Stem Cell Reports Article Hedgehog Activation Regulates Human Osteoblastogenesis Shoko Onodera, 1,2 Akiko Saito, 1 Hironori Hojo, 2,3 Takashi Nakamura, 1 Denise Zujur, 2 Katsuhito Watanabe, 4 Nana Morita, 5 Daigo Hasegawa, 4 Hideki Masaki, 6 Hiromitsu Nakauchi, 6,7 Takeshi Nomura, 5 Takahiko Shibahara, 4 Akira Yamaguchi, 8 Ung-il Chung, 2,3 Toshifumi Azuma, 1,8, * and Shinsuke Ohba 2,3,9, * 1 Department of Biochemistry, Tokyo Dental College, Tokyo 101-0061, Japan 2 Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8656, Japan 3 Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8655, Japan 4 Department of Oral and Maxillofacial Surgery, Tokyo Dental College, Tokyo 101-0061, Japan 5 Department of Oral Medicine, Oral and Maxillofacial Surgery, Tokyo Dental College, Chiba 272-8513, Japan 6 Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan 7 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5461, USA 8 Oral Health Science Center, Tokyo Dental College, Tokyo 101-0061, Japan 9 Department of Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan *Correspondence: tazuma@tdc.ac.jp (T.A.), s-ohba@nagasaki-u.ac.jp (S.O.) https://doi.org/10.1016/j.stemcr.2020.05.008 SUMMARY Two genetic diseases, Gorlin syndrome and McCune-Albright syndrome (MAS), show completely opposite symptoms in terms of bone mineral density and hedgehog (Hh) activity. In this study, we utilized human induced pluripotent stem cell (iPSC)-based models of the two diseases to understand the roles of Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogen- esis and mineralization with Hh signaling activation and upregulation of a set of transcription factors in an osteogenic culture, compared with the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh signaling activity in the osteogenic culture; impaired osteoblastogenesis was restored to the normal level by treatment with an Hh signaling-activating small molecule. These data suggest that Hh signaling is a key controller for differentiation of osteoblasts from precursors. This study may pave a path to new drug therapies for genetic abnormalities in calcification caused by dysregulation of Hh signaling. INTRODUCTION Hedgehog (Hh) signaling is a highly conserved pathway be- tween species, acting as a mitogen and morphogen in various developmental processes (Ingham and McMahon, 2001). Mouse genetics studies have highlighted the requirement of Hh signaling to specify skeletal progenitors into osteoblast precursors expressing runt-related tran- scription factor (TF) 2 (Runx2), a master TF for osteoblast development (Komori et al., 1997; Long et al., 2004). Both Gli activators and repressors affect the Hh function in this context (Joeng and Long, 2009). In adult mice, constitutive activation of Hh signaling by Patched1 (Ptch1) haploinsufficiency led to high bone mass with an acceleration of both bone formation and bone resorption (Ohba et al., 2008). Dysregulation of Hh signaling has been implicated in human diseases (Mullor et al., 2002). Mutations of PTCH1, which encodes a receptor of Hh, are known to cause Gorlin syndrome (OMIM 109400), a rare auto- somal-dominant disorder characterized by multiple tumor formation and skeletal anomalies (Gorlin, 1995; Gorlin and Goltz, 1960). Most individuals with this disease have skeletal anomalies, and ectopic calcification particu- larly in the cerebral falx is present in more than 90% of the affected individuals. Heterozygous Ptch1-deficient (Ptch1 +/ ) mice have features that recapitulate those of pa- tients with Gorlin syndrome (Goodrich et al., 1997). They have higher bone mineral density than age-matched con- trols (Ohba et al., 2008). Molecular mechanisms underlying the maintenance of human bone mass have attracted much attention due to their fundamental importance for the understanding of bone pathology. From this point of view, it is noteworthy that two different Hh-related syndromes, Gorlin syndrome and McCune-Albright syndrome (MAS) (OMIM 174800), are characterized by completely opposite features of osteo- blast differentiation. Gorlin syndrome exhibits activation of Hh signaling and enhancement of mineralization, while MAS is often accompanied with fibrous dysplasia (FD) of immature bones with poor mineralization (Albright et al., 1937; Weinstein et al., 1991). FD is a genetic, non-inherit- able rare bone disease caused by a postzygotic mosaic acti- vation mutation (R201H or R201C) of the a-subunit of stimulatory G protein (Gas) encoded by GNAS, and this mutation is also observed in MAS (Albright et al., 1937; Lichtenstein, 1938; Turan and Bastepe, 2015). FD often leads to wheelchair life due to bone deformation, fractures, and severe pain. Khan et al. (2018) elegantly described that a Gnas (R201H) knockin mouse line showed reduced mineralization in the mutant bones, and the increased osteoid surface demonstrated poor osteoblastogenesis with suppressed Hh signaling and enhanced Wnt signaling. Stem Cell Reports j Vol. 15 j 125–139 j July 14, 2020 j ª 2020 The Author(s). 125 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).