EMBRYOID BODIES DERIVED FROM HSP47-KNOCKOUT EMBRYONIC STEM CELLS ARE DEFICIENT IN BASEMENT MEMBRANE FORMATION DURING EMBRYONIC DEVELOPMENT

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Hsp47 is a molecular chaperone that specifically assists the molecular maturation of procollagen. We have previously reported that hsp47 gene knockout mice is embryonic lethal and produces immature form of type I collagen. Here, we established hsp47-knockout embryonic stem (ES) cell lines and used them to produce embryoid bodies that mimic post-implantation egg-cylinder stage embryos. Although embryoid bodies developed from wild-type ES cells form thick basement membranes between inner and outer cell layers similar to egg cylinder stage embryoes, electron microscopic analysis of embryoid bodies derived from hsp47-knockout ES cells indicated that no typical monolayer basement membrane was formed. Furthermore, immunostaining of type IV collagen, a major component of the basement membrane, suggest that distribution of type IV collagen in hsp47-knockout embryoid bodies is fragmented or dispersed whereas that in wild-type embryoid bodies is concentrated in monolayer basement membrane. Pulse-chase experiments using wild-type and hsp47-knockout ES cells treated with retinoic acid and dibutyryl cyclic AMP revealed that half-life of type IV collagen in the cell is significantly longer in hsp47-knockout ES cells than in wild-type ES cells. In addition, type IV collagen of hsp47-knockout ES cells was less efficiently secreted into culture medium compared with wild-type ES cells. These results suggest that Hsp47 plays an important role in the maturation process of type IV collagen. We are currently analyzing detailed maturation process of type IV collagen molecules in differentiating hsp47-knockout ES cells.
日本結合組織学会の論文

EMBRYOID BODIES DERIVED FROM HSP47-KNOCKOUT EMBRYONIC STEM CELLS ARE DEFICIENT IN BASEMENT MEMBRANE FORMATION DURING EMBRYONIC DEVELOPMENT

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