Proposal of patient-specific growth plate cartilage xenograft model for FGFR3 chondrodysplasia.

OBJECTIVE: FGFR3 chondrodysplasia is caused by a gain-of-function mutation of the FGFR3 gene. The disease causes abnormal growth plate cartilage and lacks effective drug treatment. We sought to establish an in vivo model for the study of FGFR3 chondrodysplasia pathology and drug testing. DESIGN: We created cartilage from human induced pluripotent stem cells (hiPSCs) and transplanted the cartilage into the subcutaneous spaces of immunodeficient mice. We then created cartilage from the hiPSCs of patients with FGFR3 chondrodysplasia and transplanted them into immunodeficient mice. We treated some mice with a FGFR inhibitor after the transplantation. RESULTS: Xenografting the hiPSC-derived cartilage reproduced human growth plate cartilage consisting of zones of resting, proliferating, prehypertrophic and hypertrophic chondrocytes and bone in immunodeficient mice. Immunohistochemistry of xenografts using anti-human nuclear antigen antibody indicated that all chondrocytes in growth plate cartilage were human, whereas bone was composed of human and mouse cells. The pathology of small hypertrophic chondrocytes due to up-regulated FGFR3 signaling in FGFR3 skeletal dysplasia was recapitulated in growth plate cartilage formed in the xenografts of patient-specific hiPSC-derived cartilage. The mean diameters of hypertrophic chondrocytes between wild type and thanatophoric dysplasia were significantly different (95% CI: 13.2-26.9; n = 4 mice, one-way analysis of variance (ANOVA)). The pathology was corrected by systemic administration of a FGFR inhibitor to the mice. CONCLUSION: The patient-specific growth plate cartilage xenograft model for FGFR3 skeletal dysplasia indicated recapitulation of pathology and effectiveness of a FGFR inhibitor for treatment and warrants more study for its usefulness to study disease pathology and drug testing.

Time-lapse observation of the dedifferentiation process in mouse chondrocytes using chondrocyte-specific reporters.

OBJECTIVE: When chondrocytes prepared from cartilage are expanded in monolayer culture, fibroblast-like cells gradually prevail. Although these prevailing fibroblast-like cells are believed to emerge because of the dedifferentiation of chondrocytes, the definite origin of the prevailing fibroblast-like cells has not been determined. We herein examined whether the prevailing non-chondrocytic cells observed after monolayer expansion culture arise from dedifferentiating chondrocytes or are the result of the overgrowth of fibroblasts that are present at the start of the culture. We also evaluated whether chondrocytes dedifferentiate because they proliferate or because they are cultured in monolayers. METHODS: Chondrocytes were prepared from Col11a2-EGFP transgenic mice and Col11a2-Cre; R26-stop(flox)-EYFP transgenic mice, which respectively express enhanced green fluorescent protein (EGFP) and Cre specifically in chondrocytes under the control of Col11a2 promoter/enhancer sequences. Col11a2-Cre; R26-stop(flox)-EYFP mice express enhanced yellow fluorescent protein (EYFP) only in cells which express or used to express Cre. We performed a time-lapse observation of the chondrocytes during monolayer expansion culture, and also observed the chondrocytes after treatment with mitomycin C. RESULTS: A time-lapse observation showed that Col11a2-EGFP chondrocytes underwent cell divisions, lost GFP fluorescence, increased cell numbers, and prevailed during the expansion culture. The observation of the Col11a2-Cre; R26-stop(flox)-EYFP chondrocytes confirmed that most of the cells after expansion in monolayer culture had been chondrocytes. Mitotically inactive chondrocytes generated by treatment with mitomycin C still underwent dedifferentiation, thus suggesting that chondrocyte dedifferentiation is not associated with cell division. CONCLUSION: The non-chondrocytic cells that prevail after the monolayer expansion culture of chondrocytes originate from chondrocytes, and are not generated by the overgrowth of fibroblasts that are present at the start of the culture. Chondrocyte dedifferentiation does not appear to be associated with cell division.

Identification of small molecular compounds and fabrication of its aqueous solution by laser-ablation, expanding primordial cartilage.

OBJECTIVE: The discovery of small molecular compounds that expand cartilage is needed. We searched for small molecular compounds that expand cartilage or enhance the actions of bone morphogenetic proteins (BMPs) on cartilage. DESIGN: Metatarsal primordial cartilage explants prepared from 14.5 days postcoitum (d.p.c.) mouse embryos were organ-cultured in the presence or absence of BMPs and/or 4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate (BPIB) and its related molecules. The perichondrium was removed from some of the cartilage explants by partial digestion with collagenase. BPIB aqueous solution was prepared by fragmenting BPIB crystals in water with laser irradiation and then added to cartilage explants in organ culture. RESULTS: We found that small molecular compounds, BPIB, available as SB431542 from Sigma and its related molecules, expand primordial cartilage explants in organ culture. These molecules are transforming growth factor-ß (TGF-ß) inhibitors, and the addition of excess TGF-ß reduced cartilage expansion induced by these molecules. The co-administration of BPIB and BMPs synergistically expanded cartilage explants. Removal of the perichondrium abolished BIPB-induced cartilage expansion but not BMP-induced cartilage-expansion, suggesting that BPIB, but not BMPs, expands cartilage through the perichondrium. Furthermore, we used the laser-ablation technique to generate BPIB aqueous solution in the presence of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) without the use of hazardous dimethyl sulfoxide (DMSO). The laser-ablation-generated BPIB aqueous solution was more stable, expanded cartilage explants more effectively than BPIB colloidal solution prepared with DMSO, and synergistically enhanced BMP-induced cartilage expansion. CONCLUSIONS: A small molecular compound, BPIB, expands primordial cartilage explants. A BPIB aqueous solution was created by laser-ablation without using DMSO and proved to be biologically active.

Separable cis-regulatory elements that contribute to tissue- and site-specific alpha 2(XI) collagen gene expression in the embryonic mouse cartilage.

Type XI collagen is a structural component of the cartilage extracellular matrix and plays an important role in skeletal morphogenesis. As a step toward defining the molecular mechanisms responsible for the regulation of type XI collagen expression, we characterized the promoter region of the mouse alpha 2(XI) collagen gene (Coll1a2). We also generated transgenic mice harboring various fragments of the promoter and the first intron of Coll1a2 linked to the Escherichia coli beta-galactosidase gene to identify the cis-acting elements responsible for tissue- and site-specific expression during development. Cloning and sequence analysis of the 5' flanking region of Coll1a2 showed that the putative 3' end of the retinoid X receptor beta gene was located 742 bp upstream of the Coll1a2 start site. This suggested that the promoter region of Coll1a2 was localized within this 742-bp sequence, which contained multiple consensus regulatory elements. Examination of the transgenic mice revealed that the longest DNA construct (containing the entire promoter and first intron sequences) directed lacZ expression in the notochord as well as in the primordial cartilage throughout the body, with the pattern of expression mimicking that of endogenous Coll1a2 transcripts. On the other hand, deletion of the upstream approximately 290 bp resulted in the elimination of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, whereas lacZ expression in the notochord and in the other primordial cartilage elsewhere was not affected. Deletion of the first intron sequence also resulted in the loss of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, as well as in the notochord. These results demonstrate that the upstream 742-bp and first intron segments of the mouse Coll1a2 gene contain the necessary information to confer high level tissue-specific expression in mouse embryos. In addition, our observations suggest the presence of site-specific cis-acting elements that control Coll11a2 gene expression in different cartilaginous components of the skeleton.

Role of CDMP-1 in skeletal morphogenesis: promotion of mesenchymal cell recruitment and chondrocyte differentiation.

Cartilage provides the template for endochondral ossification and is crucial for determining the length and width of the skeleton. Transgenic mice with targeted expression of recombinant cartilage-derived morphogenetic protein-1 (CDMP-1), a member of the bone morphogenetic protein family, were created to investigate the role of CDMP-1 in skeletal formation. The mice exhibited chondrodysplasia with expanded cartilage, which consists of the enlarged hypertrophic zone and the reduced proliferating chondrocyte zone. Histologically, CDMP-1 increased the number of chondroprogenitor cells and accelerated chondrocyte differentiation to hypertrophy. Expression of CDMP-1 in the notochord inhibited vertebral body formation by blocking migration of sclerotome cells to the notochord. These results indicate that CDMP-1 antagonizes the ventralization signals from the notochord. Our study suggests a molecular mechanism by which CDMP-1 regulates the formation, growth, and differentiation of the skeletal elements.

Splicing patterns of type XI collagen transcripts act as molecular markers for osteochondrogenic tumors.

Primary transcripts for three distinct alpha chains of the type XI collagen molecule (alpha1(XI), alpha2(XI) and alpha3[XI]) undergo tissue-specific alternative splicing during the process of osteochondrogenesis. In the present study, we analyzed the splicing patterns of type XI collagen genes in osteochondrogenic tumors as well as in various normal tissues using the reverse transcription-polymerase chain reaction method. Analysis of normal subjects revealed the coordinated expression of short alpha1(XI), alpha2(XI) and alpha3(XI) transcripts in the normal differentiated cartilage. Osteochondroma followed this pattern, reflecting the highly chondrogenic phenotype of this benign tumor. Another benign tumor, chondroblastoma, exclusively expressed the long alpha1(XI) transcript, probably reflecting the lack of a chondrogenic nature. Among malignant chondrogenic tumors, the splicing patterns of type XI collagen transcripts were more complex, showing dissociated expression of long alpha1(XI) and short alpha2(XI) mRNAs. This expression pattern may reflect heterogeneous cell populations and may also reflect various levels of cell differentiation in malignant tumors. In addition, short alpha3(XI) expression switched to the long transcript as chondrosarcomas became more aggressive. Thus, the alternative splicing of type XI collagen genes seems to be oncodevelopmentally regulated and splicing analysis may therefore be a useful marker for chondrogenic tumors.

Alternative splicing of fibronectin transcripts in osteochondrogenic tumors.

BACKGROUND: Fibronectin (FN) acts in many fundamental biological processes including cell adhesion, migration, proliferation and apoptosis. Although FN shows an ubiquitous expression pattern, alternative mRNA splicing modulates tissue-specific molecular heterogeneity at three regions: extra domain-B, extra domain-A and variable region. MATERIALS AND METHODS: In the present study, we analyzed the FN mRNA splicing in osteochondrogenic tumors as well as in various normal tissues using the RT-PCR method. RESULTS: Normal cartilage almost exclusively contained short mRNA lacking alternative exons in all regions. However, 14 osteochondrogenic tumors including osteochondroma, enchondroma, chondroblastoma, chondrosarcoma and osteosarcoma uniformly contained long mRNA with various patterns of alternative exons in the three regions. CONCLUSION: These results indicated that the lack of regulation excluding the alternative exons may be associated with tumorigenesis in chondrocytes.

Genotype phenotype correlation in achondroplasia and hypochondroplasia.

Recent studies of the fibroblast growth factor receptor 3 (FGFR3) gene have established that achondroplasia and hypochondroplasia are allelic disorders of different mutations. To determine whether the genotype could be distinguished on the basis of the phenotype, we analysed height, arm span, and skeletal radiographs from 23 patients with achondroplasia and the G380R mutation of FGFR3 and eight with hypochondroplasia and the N540K mutation. Both conditions share the classical pathological features of micromelic short stature, reduced or unchanged interpedicular distances in the lumbar spine, disproportionately long fibulae, and squared and shortened pelvic ilia. These were significantly more severe in the G380R patients than in the N540K patients. Our findings have shown a firm statistical correlation between the genotype and the phenotype, although there were a few exceptional cases in which there was phenotypic overlap between the two conditions.

Differential expression of an acidic domain in the amino-terminal propeptide of mouse pro-alpha 2(XI) collagen by complex alternative splicing.

We isolated and sequenced genomic and cDNA clones encoding the complete amino-terminal portion and the 5'-untranslated region of mouse pro-alpha 2(XI) collagen mRNA. Fourteen exons encoded the amino-terminal propeptide, which was divided into three consecutive domains (a long globular domain, an amino-terminal triple helical domain, and a telopeptide domain). The long globular domain was further divided into an upstream basic subdomain and a downstream highly acidic subdomain, as is the case for the amino-terminal propeptides of pro-alpha 1(V) and pro alpha 1(XI) collagens. We also demonstrated that the primary transcript undergoes complex alternative splicing. Three consecutive exons (exons 6, 7, and 8) encoding most of the acidic subdomain showed alternative splicing which dramatically affected the structure of the amino-terminal propeptide of pro-alpha 2(XI) collagen. Using the reverse transcription-polymerase chain reaction, we analyzed the expression of these exons in various tissues and in developing limb buds of mice. The pro-alpha 2(XI) transcripts were abundant in cartilage, but most of them lacked the 3-exon sequences encoding the acidic domain. Most of other tissues also contained mRNAs that corresponded to longer splice variants, including exons 6-8. The differential expression of specific domains of pro-alpha 2(XI) collagen may be important in modulating interactions between various components of the extracellular matrix and/or may influence heterotypic collagen assembly.

Cervical spondylotic radiculopathy involving two adjacent nerve roots. Anterior decompression through a single level intervertebral approach.

We describe a single level intervertebral approach to decompress two adjacent involved nerve roots in cases of cervical spondylosis. The operation was undertaken in 4 patients. We carried out discectomy, partial excision of the vertebral body with removal of the anteromedial part of the pedicles, removal of osteophytes and excision of the posterior longitudinal ligament, followed by an anterior interbody fusion. Fusion was achieved with the spine in normal lordosis and without complications. Pain and motor weakness was relieved in every case. This procedure can maintain movement at one additional disc level and has a better fusion rate than multilevel inter-body fusion.

Progressive degeneration of articular cartilage and intervertebral discs. An experimental study in transgenic mice bearing a type IX collagen mutation.

Transgenic mice expressing mutant alpha 1(IX) collagen were produced and found to develop progressive joint degeneration with age, as well as accelerated intervertebral disc degeneration. Radiological and histological studies showed that cervical and lumbar disc degeneration was more advanced in the transgenic mice than in control litter-mates. The changes included shrinkage or disappearance of the nucleus pulposus, and fissures in the annulus fibrosus which sometimes lead to herniation of disc material and slight osteophyte formation. These findings suggest that mutations of the type IX collagen may cause certain forms of degenerative disease in the spine as well as in joints.

A recurrent 1992delCT mutation of the type X collagen gene in a Japanese patient with Schmid metaphyseal chondrodysplasia.

We report here a recurrent frameshift mutation within the carboxyl-terminal noncollagenous domain coding region of the type X collagen gene (COL10A1) in a Japanese patient with Schmid metaphyseal chondrodysplasia. The mutation involves deletion of a CT dinucleotide from position 1992 (1992delCT), and produces a frameshift which creates a premature termination codon close to the site of the deletion. The predicted length of the mutant polypeptide is 664 amino acids, which is shorter than the wild type polypeptide (680 amino acids). A 1992delCT mutation of COL10A1 has been previously reported in one family. The independent occurrence of de novo mutation of this specific dinucleotide repeat suggests that this region is a possible mutational hot spot on COL10A1.

Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.

Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.

Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow.

OBJECTIVE: Cartilage-derived morphogenetic protein 1 (CDMP1), which is a member of the transforming growth factor-beta superfamily, is an essential molecule for the aggregation of mesenchymal cells and acceleration of chondrocyte differentiation. In this study, we investigated whether CDMP1-transfected autologous bone marrow-derived mesenchymal cells (BMMCs) enhance in vivo cartilage repair in a rabbit model. METHODS: BMMCs, which had a fibroblastic morphology and pluripotency for differentiation, were isolated from bone marrow of the tibia of rabbits, grown in monolayer culture, and transfected with the CDMP1 gene or a control gene (GFP) by the lipofection method. The autologous cells were then implanted into full-thickness articular cartilage defects in the knee joints of each rabbit. RESULTS: During in vivo repair of full-thickness articular cartilage defects, cartilage regeneration was enhanced by the implantation of CDMP1-transfected autologous BMMCs. The defects were filled by hyaline cartilage and the deeper zone showed remodelling to subchondral bone over time. The repair and reconstitution of zones of hyaline articular cartilage was superior to simple BMMC implantation. The histological score of the CDMP1-transfected BMMC group was significantly better than those of the control BMMC group and the empty control group. CONCLUSION: Modulation of BMMCs by factors such as CDMP1 allows enhanced repair and remodelling compatible with hyaline articular cartilage.

Modular arrangement of cartilage- and neural tissue-specific cis-elements in the mouse alpha2(XI) collagen promoter.

Type XI collagen, a heterotrimer specific to cartilage matrix, plays an important role in cartilage morphogenesis. We analyzed various alpha2(XI) collagen promoter-lacZ reporter gene constructs in transgenic mice to understand tissue-specific transcriptional regulation. The -530 promoter sequence was sufficient to direct reporter gene expression specifically to cartilage. Further deletion to -500 abolished reporter gene expression in cartilage but activated the expression specific to neural tissues such as brain and neural tube. An additional 47-base pair deletion resulted in random tissue expression patterns. A 24-base pair sequence from -530 to -507 of the alpha2(XI) promoter was able to switch the activity of the heterologous neurofilament light gene promoter from neural tissues to cartilage. These results suggest that the alpha2(XI) collagen gene is regulated by at least three modular elements: a basal promoter sequence distal to -453, a neural tissue-specific element (-454 to -500), and a cartilage-specific element (-501 to -530), which inhibits expression in neural tissues and induces expression in cartilage.

Activation and localization of cartilage-derived morphogenetic protein-1 at the site of ossification of the ligamentum flavum.

Localization and expression of cartilage-derived morphogenetic protein (CDMP)-1 in tissues at the site of ossification of the ligamentum flavum (OLF) were examined by immunohistochemistry and in situ hybridization. The CDMP-1 protein and messenger ribonucleic acid (mRNA) were localized in spindle-shaped cells and chondrocytes in the OLF tissues. CDMP-1 was not detected in cells in non-ossified sites. These data indicate that CDMP-1 is locally activated and localized in spindle-shaped cells and chondrocytes at the site of OLE. Given the previously reported promoting action of CDMP-1 for chondrogenesis, the current results suggest that CDMP-1 may be involved in the progression of OLF, leading to the narrowing of spinal canal and thus causing severe clinical manifestations.

A novel type X collagen gene mutation (G595R) associated with Schmid-type metaphyseal chondrodysplasia.

Metaphyseal chondrodysplasia of the Schmid type (MCDS) is a skeletal dysplasia affecting the long bone metaphyses; it is characterized by short stature, bowlegs, and coxa vara. The spine is generally not involved. Here we report a novel missense mutation of the type X collagen gene in a sporadic case of MCDS. The mutation was a heterozygous single base-pair transition of G-to-A at nucleotide 1783, which predicted a substitution of glycine by arginine at codon 595 (G595R) in the carboxyl-terminal noncollagenous domain. Interestingly, another mutation of the same codon, in which glycine is substituted by glutamic acid (G595E), was previously reported to cause spondylometaphyseal dysplasia (SMD), another group of skeletal dysplasias with involvement of the spine in addition to the long tubular bones. The novel G595R mutation identified in the present study supports the concept of type X collagenopathy.

Differential in situ expression of alpha2(XI) collagen mRNA isoforms in the developing mouse.

Type XI collagen is an essential structural component of the extracellular matrix of cartilage and plays a role in collagen fibril formation and skeletal morphogenesis. The expression of all three type XI collagen genes is not restricted to cartilage. In addition, alternative exon usage seems to increase the structural diversity and functional potential of type XI collagen during development. In order to investigate type XI collagen expression during development, we have examined alpha2(XI) and alpha1(XI) collagen genes by in situ hybridization in mice. Transcripts of the alpha2(XI) collagen gene were first detected in the notochord of mouse embryos after 11.5 days of gestation. Subsequently, alpha2(XI) mRNA was mainly found in the cartilaginous tissues of the developing limbs and axial skeleton together with transcripts of the alpha1(XI) gene. The alpha2(XI) transcripts seemed to be alternatively spliced isoforms lacking exons 6-8, which code for an acidic domain. Expression of alpha2(XI) outside the cartilage was relatively restricted, whereas expression of the alpha1(XI) gene was widespread. However, expression of alpha2(XI) transcripts containing exons 6-8 was found in non-chondrogenic tissues, including the calvarium and periosteum where intramembranous ossification occurs. These results indicate that alpha2(XI) mRNA isoforms are differentially expressed in various tissues during development. In addition, alpha2(XI) mRNA isoforms containing alternative exons are present in osteogenic cells, and their expression may be closely related to the formation of bone or cartilage.