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. Electronic supplementary material The online version of this article (doi:10.1007/s11626-015-9968-x) contains supplementary material, which is available to authorized users.


Introduction
The generation of induced pluripotent stem cells (iPSCs) by expression of exogenous defined factors provides valuable tools for transplantation therapies and regenerative medicine using patient-specific stem cells and also for understanding the mechanisms of human diseases. Human iPSCs were established originally by the introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc using a retroviral vector (Park et al. 2007;Takahashi et al. 2007). DNA-integrative retroviral and lentiviral vectors have been used widely Sachiko Yamasaki and Atsuko Hamada contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11626-015-9968-x) contains supplementary material, which is available to authorized users. because of the stable expression of transgenes owing to chromosomal insertion of the vector genome Yu et al. 2007;Lowry et al. 2008). However, iPS technology is complicated by the potential risks posed by continuous expression of transgenes and by genome integration of viral vectors. Silencing the expression of exogenous transgenes is indispensable for maintaining pluripotency (Zhou and Zeng 2013), and genome-integrating viral vectors can produce insertional mutations, which may influence differentiation potential while reactivation of the c-Myc oncogene may lead to tumorigenesis (Okita et al. 2007). For these reasons, more efficient and safer reprogramming methods have been explored to generate iPSCs carrying no exogenous genetic material.
Recently, a number of procedures have been used to generate genetically non-integrative or unmodified human iPSCs. These approaches involve chemicals or plasmid, episomal or viral vectors Woltjen et al. 2009;Jia et al. 2010;Warren et al. 2010;Zhu et al. 2010;Yu et al. 2011;). However, these approaches suffer from an extremely low efficiency in generating iPSCs, and require either chemical treatment or extended periods of transduction (Kim et al. 2009;Bernal 2013).
Sendai virus (SeV) is a member of the Paramyxovirdae family, and is an enveloped virus with a single-stranded, negative-sense, and non-segmented RNA genome (Yoshida et al. 1979;Lamb and Kolakofsky 2001;Nishimura et al. 2007). As SeV does not infect humans but is pathogenic for various animal cells with an exceptionally broad host range, various applications have been studied with SeV as a recombinant viral vector capable of transient but strong gene expression (Nakanishi and Otsu 2012). Previously, we described a replication-defective and persistent Sendai virus (SeVdp) vector in a novel gene transfer/expression system (Nishimura et al. 2011;Nakanishi and Otsu 2012). The SeVdp vector can express up to four exogenous genes simultaneously at a fixed ratio, and it can be erased quickly by interfering vectorencoded RNA polymerase. These characteristics are ideal for generating high-quality, exogenous gene-free iPSCs, and we demonstrated that SeVdp installed with Yamanaka's four factors could reprogram human tissue cells (Nishimura et al. 2011;Nakanishi and Otsu 2012).
Previously, we found that a hESF-GRO basal medium supplemented with a minimal growth factor defined serum-free culture medium hESF9 (Yamasaki et al. 2014), could generate and maintain hiPSCs. In this culture system, the effect of exogenous factors could be precisely analyzed without the confounding influences of unknown or undefined components. Therefore, with use of serum-free defined hESF9 medium and a fibronectin substrate alkaline phosphate (ALP)-positive hiPSCs were generated without feeder cells with high induction efficiencies. However, manipulation of iPSCs remained difficult because the self-renewing property of these cells was unstable. We found TGF-β1 promotes the growth of undifferentiated hiPSCs in hESF9 (Furue et al. 2008;Ohnuma et al. 2014;Yamasaki et al. 2014). However, conventional protocols for inducing hiPSCs (Park et al. 2007;Takahashi et al. 2007;Yu et al. 2007) required repetitive induction, or produced insufficient excision of integrated reprogramming vectors. Therefore, we sought an improved method to induce hiPSCs without retaining expression of reprogramming factors or integrated vector DNA for both basic studies and clinical applications. Here, we show that by using SeVdp, we efficiently generated viral/factor-free hiPSCs from dental pulp cells (DPCs) in serum-and feeder-free culture conditions. We used this new method to reprogram adult human somatic cells from cleidocranial dysplasia (CCD) into iPSCs without feeder cells. CCD (MIM #119600) is a dominantly inherited disorder caused by mutation in the gene encoding transcription factor RUNX2 (Cbfa1), which was mapped to chromosome 6p21. RUNX2 has an important role in the differentiation of osteoblasts and in the maturation of chondrocytes (Komori et al. 1997;Smith et al. 2005). The main clinical features of CCD include persistently open skull sutures with bulging calvaria, hypoplasia or aplasia of the clavicles permitting abnormal facility in opposing the shoulders, wide pubic symphysis, short middle phalanx of the fifth fingers, dental anomalies, and often vertebral malformation. RUNX2 has a primary role in the differentiation of osteoblasts and hypertrophy of cartilage at the growth plate, cell migration, and vascular invasion of bone (Cohen 2009). To our knowledge, this is the first report of the establishment of CCD-specific iPS cells. Here, we report molecular and phenotypic profiles of CCD in tissues differentiated from hiPSCs derived from patient's dental pulp cells that carry a heritable mutation in RUNX2. The teratoma-containing cartilage from CCD-iPSCs revealed abnormality by histological analysis. Patient-derived iPSCs could provide somatic cells, which cannot be directly obtained from patients, and this advantage may contribute to understanding mechanisms of the disease and lead to the development of a new field of disease modeling or useful tools for exploring disease mechanisms.

Materials and Methods
Ethics statement and cell culture of dental pulp cell. This study was approved by the Ethics Committee of Human Genome/Gene Analysis Research at Hiroshima University (approval number: hi-58). We obtained human third molars from healthy volunteers or supernumerary tooth from CCD patient (17-year-old girl) at Hiroshima University Hospital after obtaining informed consent for the use of dental pulp cells (DPCs) to generate iPSCs in accordance with the approved guidelines. The dental pulp tissues obtained aseptically from extracted human third molars or supernumerary tooth were minced into small clumps and cultured on type I collagen-coated dishes in RD6F serum-free medium (Yamasaki et al. 2014). Primary human DPCs were isolated and cultured at 37°C in a humid atmosphere of 95% air/5% CO 2 . For subculturing, DPCs were harvested in 0.05% trypsin-0.02% ethylenediamine tetraacetic acid (EDTA) in Ca 2+and Mg 2+ -free phosphate-buffered saline (CMF-PBS), and the trypsin was inactivated with 0.1% soybean trypsin inhibitor (Sigma Aldrich, St. Louis, MO).
Induction of hiPSCs with SeVdp under completely defined culture. Human DPCs cultured in a 12-well plate at a density of 1×10 5 cells in RD6F serum-free medium were infected with SeVdp (KOSM) vector at MOIs of 3, 6, and 9, at room temperature for 2 h then at 37°C for overnight in a humid atmosphere of 95% air/5% CO 2 in RD6F medium. Then, these infected cells were trypsinized and seeded on fibronectin (2 μg/cm 2 ) (Sigma Aldrich)-coated dishes at 1.0×, 1.2×, 1.5×, 1.8×, and 2.0×10 4 cells per six-well plate in hESF9-medium (Furue et al. 2008;Ohnuma et al. 2014;Yamasaki et al. 2014) at 38°C in a humid atmosphere of 95% air/5% CO 2 . The medium was changed every other day. Within 20 d after transduction, iPS colonies were picked based on human ES cell-like colony morphology. The picked colonies were mechanically dissociated into small clumps and subsequently expanded and maintained on fibronectin-coated dishes in hESF9 with TGF-β1 (2 ng/ml) (hESF9T) or activin A (10 ng/ml) (hESF9a) at 37°C, 95% air/5% CO 2 as described previously (Hayashi et al. 2011;Yamasaki et al. 2014). We defined this stage as passage 1. The medium was changed every day with hESF9T or hESF9a medium. Reprogramming efficiency was determined as the positive number of total ALP positive colonies per total number of infected cells. At the same time, transduced DPCs were seeded on mitomycin-C-treated mouse embryonic fibroblast (MEF) cells with KSR-based conditions Yu et al. 2007) as a control.
RNA isolation and reverse transcription gene expression. Total RNA was extracted from iPSCs using the Illustra RNA spin Mini Isolation kit (GE Healthcare UK Ltd, Buckinghamshire, England) according to manufacturer's instruction. cDNA was synthesized from 1 μg of total RNA using High capacity RNA-to cDNA master mix (Applied Biosystems, Carlsbad, CA) and RT-PCR reactions were performed with KOD-FX neo (Toyobo, Japan) using primers described in Supplementary  Table S2. PCR products were size-fractionated using 1.5% agarose gel electrophoresis. DNA markers were used to confirm the size of the fragments.
Verification of removal of SeVdp-iPS genome. Human iPSCs generated with SeVdp (SeV-iPSCs) and cultured in serumand feeder-free culture conditions were harvested with a trypsin-EDTA, then total RNA was extracted. We carried out RT-PCR for detecting SeVdp NP mRNA.
PCR analysis of genomic DNA and mutation analysis. Genomic DNA was used for analysis of viral integration. iPSCs using QIAamp ® DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Genomic DNA was used for PCR reactions to check for viral integration using KOD-FX neo (Toyobo). Specific primer sets were used that detect only the transgene and not the endogenous gene. Reactions were performed with pMXs primers described in Supplementary Table S2.
Mutational analysis of RUNX2 gene was performed by using specific primers franking the eight coding exons and their splice junctions (Quack et al. 1999). PCR products purified using PCR purification kit (QIAGEN) and sequenced directly using CEQ8000 sequencer (Beckman coulter, Brea, CA).
In vitro differentiation of integration-free iPSCs. Human iPSCs generated and maintained in serum-and feeder-free culture conditions with SeVdp (SeV-iPSCs or CCD-iPSCs) were differentiated in vitro by the formation of embryoid bodies (EBs) as described previously (Yamasaki et al. 2014). Briefly, undifferentiated hiPSCs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum for 4 d in low-attachment 96 well plates. After 4 d in suspension culture, aggregated EBs were harvested onto gelatin-coated dishes for another 10 d. Then, the EBs were fixed and stained with hematoxylin/eosin (H-E) staining and Alcian Blue/PAS staining. Histological findings were evaluated using a Nikon ECLIPSE E800 microscope (Nikon Corporation, Tokyo, Japan) and photographed using Leica DC500 (Leica Microsystems AG, Wetzlar, Germany).
Assay for teratoma formation. SeV-iPSCs maintained in serum-and feeder-free culture conditions were injected subcutaneously into dorsal flank of SCID (CB17/Icr-Prkdc scid / CrlCrlj) mice (1×10 6 cells/50 μl of the cell suspension). Around 10 weeks after the injection, tumors were surgically dissected, weighed, fixed in PBS containing 4% paraformaldehyde, and embedded in paraffin. Each section was stained with hematoxylin/eosin staining, Alcian Blue/PAS staining, and toluidine blue staining. Histological findings were evaluated as described above. All animal experiments in this study were strictly followed a protocol approved by the Institutional Animal Care and Use Committee of Hiroshima University (approval number: A-11-140).
Chondrogenic differentiation. For in vitro chondrogenic differentiation, CCD-iPSCs were transferred to low-attachment 96-well plates with hMSC Chondrogenic Differentiation medium (Lonza, Basel, Switzerland) supplemented with dexamethasone, ascorbate, ITS supplement, GA-1000, sodium pyruvate, proline, L-glutamine, and TGF-β3 (R&D Systems), at a cell density of 1×10 5 cells/well in 200 μl. After 3 d in suspension culture, embryoid bodies were transferred to 24well low-attachment plates. Medium was changed every 3 d. After 10 weeks, particles were fixed in 4% (w/v) paraformaldehyde and embedded in paraffin and the content of sulfated glycosaminoglycans (GAGs) was investigated by Alcian blue/PAS staining (Quarto et al. 2012;Yamashita et al. 2014;Okada et al. 2015). The data are representative of three independent experiments.
Short tandem repeat DNA analysis. Genomic DNA was used for PCR with Powerplex 16 system (Promega Corporation, Madison, WI) and analyzed by ABI PRISM 3100 Genetic analyzer and Gene Mapper v3.5 (Applied Biosystems).
Karyotyping. Standard Q-banding chromosome analysis was carried out using quinacrine and Hoechst 33258 as described previously (Murakami et al. 2013;Lin et al. 1978). Approximately 50 separate metaphase spreads of SeV-iPSCs maintained in serum-and feeder-free culture conditions were examined using a Zeiss Axio Imager microscope (Carl Zeiss) and mapped.

Results
Generation of integration-free hiPSCs in serum-and feederfree culture. To avoid transgene integration, we used a SeVdp (KOSM) vector, which does not integrate into the host genome and is not pathogenic for humans. Moreover, providing a significant advantage for cell reprogramming, transcription factors (Klf4, Oct4, Sox2, and c-Myc) were incorporated into a single vector allowing these genes to be expressed reproducibly at fixed ratios (Fig. 1A). We carefully dissected dental pulp tissues from third molar teeth and cultured dental pulp cells (DPCs) in a serum-free defined medium designated RD6F (Sato et al. 1987;Myoken et al. 1989) (Fig. 1B,  Supplementary Fig. S1A). DPCs less than two passages were used for hiPS cell reprogramming with the SeVdp (KOSM) vector at MOIs of 3, 6, and 9 in completely feeder-and serum-free culture conditions ( Supplementary  Fig. S2B). The infected DPCs were seeded on fibronectincoated plate at 1.0×10 4 cells per six-well plate in hESF9 medium at 38°C in a humid atmosphere of 95% air/5% CO 2 . After 7 d, ES-like tightly packed colonies appeared. Continuously, human iPS cell colonies appeared at 15 d after transfection. After 20 d in culture, individual iPS cell colonies were picked and subsequently passaged and maintained in hESF9T or hESF9a medium (Yamasaki et al. 2014;Ohnuma et al. 2014;Hayashi et al. 2011) in dishes coated with fibronectin at 37°C in a humid atmosphere of 5% CO 2 (Fig. 1C). On 30 d in culture, alkaline phosphatase (ALP)-positive hiPS cell colonies were counted; approximately 24∼37 colonies were observed at MOI 3 (the efficiency of reprogramming was 0.30%) and 25∼42 colonies at MOI 6 (0.31%) and 15∼36 colonies at MOI 9 (0.25%) per six-well plate ( Supplementary Fig. S2C).
Generation of integration-free CCD-iPSCs. A CCD patient was diagnosed with mutation of RUNX2 (674G > A, R225Q), leading to a clinical phenotype (Fig. 2). We obtained CCD-DPCs from supernumerary tooth (Fig. 3A). CCD-DPCs were reprogrammed into iPSCs with SeVdp (KOSM) vector at MOI of 6 in completely serum-free culture conditions. The infected CCD-DPCs were seeded on fibronectin-coated plate at 1.0×10 4 cells per six-well plate in hESF9 (Fig. 3B). After 22 d in culture, individual iPS cell colonies were picked and subsequently passaged and maintained in hESF9based medium (Fig. 3C). The efficiency of reprogramming was 0.15% in hESF9.
Missense mutations. Missense mutations were detected in exon3 of Runnx2 (Fig. 2B). The dental pulp cell and the CCD-iPSCs were both shown to carry a missense mutation (647G > A) in runt domain of RUNX2, which was responsible for arginine to glutamine substitution [R225Q] (Fig. 5D).
Dysplasia of cartilage in CCD-iPSCs-induced teratomas of SCID mice. To understand pathogenesis of CCD-iPSCs, we evaluated their chondrogenic differentiation in teratomas of SCID mice. We examined the cartilage formed in teratomas. A histological analysis of the teratomas derived from CCD-iPSCs showed specific cartilage formation that the cartilage matrix tended to insufficiently compare to the teratomas generated from control SeV-iPSCs (SeV-H-iPS, SeV-M-iPS) confirmed by stain with Alcian blue/PAS and toluidine blue (Fig. 6). Impaired chondrogenic differentiation in CCD-iPSCs. To identify the differentiation ability of CCD-iPSCs, we evaluated their chondrogenic differentiation. The results of the histological analysis of the pellet culture of chondrogenically differentiated CCD-iPSCs indicated striking difference in chondrogenesis confirmed by Alcian blue/PAS staining (Fig. 7). The particle of CCD-iPSCs indicated lack of cartilaginous elements.
mutations. Both of these characteristics posed significant potential safety issues for future clinical uses of hiPS cells. The SeV vector described here expresses reprogramming genes without chromosomal integration. Defective and persistent Sendai virus (SeVdp) vectors are recognized as a superior tool for iPS cell generation because of their remarkably high potential and simplicity (Nishimura et al. 2011). As the four reprogramming genes are incorporated in a single vector, simultaneous delivery of four exogenous genes led to stable and reproducible expression of the genes at a pre-determined ratio without chromosomal integration. The rapid and complete loss of the vector genome after several passages makes feasible applications in the fields of gene therapy and the development of patient-specific therapeutic reagents.
In a previous study, we have reported generation of hiPSCs from adult human dental pulp cells (DPCs) and Figure 5. Differentiation ability of CCD-iPSCs derived from DPCs in serum-, feeder-and integrationfree defined culture conditions. (A) Differentiation was performed using embryoid body formation, and the differentiated iPSCs (CCD-SeV-iPS1 clone8) were fixed and reacted with antibodies. Shown were immunocytochemistry of Nestin, βIII-tubulin, MAP-2, αsmooth muscle actin (α-SMA), and α-fetoprotein (AFP). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies (green). Nucleuses were stained with DAPI.   fibroblast using retrovirus and successfully maintained hiPSCs in an undifferentiated state for long-term period using serum-free defined medium without feeder cells (Yamasaki et al. 2014). Here, we described that integrationfree hiPSCs could be generated from human DPCs using this complete serum-free culture system. Under this culture system, we successfully generated genome integration-free iPSCs from DPCs using SeVdp vector installed with Klf4, Oct4, Sox2, and c-Myc. These integration-free iPSCs could be generated from DPCs in 2∼3 weeks with SeVdp (KOSM) at a high efficiency, and exhibited similar morphology and cellular characteristics to those of established in conventional serum-supplemented culture conditions. These generated iPSCs could differentiate into several cell types such as neuronal cells, muscle cells, cartilage tissues, and intestinal epithelium.
We have derived human iPSCs from cleidocranial dysplasia (CCD) donors to understand the mechanisms underlying this disease. CCD is an autosomal dominant inheritance caused by heterozygous mutation in the Runt-related transcription factor 2 (RUNX2) gene on chromosome 6p21 (Zheng et al. 2005;Komori 2007). The main clinical features of CCD include persistently open skull sutures with bulging of the calvaria, hypoplasia, or aplasia of the clavicles permitting abnormal facility in opposing shoulders, short middle phalanx of the fifth fingers, abnormal dentition, and often vertebral malformation (Otto et al. 1997;Quack et al. 1999;Bufalino et al. 2011). RUNX2 is an important regulator of osteoblast differentiation, chondrocyte maturation, especially hypertrophy of cartilage at growth plate (Komori et al. 1997;Sugawara et al. 2011). This gene encodes a transcription factor, which has a DNA-binding Runt domain. Most CCD patients are reported to have mutations in this domain (Komori et al. 1997). In RUNX2-deficient mice, chondrocyte differentiation was severely disturbed with a lack of osteoblasts and bone formation (Komori et al. 1997;Yoshida et al. 2002). The mutations consist of missense, nonsense, frameshift, and splice mutations (Cohen 2009). These mutations of the RUNX2 gene affect its downstream target (Otto et al. 2003). However, the effect of RUNX2 mutations from CCD patients is still unclear.
In this study, donors showed features characteristic of CCD such as partially absent clavicles, persistent open anterior fontanelle, short stature, delayed eruption of permanent teeth, and supernumerary teeth (Banshodani et al. 2007). They also carried the missense mutation 674G>A in exon 3 of the RUNX2, which was predicted to substitute an arginine with a glutamine residue at codon 225 of the RUNX2 protein (Zheng et al. 2005). This mutation has been frequently identified and reported in several previous studies (Quack et al. 1999;Zhou et al. 1999;Otto et al. 2002;Kim et al. 2006;Xuan et al. 2008;Wu et al. 2014). Amino acid R225 is located in the c-terminal region of the Runt domain, which has been shown to be associated with severe parietal bone dysplasia (Cunningham et al. 2006). Substitution of R225 with glutamine is predicted to impair DNA binding to RUNX2. The Runt domain mediates binding to CBFβ, an unrelated partner protein that does not interact directly with DNA but enhances the DNA-binding affinity of the RUNX protein (Bartfeld et al. 2002). The mutations at this codon interfere with nuclear localization (NLS) and abolish DNA binding. Therefore this mutation (R225Q) may affect the DNA-binding activity of RUNX2 (Quack et al. 1999;Yoshida et al. 2002). Lack of nuclear RUNX2 accumulation might be the causes of haploinsufficiency in CCD.
In our studies, altered chondrocyte hypertrophy in teratomas formed by human CCD-iPSCs was found by histological analysis. Cartilage matrix revealed sparse and cytoplasm tended to be swelling and lack of normal hypertrophic chondrocytes with aggregation of small round cell has also been observed in the teratomas generated from CCD-iPSCs compared with healthy control. These findings suggest that this phenomenon may occur by inhibitory mechanisms of proliferation and differentiation of growth cartilage cells into hypertrophic chondrocytes mediated by RUNX2 mutation. This histological analysis of teratomas suggests that the CCD-iPSCs model could mimic the pathology of CCD to some extent. Inhibition of chondrocyte differentiation and lack of osteoblasts and abnormal endochondral ossification might occur as a result of the disequilibrium of chondrocytes maturation in growth plate in CCD. Moreover, abnormal chondrocyte hypertrophy may associate with downregulation of RUNX2 transcriptional targets.
CCD-iPSCs might be effective for screening small molecules that target different stages of human chondrogenesis, osteogenesis, tooth formation, and morphogenesis. They could help facilitate identification of the triggers of chondrogenic maturation and bone formation. In addition, CCD-iPSCs provide the possibility to create important human cell types that could not be directly obtained from CCD patients. CCD-iPSCs would also provide interesting human-specific perspectives complementary to the in vivo studies of the CCD mouse model. The mechanisms of tooth morphogenesis and formation are known to be strictly governed by cellular communications and the genetic control regulating signal pathways (Thesleff 2006;Xuan et al. 2010). Mutational and functional analyses in our CCD-iPSC model will help to identify the distinct effects of RUNX2 on chondrogenesis, osteogenesis, and tooth morphogenesis in the pathogenesis of CCD.
Our simple defined serum-and feeder-free culture system consisting of basal medium, defined components and growth factors along with a SeVdp vector can be helpful to understand specific signaling pathways and molecular mechanisms of cell reprogramming. In addition, our results suggest that generating patient-specific iPSCs with integration-free and serum-free methods could eliminate the possibility of complications resulting from persistent transgene expression. iPSC-derived cell types could be used to identify specific roles for RUNX2 in chondrocytes formation and to identify new therapeutic targets that would complement strategies to modulate the signaling properties of the RUNX2. Moreover, in this defined serum-free culture system, microRNAs and small RNAs produced by the iPSCs themselves could be easily purified for functional studies. In practice, we have generated a variety of disease-specific iPSCs from peripheral blood mononuclear cells using this culture method (data not shown).

Conclusions
We have successfully generated transgene-free hiPSCs from human DPCs using SeVdp vector and maintained in an undifferentiated state in complete feeder-and serum-free defined culture. Using this culture system, we have derived diseasespecific iPS cells from CCD patients. CCD-iPSCs are a valuable in vitro human model for understanding the mechanisms involving human chondrocytes in bone and tooth formation. Further characterization of the cells in the serum-, feeder-, and integration-free culture would be beneficial to clarify the molecular mechanism involved in the disease.