Figure 1.
Clonogenicity, cell proliferation capacity and stem cell marker expression of SHED-Cryo.
(A) Histology of cryopreserved dental pulp tissue of exfoliated deciduous teeth. Black arrowheads: blood vessel, white arrowheads: nerve fibers. H&E staining. (B) Localization of MSC markers in the cryopreserved deciduous pulp tissues. Yellow arrows: STRO-1-positive cells, black arrows: CD146-positive cells. BV: blood vessel, Control: subclass-matched antibody staining. (C–E) CFU-F assay. Formation of a clonogenic cell cluster from a single attached cell (C). Images of attached colonies of SHED-Cryo. Toluidine blue staining (D). Comparison of CFU-F number (E). (F, G) Cell proliferation assay. Immunostaining of BrdU-positive nuclei. (F). Comparison of cell proliferation (G). (H, I) Flow cytometry of MSC markers in SHED-Cryo. Representative histograms (H). Comparison of STRO-1, CD146, CD73 and CD105. Black columns: SHED-Cryo, white columns: SHED-Fresh (I). (J, K) Gene expression of embryonic stem and neural crest cell markers. MW: molecular weight markers (J). Comparative analysis of NANOG, octamer 4 (OCT4), NESTIN, NOTCH1 and low affinity nerve growth factor receptor (LNGFR) (K). (L) Flow cytometry of Nestin in SHED-Cryo. A, B: n = 3. C–L: n = 5 for all group. A–E: Bar = 30 µm (A), 5 µm (B, C, F), 1 mm (D, left) 25 µm (D, middle and right). G, I, K: ***P<0.005. ns: no significance. The graph bars represent mean±SD.
Figure 2.
(A–C) Dentinogenic/osteogenic differentiation capacity. Images of Alizarin Red staining (A) and alkaline phosphatase (ALP) activity (B) of SHED-Cryo. Comparison of Alizarin Red-positive (Alizarin Red+) area (A), ALP activity (B) and odontoblast/osteoblast-specific genes, runt-related gene 2 (RUNX2), ALP, osteocalcin (OCN), and dentin sialophosphoprotein (DSPP) (C). (D) Chondrogenic differentiation capacity. Comparison of chondrocyte-specific genes, SOX9, aggrecan (AGG) and type X collagen (ColX). (E, F) Adipogenic differentiation assay. A representative image of Oil Red-O staining and comparison of Oil Red-O accumulation (E). Comparison of adipocyte-specific genes lipoprotein lipase (LPL) and peroxisome proliferator activated receptor-gamma2 (PPARgamma2) (F). (G) Hepatogenic differentiation capacity. Comparison of hepatocyte-specific gene albumin (ALB). (H) Endothelial cell differentiation assay. Comparison of endothelial cell markers CD31 and CD34. (H) Neural cell differentiation assay. Comparison of neural cell markers neurofilament M (NFM) and tubulin betaIII (betaIII). A–I: n = 5 for all group. ns: no significance. The graph bars represent mean±SD.
Figure 3.
Tissue regeneration capability, self-renewal potency, heterogeneity and in vitro immunomodulatory functions of SHED-Cryo.
(A–D) Images of primary transplant tissues of SHED-Cryo. H&E staining (A). Comparison of newly formed-mineralized tissue (B). Immunofluorescence with anti-human specific mitochondria (hMt) (C) and anti-STRO-1/human CD146 (hCD146) (D) antibodies. (E, F) Purity of hCD146 antibody-sorted cells from primary transplants. Flow cytometry with hCD146 and mouse CD146 (mCD146). (E). Immunocytochemistry with hCD146 antibody of sorted cell-derived CFU-F (F). (G, H) in vivo self-renewal assay. Images of secondary transplant tissues. H&E staining (G). Immunofluorescence with anti-hCD146/anti-Mt antibodies. (H). (I) Comparison of population doubling (PD) scores. (J) Comparison of telomerase activity. (K) Single-colony-derived cell assay with 17 single cell colonies from a cryopreserved deciduous pulp tissues. (L) In vitro direct immunosuppressive effects of SHED-Cryo on human Th17 cells. A–J, L: n = 5 for all group. A, C, D, F, H: B: bone, BM: bone marrow, CT: connective tissue, D: dentin, DP: dental pulp, HA: HA/TCP, I: HEK: HEK293 cells, H.I. HEK: heat inactivated HEK, H.I. SHED-Cryo: heat inactivated SHED-Cryo, H.I. SHED-Fresh: heat inactivated SHED-Fresh. C, D, H: Dot lined areas: mineralized tissue. Nuclei are counterstained with DAPI. B, H, I, L: ***P<0.005, ns: no significance. The graph bars represent mean±SD.
Figure 4.
Systemic SHED-Cryo-transplantation improves lifespan and SLE-like disorders in MRL/lpr mice.
(A) Kaplan-Meier survival curve of MRL/lpr mice. (B) ELISA of serum levels of autoantibodies ANA and anti-dsDNA IgG and IgM antibodies. (C) Histopathology of kidneys. G and dot-circled area: glomerular. HE: H&E staining, TC: Gomori trichrome staining, PAS: Periodic acid-Schiff staining, C3: Immunofluorescence of Complement C3. DAPI staining. (D) Levels of serum albumin and creatinine and urine C3 and protein. A–D: MRL/lpr: control group, SHED-Cryo: SHED-Cryo-transplant group, SHED-Fresh: SHED-Fresh-transplant group. A: n = 7, B–D: n = 5 for all group. B, D: *P<0.05, **P<0.01, ***P<0.005, ns: no significance. The graph bars represent mean±SD.
Figure 5.
SHED transplantation suppresses circulating and local levels of Th17 cells in MRL/lpr mice.
(A, B) Flow cytometry of peripheral CD4+IL17+IFNgamma− Th17 cells. (C) Serum levels of IL-17. (D) Homing of systemically infused CFSE-labeled SHED-Cryo and SHED-Fresh to lymph node (LN) and kidney of MRL/lpr Mice after 1- (Day 1), 2- (Day 2) or 7- (Day 7) day transplantation. Dot-circled area: glomerular. (E) ELISA of IL-17 an IL-6 levels in lymph node and kidney. A–E: n = 5 for all group. MRL/lpr: control group, SHED-Cryo: SHED-Cryo-transplant group, SHED-Fresh: SHED-Fresh-transplant group. B, C, E: *P<0.05, **P<0.01, ***P<0.005, ns: no significance. The graph bars represent mean±SD.
Figure 6.
SHED-Cryo transplantation ameliorates osteoporotic bone disorder in MRL/lpr mice. (A, B)
MicroCT analysis of tibiae. BMD (A). Trabecular parameters, bone volume ratio to tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) along with increased trabecular separation (Tb.Sp) (B). (C, D) MicroCT (C) and histological (D) images of trabecular bone structures of tibiae. H&E staining (D). (E, F) In vivo osteoclast activity. TRAP staining (E). ELISA of serum sRANKL and C-terminal telopeptides of type I collagen (CTX) (F). (G) Ex vivo sRANKL-induced osteoclastogenesis. TRAP+ cells: TRAP-positive osteoclast-like cells. (H) Ex vivo osteogenic capacity. Alizarin red-positive (AR+) area after four-week induction. A–H: n = 5 for all groups. A–F: MRL/lpr: control group, SHED-Cryo: SHED-Cryo-transplant group, SHED-Fresh: SHED-Fresh-transplant group, G, H: BM-MRL/lpr: control MRL/lpr mice-derived bone marrow cells, BM-SHED-Cryo: SHED-Cryo-transplanted mice-derived bone marrow cells, BM-SHED-Fresh: SHED-Fresh-transplanted mice-derived bone marrow cells. A, B, E, F: *P<0.05, ***P<0.005 (vs. MRL/lpr). G, H: *P<0.05, **P<0.01, ***P<0.005 (vs. BM-MRL/lpr), ns: no significance. A, B, E–H: The graph bars represent mean±SD.
Figure 7.
SHED-Cryo are capable of repairing critical calvarial bone defects in immunocompromised mice.
(A) MicroCT images of mouse calvariae. Left panels: cranial images, middle panels: saggital images, right panels: images of red-bowed area in riddle panels. (B) Histology of bone regeneration in mouse calvariae. Left panels: edge parts of the defect area (yellow-boxed area in Figure 7A). H&E staining; middle panels: middle parts of the defect area (red red-bowed area in Figure 7A). H&E staining; right panels: immunofluorescence with anti-human CD146 antibody (hCD146). DAPI staining. CB, yellow dot-circled area: calvarial bone, HA: HA/TCP, RB: regenerated bone, RBM: regenerated bone marrow. (C) Regenerated bone area in the defect area. (D) Distribution of osteoclasts. Arrowheads: TRAP-positive cells. TRAP staining. A–D: n = 5 for all groups. Control: control (non-defect) group, CD: calvarial defect group, CD+HA: HA/TCP-implanted group, CD+HA+SHED-Fresh: SHED-Fresh-implanted group, CD+HA+SHED-Cryo: SHED-Cryo-implanted group. C: ***P<0.005, ns: no significance. The graph bars represent mean±SD.