intracellular pro-insulin and insulin expressions for at least 14 days. Functional analysis revealed a glucose-responsive C-peptide secretion of ALG- and ALGPA-encapsulated hDPSC-derived IPCs at 14 days post-encapsulation.

Conclusion: ALG and ALGPA encapsulations efficiently preserved the viability and functionality of hDPSC-derived IPCs in vitro and could be the potential transplantation platform for further clinical application.

Keywords: Alginate; Dental pulp stem cells (DPSCs); Diabetes mellitus; Encapsulation; Insulin-producing cells (IPCs); Pluronic F127.

Fig. 1

Bead diameter and morphology evaluation of ALG and ALGPA. Bead diameters and morphological appearances of ALG and ALGPA generated by manual extrusion through different sizes of needle (22G, 24G and 26G) at day 0, 7, 14 and 28 were illustrated. Bars indicated statistical relationship, p < 0.05 or not significant (ns)

Fig. 2

Glucose and insulin diffusion efficiency of ALG and ALGPA. The percentages of glucose diffusion a and insulin diffusion b across ALG and ALGPA as determined by modified trans-well diffusion assay were illustrated. The asterisks indicated significant difference, comparing with the initial condition at 15 min (glucose diffusion) and 30 min (insulin diffusion) (p < 0.05)

Fig. 3

Morphology and viability evaluation of encapsulated hDPSCs using ALG and ALGPA. ALG- and ALGPA-encapsulated hDPSC morphologies were evaluated under light microscope a. The viability of encapsulated hDPSCs was also determined by alamarBlue™ assay b and live/dead staining c. The asterisks indicated significant difference, comparing with initial condition at 2 h (p < 0.05)

Fig. 4

Morphology and characterization of hDPSCs. Morphological features of hDPSCs with low and high magnification were evaluated under light microscope a. The mRNA expression of stemness property genes (REX1, NANOG and OCT4) and proliferation gene (Ki67) were determined by RT-qPCR b. Expression of surface marker reflecting MSC property was also determined using flow cytometry c

Fig. 5

In vitro differentiation of hDPSCs toward IPCs. Different morphological appearances of hDPSCs after induction using three-stage differentiation protocol were illustrated at day 3, 5, 7 and 10 a. The total colony number b and the colony size distribution c were also investigated. The expression of pro-insulin and insulin were evaluated by immunocytochemistry staining d

Fig. 6

Morphology and viability evaluation of encapsulated hDPSC-derived IPCs using ALG and ALGPA. Encapsulated hDPSC-derived IPCs morphologies in both ALG and ALGPA were ilustrated a. The viability evaluation of encapsulated hDPSC-derived IPCs was also determined by live/dead staining b

Fig. 7

Pancreatic gene expression analysis of encapsulated hDPSC- derrived IPCs using ALG and ALGPA. The mRNA expression of pancreatic endoderm marker (NGN3) a, pancreatic islet markers (NKX 6.1, MAFA, ISL-1, GLUT-2 and INSULIN) b and pancreatic related markers (GLP-1R and GLUCAGON) c by ALG- and ALGPA-encapsulated hDPSC-derived IPCs were determined by RT-qPCR at day 7 and 14 post encapsulation. The asterisks indicated significant difference, comparing with undifferentiated cell (p < 0.05)

Fig. 8

Pancreatic protein expression and functional analyses of encapsulated hDPSC-IPCs using ALG and ALGPA. The expression of pro-insulin and insulin by ALG- and ALGPA-encapsulated hDPSC-IPCs were evaluated by immunocytochemistry staining at day 7 and 14 post-encapsulation a. C-peptide secretion was also determined by glucose-stimulated C-peptide secretion (GSCS) assay b. The asterisks indicated significant difference, comparing with normal and glucose 5.5 mM in KRBH (p < 0.05)