Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC-Derived Chondrocytes

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to the formation of growth plate structures. These observations

might support previous reports describing stem cell populations

in proliferating chondrocytes.(43,44) Although the difference was

statistically significant only in the case of 414C2, the proportion

of the bone area in each tissue was larger in COL X-deficient tissues than in parental tissues, suggesting the acceleration of differentiation. The ectopic expression of SOX9 in hypertrophic

chondrocytes downregulates the expression of COL10A1, resulting in the elongation of the hypertrophic zone and reduction of

the proliferating zone,(45) which may be caused by the accelerated differentiation. One of the proposed functions of COL X is

the compartmentalization of matrix components to regulate

proper spatiotemporal differentiation,(11) and the loss of COL X

may induce an improper distribution of matrix molecules to alter

the differentiation process.

Cells at day 28 are already positive for IHH, a marker for prehypertrophic chondrocytes and also for RUNX2. No proliferating

chondrocytes were observed in collected pellets, suggesting

that most cells at day 28 in vitro may lose the capacity to proliferate and only the differentiation process continued in vivo.

The presence of HNA-positive osteoblasts and osteocytes in

these samples, however, suggested that undifferentiated cells

with osteogenic potentials existed in day 28 pellets or that

hypertrophic chondrocytes transdifferentiated into osteogenic

cells. No clear difference was observed between wild-type- and

mutant-derived pellets in these experiments, suggesting that

the effect of COL X loss happens only at early stages.

There are several limitations in our hiPSC differentiation system. Most importantly, it lacks the well-organized regulatory network in growth plates such as the IHH-PTHrP pathway,(1) which

may result in a nonphysiological differentiation process.

Mechanical factors, which are another important factor for the

metabolic regulation of the growth plate,(2) are also missing. Further modifications of our system are required to faithfully recapitulate the physiological process of chondrocyte differentiation.

In conclusion, COL X deficiency is dispensable for columnar

differentiation processes including hypertrophic differentiation

and the endochondral ossification of chondrocytes induced

from hiPSCs. COL X deficiency may have, however, an effect on

facilitating the differentiation process through an unknown

mechanism. The combination of gene-edited human iPSCs and

in vitro and in vivo assay systems for hypertrophic differentiation

and growth plate-like structures is useful for investigating the

role of molecules involved in the differentiation and also the

molecular mechanism of hypertrophy.

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COL X IS DISPESABLE FOR HYPERTROPHY

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