Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model

Abstract

BackgroundRunt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models.MethodsTwo cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis.ResultsMutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect.ConclusionsTaken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0754-4) contains supplementary material, which is available to authorized users.

Effects of alloplastic graft material combined with a topical ozone application on calvarial bone defects in rats

“…Many clinical studies have been performed on calvarial defect models to evaluate bone regeneration related to different diseases (43)(44)(45)(46). Histological techniques have been used to demonstrate tissue damage (47)(48)(49).…”

Induced Pluripotent Stem Cells in Dental and Nondental Tissue Regeneration: A Review of an Unexploited Potential

“…The osteoinductive properties of iPSC-derived bone cells and their capability in treating bone defects were further assessed in vivo by their implantation into a severe combined immunodeficiency (SCID) mouse model. Bone formation was confirmed four weeks following implantation by soft X-ray images [43], X-ray microcomputed tomography (μCT) [55], cone beam computed tomography imaging [49], and histological tissue specimens [43,[47][48][49][50][51][52]. In a cleidocranial dysostosis model, the mutation in RUNX2 gene was repaired in iPSCs derived from mucosal tissues of affected patients.…”

“…To induce osteogenic differentiation of iPSCs, a variety of agents were proposed in isolation or combination, including osteogenic media, ascorbic acid, b-glycerophosphate, dexamethasone, bone morphogenetic proteins (BMPs), and vitamin D 3 [43][44][45][46]. Osteogenic differentiation is followed by proper characterization of generated bone cells through their expression of osteogenesis-related genes (RUNX2, osteopontin (OPN), osterix (OSX), osteocalcin (OCN), and collagen type I (COL1A1)) [47][48][49][50] in addition to the evaluation of in vitro mineralization and alkaline phosphatase (ALP) activity [51,52]. Osteogenic potential of human iPSCs was demonstrated on polymeric nanofibrous polyethersulfone (PES) scaffold with upregulated expressions of osteogenic genes and alkaline phosphatase activity in vitro [48,53].…”

“…The reverted cells revealed marked upregulation of osteoblast differentiation markers after being cultured in OM for nine days. Loading the differentiated osteoblasts originating from iPSCs with a corrected mutation on a peptide nanofiber scaffold and implanting them into SCID rats' calvarial bone defects revealed reossification four weeks after transplantation with a significant increase in bone volume and bone mineral content [52]. Similarly, osteogenic cells differentiating from EB derived from iPSCs showed positive results in bone regeneration and healing following implantation in the rats' critical-sized calvarial defect [53,56,57] and long bone segmental defect rat model [57] after being loaded on polymeric nanofibrous PES scaffold [53], fibrin glue scaffold [57], hydroxyapatite (HA)/b-tricalcium phosphate scaffold [57], or self-assembling peptide nanofiber hydrogel scaffold [56].…”

“…Transduction of repaired, edited, and/or modified genes in iPSCs could be a beneficial tool for treating various disorders. In this context, repairing RUNX2 gene mutation in iPSCs derived from cleidocranial dysostosis patients [52] as well as transducing nuclear matrix protein SATB2 [104] and Alox5 gene into iPSCs promoted osteodifferentiation [115]. Besides, Pax9 and BMP-4 gene transfection into iPSC-derived NCLCs promoted odontoblast-like cell differentiation [141] and attained a long-term effect of these factors rather than the short-term effect acquired following their local application [113].…”