Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells

Nakajima, Taiki; Shibata, Mitsuaki; Nishio, Makoto; Nagata, Shinji; Alev, Cantas; Sakurai, Hiroyuki; Toguchida, Junya; Ikeya, Motoji

doi:10.1242/dev.165431

Cited by 52 publications

(93 citation statements)

References 76 publications

(115 reference statements)

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“…Thus, a phenotypic screening focused on fibroproliferation could shed light on novel mechanisms of FOP. Three studies have identified the cell of origin of HO in FOP model mice ( Agarwal et al., 2017 , Dey et al., 2016 , Lees-Shepard et al., 2018 ), and iMSCs or paraxial mesoderm-derived MSC-like cells can be induced from FOP-iPSCs ( Hino et al., 2015 , Hino et al., 2017 , Matsumoto et al., 2015 , Nakajima et al., 2018 ); therefore, these cells could be applicable to future phenotypic screenings for the inhibition of stage 2A. Combination therapy targeting multiple phases could be the best strategy for controlling the HO of FOP.…”

Section: Discussionmentioning

confidence: 99%

An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva

et al. 2018

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SummaryFibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.

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Section: Discussionmentioning

confidence: 99%

An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva

et al. 2018

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“…The induced mouse EpiLCs were further cultured in CDMi 40 containing SB431542 (10 μM), DMH1 (2 μM), CHIR99021 (10 μM), and bFGF (20 ng/ml) for 2 days to induce mouse PSM cells. To induce human PSM cells, our 1 step induction protocol 9 was mainly used. Human iPSCs were seeded on a 35 mm dishes coated with iMatrix-511 silk or matrigel and cultured for 4 days.…”

Section: Methodsmentioning

confidence: 99%

“…1a), as other groups have recently reported 7–12 . In essence, our PSM induction protocol is based on activation of WNT and FGF signaling as well as inhibition of TGFβ and BMP signaling 9,12 . Prior to the PSM induction, mouse ESCs, which are in the naïve pluripotent state, were pretreated with ACTIVIN A and bFGF and converted to mouse epiblast-like cells (EpiLCs) that possess primed pluripotency as human iPSCs do.…”

Section: Mainmentioning

confidence: 99%

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters

Matsuda

Hayashi

García‐Ojalvo

et al. 2019

Preprint

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1While the mechanisms of embryonic development are similar between mouse and human, 2 the tempo is in general slower in human. The cause of interspecies differences in 3 developmental time remains a mystery partly due to lack of an appropriate model system 1 . 4 Since murine and human embryos differ in their sizes, geometries, and nutrients, we use 5 in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells 6 between the species in similar culture conditions. As an example of well-defined 7 developmental time, we focus on the segmentation clock, oscillatory gene expression that 8 regulates the timing of sequential formation of body segments [2][3][4] . In this way we 9 recapitulate the murine and human segmentation clocks in vitro, showing that the species-10 specific oscillation periods are derived from cell autonomous differences in the speeds of 11 biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and 12 human PSCs displayed the oscillatory expression of HES7, the core gene of the 13 segmentation clock 5,6 , with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours 14 (human PSM). Swapping HES7 loci between murine and human genomes did not change 15 the oscillation periods dramatically, denying the possibility that interspecies differences 16 in the sequences of HES7 loci might be the cause of the observed period difference. 17Instead, we found that the biochemical reactions that determine the oscillation period, 18 such as the degradation of HES7 and delays in its expression, are slower in human PSM 19 compared with those in mouse PSM. With the measured biochemical parameters, our 20 mathematical model successfully accounted for the 2-3-fold period difference between 21 mouse and human. We further demonstrate that the concept of slower biochemical 22 reactions in human cells is generalizable to several other genes, as well as to another cell 23 type. These results collectively indicate that differences in the speeds of biochemical 24 reactions between murine and human cells give rise to the interspecies period difference 25 of the segmentation clock and may contribute to other interspecies differences in 26 developmental time.27 28 Main 29 To compare murine and human segmentation clocks in vitro, we induced PSM-like cells 30 from mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells 31 (iPSCs) (Fig. 1a), as other groups have recently reported 7-12 . In essence, our PSM 32 induction protocol is based on activation of WNT and FGF signaling as well as inhibition 33 of TGFβ and BMP signaling 9,12 . Prior to the PSM induction, mouse ESCs, which are in 34 the naïve pluripotent state, were pretreated with ACTIVIN A and bFGF and converted to 35 mouse epiblast-like cells (EpiLCs) that possess primed pluripotency as human iPSCs do.36 3To visualize the segmentation clock in the induced PSM, we introduced a HES7 promoter-1 luciferase reporter 13,14 , detecting clear synchronized oscillations of HES7 expression in 2 both murine a...

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“…After additional 3 days culture, the medium was changed into CDMi without inhibitors for measurement with Kronos Dio Luminometer (Atto). This (modified) one-step protocol 39 was used for Fig. 1g and 2d.…”

Section: Methodsmentioning

confidence: 99%

Modeling the Human Segmentation Clock with Pluripotent Stem Cells

Matsuda

Yamanaka

Uemura

et al. 2019

Preprint

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32 33 Keywords: segmentation clock, somitogenesis, human mesoderm development, induced 34 pluripotent stem cells, step-wise in vitro mesoderm differentiation, disease modeling 35 Pluripotent stem cells (PSCs) have increasingly been used to model different aspects 36 of embryogenesis and organ formation 1 . Despite recent advances in the in vitro 37 induction of major mesodermal lineages and mesoderm-derived cell types 2,3 , 38 experimental model systems that can recapitulate more complex biological features 39 of human mesoderm development and patterning are largely missing. Here, we 40 utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of 41 presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human 42 somitogenesis. We focused initially on modeling the human segmentation clock, a 43 major biological concept believed to underlie the rhythmic and controlled 44 emergence of somites, which give rise to the segmental pattern of the vertebrate 45 axial skeleton. We succeeded to observe oscillatory expression of core segmentation 46 clock genes, including HES7 and DKK1, and identified novel oscillatory genes in 47 human iPSC-derived PSM. We furthermore determined the period of the human 48 segmentation clock to be around five hours and showed the presence of dynamic 49 traveling wave-like gene expression within in vitro induced human PSM. Utilizing 50 CRISPR/Cas9-based genome editing technology, we then targeted genes, for which 51 mutations in patients with abnormal axial skeletal development such as 52 spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic 53 dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like 54iPSC knock-out lines as well as patient-derived iPSCs together with their genetically 55 corrected isogenic controls revealed gene-specific alterations in oscillation, 56 synchronization or differentiation properties, validating the overall utility of our 57 model system, to recapitulate not only key features of human somitogenesis but also 58 to provide novel insights into diseases associated with the formation and patterning 59 of the human axial skeleton. 60 We initially aimed to mimic and recreate in vitro the signaling events responsible 61for the step-wise emergence of PSM and its derivatives during embryonic development, 62as also recently attempted by others 2,4,5 , via selective activation or inhibition of 63 appropriate signaling pathways, using human iPSCs as starting material (Fig. 1a). We 64 characterized the ability of our in vitro induced human PSM cells to differentiate into 65 somitic mesoderm and its two main derivatives, sclerotome and dermomyotome, which 66give rise to bone and cartilage of the axial skeleton and skeletal muscle and dermis of the 67 emerging embryo respectively. RNA-sequencing (RNA-seq) analysis and subsequent 68 characterization of in vitro derived human PSM samples revealed, that at each step of our 69 induction and differentiation protocol, markers expected to be pr...

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Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells

Cited by 52 publications

References 76 publications

An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva

An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters

Modeling the Human Segmentation Clock with Pluripotent Stem Cells

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