Induced pluripotent stem cells from homozygous Runx2-deficient mice show poor response to vitamin D during osteoblastic differentiation.

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.

Induction of integration-free human-induced pluripotent stem cells under serum- and feeder-free conditions.

Human-induced pluripotent stem cells (hiPSCs) have shown great potential toward practical and scientific applications. We previously reported the generation of human dental pulp stem cells using non-integrating replication-defective Sendai virus (SeVdp) vector in feeder-free culture with serum-free medium hESF9. This study describes the generation of hiPSCs from peripheral blood mononuclear cells to increase the donor population, while reducing biopsy invasiveness. From 6-d-old primary culture of peripheral blood mononuclear cells (PBMCs) with IL-2, hiPSCs were established using SeVdp(KOSM)302L with recombinant Laminin-511 E8 fragments under serum-free condition. The established PBMC-derived hiPSCs showed pluripotency and differentiation ability both in vivo and in vitro. In addition, we evaluated microarray data from PBMC- and dental pulp-derived hiPSCs. These hiPSCs will be beneficial for characterizing the molecular mechanisms of cellular differentiation and may provide useful substrates for developing cellular therapeutics.

Induction of Noonan syndrome-specific human-induced pluripotent stem cells under serum-, feeder-, and integration-free conditions.

Noonan syndrome is an autosomal dominant developmental disorder. Although it is relatively common, and its phenotypical variability is well documented, its pathophysiology is not fully understood. Previously, with the aim of revealing the pathogenesis of genetic disorders, we reported the induction of cleidocranial dysplasia-specific human-induced pluripotent stem cells (hiPSCs) from patient's dental pulp cells (DPCs) under serum-free, feeder-free, and integration-free conditions. Notably, these cells showed potential for application to genetic disorder disease models. Furthermore, using similar procedures, we reported the induction of hiPSCs derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. These methods are beneficial, because they are carried out without invasive and painful biopsies. Using those procedures, we reprogrammed DPCs and PBMCs that were derived from a patient with Noonan syndrome (NS) to establish NS-specific hiPSCs (NS-DPC-hiPSCs and NS-PBMC-hiPSCs, respectively). The induction efficiency of NS-hiPSCs was higher than that of WT-hiPSCs. We hypothesize that this was caused by high NANOG expression. Here, we describe the experimental results and findings related to NS-hiPSCs. This is the first report on the establishment of NS-hiPSCs and their disease modeling.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction.

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype-phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities.

Generation of cleidocranial dysplasia-specific human induced pluripotent stem cells in completely serum-, feeder-, and integration-free culture.

Human pluripotent stem cells hold great promise for their practical and scientific potentials. To improve understanding of self-renewal and differentiation, we previously reported a defined serum-free medium hESF9 could generate and maintain human induced pluripotent stem cells (iPSCs) in serum- and feeder-free culture conditions using retroviral vectors. To avoid the unpredictable side effects associated with retrovirus integration, we report here the successful generation of hiPSCs from dental pulp cells with a non-integrating replication-defective and persistent Sendai virus (SeVdp) vector expressing four key reprogramming genes. We found that hESF9 medium in combination with fibronectin are effective for generating and maintaining hiPSCs with SeVdp (KOSM). Using this system, pluripotent and self-renewing hiPSCs could be easily and stably generated and propagated. With this system, we successfully generated hiPSCs from cleidocranial dysplasia (CCD) caused by a heterozygous germ-line mutation of runt-related protein2 (RUNX2), which has an important role in the differentiation of osteoblasts and maturation of chondrocytes. This is the first report of the establishment of CCD-specific iPSCs. The cartilage in the teratomas of CCD-iPSCs showed abnormalities. These CCD-iPSCs would be beneficial to clarify the molecular mechanism and for development of medical applications. Moreover, it brings new pathophysiological role of RUNX2 in the differentiation of the human chondrocytes and osteocytes.

Novel mucosal insulin delivery systems based on fusogenic liposomes.

PURPOSE: Fusogenic liposomes (FLs) are unique delivery vehicles capable of introducing their contents directly into the cytoplasm with the aid of envelope glycoproteins of Sendai virus (SeV). The objective of this study was to evaluate the potential of FL to improve the mucosal absorption of insulin from rat intestinal membranes. METHOD: The FLs containing insulin were prepared by fusing insulin-loaded liposomes with inactivated SeV particles and were administered directly into the ileal, the colonic, and the rectal loops (10 IU/kg). RESULTS: The FL successfully enhanced the insulin absorption and induced a significant hypoglycemic effect following the colonic and the rectal administration without detectable mucosal damage. This enhancing effect of insulin absorption was further improved by increasing the amount of insulin loaded in the FL and by coencapsulating insulin-degrading enzyme inhibitor. In contrast, the insulin absorption was not increased by the ileal administration of FL because the mucous/glycocalyx layers overlaid on the ileal epithelium impede the fusion of FL to the intestinal mucosa. CONCLUSION: Our results indicated that FL is a useful carrier for improving the absorption of poorly absorbable drugs, such as insulin, via the intestinal tract.