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Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo.

ABSTRACT: Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties through the forced expression of transcription factors. Herein, we investigated the in vitro hypertrophic differentiation and in vivo tissue formation capacity of induced chondrogenic cells (iChon cells) obtained through direct reprogramming. In vitro hypertrophic differentiation was detected in iChon cells that contained a doxycycline-inducible expression system for Klf4, cMyc, and Sox9. Furthermore, endochondral bone formation was detected after implantation in nude mice. The bone tissue was derived entirely from host origin, whereas cartilage tissue contained cells from both host and donor. The results obtained highlight the promise of cellular reprogramming for the creation of functional skeletal cells that can be used for novel bone healing strategies.

SUBMITTER: Tam WL

PROVIDER: S-EPMC3920914 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo.

Tam Wai Long WL O Dorien F DF Hiramatsu Kunihiko K Tsumaki Noriyuki N Luyten Frank P FP Roberts Scott J SJ

Cellular reprogramming 20140201 1

Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties throug ...[more]

PMID: 24459991

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Project description:Collagen X is a non-fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss of Col10a1 gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous (COL10A1 +/-) or homozygous (COL10A1 -/-) deletions of COL10A1 gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet-derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger in COL10A1 -/- tissues. Prehypertrophic pellet-derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental- and mutant-derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating-phase genes and a higher expression of calcification-phase genes in COL10A1 -/- pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC-derived chondrocytes, though it may facilitate the differentiation process. Thus, COL10A1 -/- iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Project description:BackgroundIn healthy articular cartilage, chondrocytes are found along arcades of collagen fibers as Single Strings. With onset of cartilage degeneration this pattern changes to Double Strings. In the course of osteoarthritis Small Clusters, and finally Big Clusters form. In highly degenerated articular cartilage, another poorly understood pattern is found where chondrocyte morphology differs considerably, and the distribution of cells is diffuse. Progression of osteoarthritis is accompanied by key processes such as chondrocyte proliferation, apoptosis, hypertrophic differentiation, inflammation, and angiogenesis. The aim of this exploratory study was to identify biomarkers for these processes in the context of spatial cellular organizational changes in articular cartilage.MethodsCartilage explants (n = 166 patients) were sorted according to their predominant cellular pattern. Quantitative or semi-quantitative analysis of 39 biomarkers were performed by multiplex assay (31) or ELISA (8), and qualitative analysis on 12 immunohistochemical markers.ResultsHypertrophic differentiation (e.g. type-X collagen, osteopontin, osteocalcin and interleukin-6) and angiogenesis were associated with changes in chondrocyte organisation. First changes take place already at the transition from Single Strings to Double Strings. Drastic changes in the appearance of numerous biomarkers are found at the transition from Big Clusters to Diffuse.ConclusionKey processes in osteoarthritis and their biomarkers seem to depend on the spatial distribution of chondrocytes in articular cartilage. Abrupt changes in biomarker occurrence were observed between Big Clusters and Diffuse insinuating that the Diffuse pattern is composed of a different cell population or at least a different form of chondrocyte morphology.The translational potential of this articleIn situ identification of the different spatial chondrocyte patterns by fluorescence microscopy has already been established in the recent past. Analysing human in-situ cartilage explants rather than isolated OA chondrocytes closes the gap between in vitro and in vivo studies and as such, stretches a big step towards translation of the observed findings. The direct association between tissue biomarker profile and cellular arrangements representing different states of OA sheds new light on the molecular and cellular physiopathology, especially in the context of larger processes such as angiogenesis, cellular proliferation, differentiation, and apoptosis. This also opens an interesting perspective for future investigation of such biomarkers and processes in clinical studies.

Project description:Following penetrating injury of the skin, a highly orchestrated and overlapping sequence of events helps to facilitate wound resolution. Inflammation is a hallmark that is initiated early, but the reciprocal relationship between cells and matrix molecules that triggers and maintains inflammation is poorly appreciated. Elastin is enriched in the deep dermis of skin. We propose that deep tissue injury encompasses elastin damage, yielding solubilized elastin that triggers inflammation. As dermal fibroblasts dominate the deep dermis, this means that a direct interaction between elastin sequences and fibroblasts would reveal a proinflammatory signature. Tropoelastin was used as a surrogate for elastin sequences. Tropoelastin triggered fibroblast expression of the metalloelastase MMP-12, which is normally expressed by macrophages. MMP-12 expression increased 1056 ± 286-fold by 6 h and persisted for 24 h. Chemokine expression was more transient, as chemokine C-X-C motif ligand 8 (CXCL8), CXCL1, and CXCL5 transcripts increased 11.8 ± 2.6-, 10.2 ± 0.4-, and 8593 ± 996-fold, respectively, by 6-12 h and then decreased. Through the use of specific inhibitors and protein truncation, we found that transduction of the tropoelastin signal was mediated by the fibroblast elastin binding protein (EBP). In silico modeling using a predictive computational fibroblast model confirmed the up-regulation, and simulations revealed PKA as a key part of the signaling circuit. We tested this prediction with 1 ?M PKA inhibitor H-89 and found that 2 h of exposure correspondingly reduced expression of MMP-12 (63.9±12.3%) and all chemokine markers, consistent with the levels seen with EBP inhibition, and validated PKA as a novel node and druggable target to ameliorate the proinflammatory state. A separate trigger that utilized C-terminal RKRK of tropoelastin reduced marker expression to 65.0-76.5% and suggests the parallel involvement of integrin ?V?3. We propose that the solubilization of elastin as a result of dermal damage leads to rapid chemokine up-regulation by fibroblasts that is quenched when exposed elastin is removed by MMP-12.

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Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo.

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Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo.

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