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Inhibition of Human Dental Pulp Stem Cell Differentiation by Notch Signaling

¹ Department of Special Dental Service, School and Hospital of Stomatology, Peking University, Beijing, China;
² Laboratory of Molecular Signaling, Division of Oral Biology and Medicine, UCLA School of Dentistry, 10833 Le Conte Ave., Los Angeles, CA 90095, USA; and
³ Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA

View larger version (37K): [in this window] [in a new window]   Figure 1. Generation of Jagged-1-expressing DPSCs. (A) DPSCs were stably infected with retroviruses expressing HA-Jagged-1 or control vector. Fifty-µg aliquots of protein extracts isolated from both DPSC/V and DPSC/Jag cells were probed with monoclonal antibodies against HA. (B) DPSCs expressed Notch1. DPSCs were induced to differentiate for 0, 3, 7, and 14 days, and total RNA was isolated. Notch mRNA was determined by RT-PCR. GAPDH was used as internal control. (C) Both DPSC/V and DPSC/Jag cells were grown in differentiation-inducing media for 0 and 7 days. Fifty-µg aliquots of protein extracts from these cells were probed with monoclonal antibodies against Notch1-ICD.

View larger version (35K): [in this window] [in a new window]   Figure 2. The activation of Notch signaling by Jagged-1 inhibits ALP activity in DPSCs. (A) Both DPSC/V and DPSC/Jag cells were treated with differentiation-inducing media for 2 wks. ALP activity was stained with 0.25% naphthol AS-BI phosphate and 0.75% Fast Blue BB. (B) ALP activity assay was determined with an ALP assay kit according to the manufacturer’s instructions. (C) Over-expression of Jagged-1 did not affect cell proliferation. Both DPSC/V and DPSC/Jag cells were treated with differentiation-inducing media for the indicated times. Cell numbers were counted. (D) Over-expression of Jagged-1 did not affect cell survival. Cell viability was determined with the Trypan blue exclusion assay.

View larger version (72K): [in this window] [in a new window]   Figure 3. The activation of Notch signaling by Jagged-1 inhibits the odontoblastic differentiation of DPSCs in vitro. (A) Both DPSC/V and DPSC/Jag cells were treated with differentiation-inducing media for 0, 2, and 3 wks. Cells were fixed with 4% paraformaldehyde and stained with 2% Alizarin red. (B) Cells were de-stained with 10% cetylpyridinium chloride in sodium phosphate buffer. The concentration was determined by absorbance measurement at 562 nm on a multiplate reader. We performed Student’s t test to determine statistical significance. *p < 0.01. (C) Both DPSC/V and DPSC/Jag cells were treated with differentiation-inducing media for 0, 1, and 2 wks. Aliquots (50 µg) of protein extracts were probed with polyclonal antibodies against DSP. For loading control, the membrane was stripped and re-probed with monoclonal antibodies against -tubulin. (D) The activation of Notch signaling by Jagged-1 inhibits the formation of mineralized tissues by DPSCs in vivo. Both DPSC/V and DPSC/Jag cells were transplanted subcutaneously into the dorsal surfaces of 10-week-old immunocompromised beige mice. Eight wks after transplantation, the transplants were sectioned and stained with H&E. The transplants of DPSC/V (a and b), but not of DPSC/Jag (c and d), generated mineralized tissues in mice. Scale bar, 100 µm.

View larger version (55K): [in this window] [in a new window]   Figure 4. Notch-ICD inhibits DPSC differentiation. (A) DPSCs were stably infected with retroviruses expressing HA-Notch-ICD or control vector. (B) Both DPSC/V and DPSC/ICD cells were induced to differentiate for 7 days. ALP activity was stained with 0.25% naphthol AS-BI phosphate and 0.75% Fast Blue BB. (C) ALP activity assay was determined with an ALP assay kit. (D) Both DPSC/V and DPSC/Jag cells were induced to differentiate for 2 wks and stained with 2% Alizarin red. (E) Cells were de-stained, and calcium concentration was determined by absorbance measurement at 562 nm in a multiplate reader. We performed Student’s t test to determine statistical significance. *p < 0.01.

Journal of Dental Research, Vol. 87, No. 3, 250-255 (2008)
DOI: 10.1177/154405910808700312


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