Fabrication of Hard Tissue Constructs from Induced Pluripotent Stem Cells for Exploring Mechanisms of Hereditary Tooth/Skeletal Dysplasia

Abstract

Tooth/skeletal dysplasia, such as hypophosphatasia (HPP), has been extensively studied. However, there are few definitive treatments for these diseases owing to the lack of an in vitro disease model. Cells differentiated from patient-derived induced pluripotent stem cells (iPSCs) demonstrate a pathological phenotype. This study aimed to establish a method for fabricating hard tissue-forming cells derived from human iPSCs (hiPSCs) for the pathological analysis of tooth/skeletal dysplasia. Healthy (HLTH) adult-derived hiPSCs were cultured in a hard tissue induction medium (HM) with or without retinoic acid (RA) under 3D culture conditions, and mineralization and expression of dentinogenesis- and osteogenesis-related markers in 3D hiPSC constructs were evaluated. hiPSCs derived from patients with hypophosphatasia were also cultured in HM with RA. HLTH-derived hiPSCs formed mineralized 3D constructs and showed increased expression of dentinogenesis- and osteogenesis-related markers; addition of RA promoted the expression of these markers in hiPSC constructs. HPP-derived hiPSC constructs showed lower mineralization and expression of dentinogenesis- and osteogenesis-related markers than HLTH-derived hiPSCs, indicating an impaired ability to differentiate into odontoblasts and osteoblasts. This method for fabricating 3D hiPSC constructs allows for simultaneous assessment of dentinogenesis and osteogenesis, with HPP-derived hiPSC constructs recapitulating pathological phenotypes.

Keywords: dentinogenesis; hypophosphatasia; induced pluripotent stem cell; osteogenesis; pathological phenotype; skeletal dysplasia; three-dimensional construct; tooth dysplasia.

Figure 1

Effects of retinoic acid on mineralization of 3D human induced pluripotent stem cell (hiPSC) constructs during hard tissue induction. (a) Cell culture and 3D hard tissue induction methods for hiPSCs. “ES medium” is growth medium for hiPSCs. “Day 0” refers to the day when hard tissue induction commences. (b) Expression of mesoderm marker TBXT at Day 0 was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Representative hematoxylin and eosin (HE) and von Kossa staining images of 3D hiPSC constructs, cultured in HM with or without RA under shaking conditions, on Day 10 and Day 20 (scale bars: 100 μm). Student’s t-test. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Figure 2

Effects of retinoic acid on expression of dentinogenesis- and osteogenesis-related genes of 3D hiPSC constructs during hard tissue induction. (a) Expression of phosphate metabolism-related genes such as dentin matrix acidic phosphoprotein 1 (DMP1) and phosphate regulating endopeptidase homolog X-linked (PHEX) was determined by quantitative real-time RT-PCR analysis (n = 3). (b) Expression of ECM-related genes such as dentin sialophosphoprotein (DSPP) and collagen type 1 alpha 1 (COL1A1) was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Gene expression of regulators of WNT signaling, such as WNT inhibitory factor 1 (WIF1) and sclerostin (SOST), was determined by quantitative real-time RT-PCR analysis (n = 3). (d) Gene expression of transcription factors such as msh homeobox 1 (MSX1), sp7 transcription factor (SP7), paired box gene 9 (PAX9), and LIM homeobox domain 6 (LHX6) was determined by quantitative real-time RT-PCR analysis (n = 3). Student’s t-test at the same time point. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Figure 2

Effects of retinoic acid on expression of dentinogenesis- and osteogenesis-related genes of 3D hiPSC constructs during hard tissue induction. (a) Expression of phosphate metabolism-related genes such as dentin matrix acidic phosphoprotein 1 (DMP1) and phosphate regulating endopeptidase homolog X-linked (PHEX) was determined by quantitative real-time RT-PCR analysis (n = 3). (b) Expression of ECM-related genes such as dentin sialophosphoprotein (DSPP) and collagen type 1 alpha 1 (COL1A1) was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Gene expression of regulators of WNT signaling, such as WNT inhibitory factor 1 (WIF1) and sclerostin (SOST), was determined by quantitative real-time RT-PCR analysis (n = 3). (d) Gene expression of transcription factors such as msh homeobox 1 (MSX1), sp7 transcription factor (SP7), paired box gene 9 (PAX9), and LIM homeobox domain 6 (LHX6) was determined by quantitative real-time RT-PCR analysis (n = 3). Student’s t-test at the same time point. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Figure 3

Effects of retinoic acid on dentinogenesis- and osteogenesis-related protein expression of 3D hiPSC constructs after hard tissue induction. Three-dimensional hiPSC constructs were assessed on Day 20 of hard tissue induction with or without RA by immunohistochemical staining for DMP1, DSPP, WIF1, and MSX1 (scale bars: 100 μm).

Figure 4

Mineralization of 3D constructs fabricated from hiPSCs from a patient with hypophosphatasia (HPP) after hard tissue induction. (a) Representative bright-field microscopic images of healthy (HLTH) adult- and HPP-derived hiPSCs (scale bars: 200 μm). (b) Expression of TBXT at Day 0 was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Representative HE and von Kossa staining images of 3D constructs fabricated from HLTH and HPP-derived hiPSCs, cultured in HM with RA for 20 days (scale bars: 100 μm). Student’s t-test at the same time point. There were no significant differences between the groups at the same time points. Data are presented as mean ± SD; p < 0.05 was considered significant.

Figure 5

Expression of dentinogenesis- and osteogenesis-related genes of 3D constructs fabricated from HPP patient-derived hiPSCs after hard tissue induction with RA. (a) Gene expression of DMP1 and PHEX was determined by quantitative real-time RT-PCR analysis (n = 3). (b) Gene expression of DSPP and COL1A1 was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Gene expression of WIF1 and SOST was determined by quantitative real-time RT-PCR analysis (n = 3). (d) Gene expression of MSX1, SP7, PAX9, and LHX6 was determined by quantitative real-time RT-PCR analysis (n = 3). Student’s t-test at the same time point. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Figure 5

Expression of dentinogenesis- and osteogenesis-related genes of 3D constructs fabricated from HPP patient-derived hiPSCs after hard tissue induction with RA. (a) Gene expression of DMP1 and PHEX was determined by quantitative real-time RT-PCR analysis (n = 3). (b) Gene expression of DSPP and COL1A1 was determined by quantitative real-time RT-PCR analysis (n = 3). (c) Gene expression of WIF1 and SOST was determined by quantitative real-time RT-PCR analysis (n = 3). (d) Gene expression of MSX1, SP7, PAX9, and LHX6 was determined by quantitative real-time RT-PCR analysis (n = 3). Student’s t-test at the same time point. Data are presented as mean ± SD; * p < 0.05 was considered significant.

Figure 6

Expression of dentinogenesis- and osteogenesis-related proteins in 3D constructs fabricated from HPP-derived hiPSCs after hard tissue induction with RA. 3D hiPSC constructs were assessed on Day 20 of hard tissue induction with RA by immunohistochemical staining for DMP1, DSPP, WIF1, and MSX1 (scale bars: 100 μm).