Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
Department of Orthodontics, Tokyo Dental College, Tokyo, Japan.
Stem Cell Res Ther. 2018 Jan 22;9(1):12. doi: 10.1186/s13287-017-0754-4.
Runt-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.
Two 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.
Mutation 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.
Taken 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.
runt 相关转录因子 2（RUNX2）杂合子不足会导致颅锁骨发育不全（CCD），其特征是额外的牙齿、身材矮小、锁骨发育不良和骨质疏松症。目前，除了药物治疗外，还进行间充质干细胞（MSC）移植治疗作为骨质疏松症的治疗策略。然而，由于 RUNX2 功能受损导致 MSC 分化为成骨细胞的能力降低，CCD 患者的 MSC 为基础的骨质疏松症治疗变得困难。在这里，我们研究了是否可以通过修复 CCD 患者来源的诱导多能干细胞（iPSC）中的 RUNX2 突变来建立正常的 iPSC，使 iPSC 适当分化为成骨细胞，以及是否适当反转的 iPSC 衍生的成骨细胞的植入会导致免疫缺陷大鼠颅盖骨骨缺损模型中更好的再生。
使用逆转录病毒载体（OCT3/4、SOX2、KLF4 和 c-MYC）或 Sendai 病毒 SeVdp 载体（KOSM302L）生成了两例 CCD 患者来源的诱导多能干细胞（CCD-iPSC）。使用可编程核酸酶、成簇的规律间隔的短回文重复序列（CRISPR）/Cas 衍生的 RNA 指导的内切酶建立了纠正 CCD-iPSC 突变的重排 iPSC。使用定量逆转录酶聚合酶链反应分析成骨细胞特异性标志物的 mRNA 表达。将 iPSC 衍生的成骨细胞移植到大鼠颅盖骨骨缺损中，并使用微计算机断层扫描分析和组织学分析评估骨再生。
突变分析表明，两者都包含无意义突变：一个在 exon1 的起始处，另一个在核基质靶向信号的起始处。源自 CCD-iPSC（CCD-OBs）的成骨细胞表达几种成骨细胞分化标志物的水平较低，将这些成骨细胞移植到严重联合免疫缺陷大鼠的颅盖骨骨缺损中，显示出较差的再生。然而，重排 iPSC 改善了异常的成骨细胞分化，从而使这些成骨细胞更好地植入大鼠颅盖骨骨缺损中。
总之，这些结果表明，患者特异性 iPSC 技术不仅可以提供一个有用的疾病模型来阐明 RUNX2 在成骨细胞分化中的作用，而且还提出了一个诱人的前景，即重排 iPSC 可能为 CCD 提供一种实用的医学治疗方法。
