Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

Wilson’s disease (WD) is an inherited metabolic disease arising from ATPase copper transporting beta gene (ATP7B) mutation. Orthotoropic liver transplantation is the only radical treatment of fulminant WD, although appropriate donors are lacking at the onset of emergency. Given the hepatogenic capacity and tissue-integration/reconstruction ability in the liver of stem cells from human exfoliated deciduous teeth (SHED), SHED have been proposed as a source for curing liver diseases. We hypothesized the therapeutic potential of SHED and SHED-converted hepatocyte-like- cells (SHED-Heps) for fulminant WD. SHED and SHED-Heps were transplanted into WD model Atp7b-mutated Long-Evans Cinnamon (LEC) rats received copper overloading to induce a lethal fulminant liver failure. Due to the superior copper tolerance via ATP7B, SHED-Hep transplantation gave more prolonged life-span of fulminant LEC rats than SHED transplantation. The integrated ATP7B-expressing SHED-Heps showed more therapeutic effects on to restoring the hepatic dysfunction and tissue damages in the recipient liver than the integrated naïve SHED without ATP7B expression. Moreover, SHED-Heps could reduce copper-induced oxidative stress via ATP7B- independent stanniocalcin 1 secretion in the fulminant LEC rats, suggesting a possible role for paracrine effect of the integrated SHED-Heps. Taken together, SHED-Heps offer a potential of functional restoring, bridging, and preventive approaches for treating fulminant WD.


Isolation and culture of stem cells from exfoliated deciduous teeth (SHED)
Dental pulp tissues from human deciduous teeth were digested with 0.3% collagenase type I (Worthington Biochemicals, Lakewood, NJ) and 0.4% dispase II (Sanko Junyaku Co., Ltd., Tokyo, Japan) for 60 min at 37°C. The obtained cells were seeded on culture flasks. After three hours of the seeding, the cultures were washed with sterilized phosphate-buffered saline (PBS).
The adherent cells were grown with a growth medium containing of 15% fetal bovine serum (Equitech-Bio, Kerrville, TX), 100 µM L-ascorbic acid 2-phosphate (Wako Pure Chemicals, Osaka, Japan), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), and premixed antibiotics containing 100 U/mL penicillin and 100 μg/ml streptomycin (Nacalai Tesque) in alpha Modification of Eagle's Medium (Thermo Fisher Scientific, Waltham, MA). The isolated cells formed attached colonies consisting of spindle-shaped cells on plastic culture flasks. The adherent colony-forming cells were passaged and were sub-cultured. The medium was changed twice a week. The passaged 3 (P3) cells were determined the characteristics as MSCs as described below.

Characterization of SHED
The characterization of SHED was determined by previous standard 1,2 according to previous reports. 3,4 Adherent colony-forming capacity: Adherent colony-forming capacity of SHED was evaluated by colony-forming unit fibroblasts assay. 5 Isolated cells were seeded and were cultured in the growth medium for 16 days. The flasks were treated with 4% paraformaldehyde and 0.1% toluidine blue in PBS for 18 h. Attached colonies containing >50 cells were observed under a microscopy.
Cell proliferation capacity: P3 SHED were cultured in the growth medium for 2 days and were examined using a bromodeoxyuridine incorporation assay kit (Thermo Fisher Scientific).
Population doubling assay: Cells were seeded on T-75 culture flasks. When the cells reached at sub-confluent condition, the cells were passed. These steps were repeated until the cells lost dividing capability. Finally, the population doubling score was calculated.
Telomerase activity test: SHED (P3, 5x10 3 ) were used for measuring the telomerase activity by a telomere repeat amplification protocol assay using a quantitative telomerase detection kit (Allied Biotech, Inc., Ijamsville, MD). The average starting quantity of fluorescence units was used to compare the telomerase activity among the samples.
Expression of cell surface markers: Flow cytometric analysis of SHED (P3) was performed to examine the expression of human CD146, CD105, CD73, CD45, CD34, CD14, CD11b, and human leukocyte antigen DR (HLA-DR). The number (percentage) of positive cells was determined on a FACSVerse flow cytometer (BD Bioscience, Franklin Lake, NJ) and were analyzed using FACSuite software (BD Bioscience) in comparison with the corresponding control cells stained with corresponding isotype-matched antibody in which a false-positive rate of less than 1% was accepted.
In vitro multipotent assay: (1) in vitro osteogenic induction assay. P3 SHED were grown in the growth medium until confluent condition and induced with an osteogenic medium supplemented with 1.8 mM potassium dihydrogen phosphate (Sigma-Aldrich, St. Louis, MO) and 10 nM dexamethasone (Sigma-Aldrich). The osteogenic medium was changed twice a week. The cultures were also stained with 1% Alizarin Red-S at 4 weeks post induction and analyzed mineralized nodule formation.

References
Supplementary Table S1. The list of specific antibodies.    QRT-PCR assay demonstrates the expression of fulminant hepatitis-associated genes in the liver tissues after 4 weeks of SHED-Hep-transplantation in fulminant LEC rats. Cdkn1a, cyclin dependent kinase inhibitor 1A; Hgf, hepatocyte growth factor; Il-6, interleukin 6; Tgfb, transforming growth factor beta; Tnfa, tumor necrosis factor alpha. LEA, control LEA rats; LEC, non-transplanted fulminant LEC rats; SHED-Hep-T, SHED-Hep-transplanted fulminant LEC rats. n = 3 for all groups. * P < 0.05, ** P < 0.01, and *** P < 0.005. ns, no significance. The results are shown as the ratio to the expression of each gene in control LEA rats. Graph bars show the means SD.  The results are shown as the ratio to the expression of ROS in HepG2 treated without H 2 O 2 (b) and HepG2 treated with H 2 O 2 (e). c, d: The results are shown as the ratio to the expression of STC1 in SHED (c) and siCTRL-treated SHED (d). d, e: siSTC1, STC1 siRNA pre-treatment; siCTRL, scrambled control siRNA pre-treatment. (c) Biochemical assay shows the serum levels of ALT and AST after 4 weeks of the transplantation. SHED-Hep-T, SHED-Hep-transplantation; siSTC1, STC1 siRNA pre-treatment; siCTRL: scrambled control siRNA pre-treatment. a-c: n = 3 for all groups. * P < 0.05, ** P <0 .01, and *** P < 0.005. ns, no significance. Graph bars show the means SD.