Adamts-9 is involved in mesoderm differentiation of human embryonic stem cells

Abstract

A Disintegrin-like And Metalloproteinase with Thrombospondin (TSP)-Type I sequence motifs 9 (ADAMTS9) is widely expressed in mouse and human fetal tissues. ADAMTS9 null mice cannot survive beyond E7.5 and its haploinsufficiency is associated with cardiac and aortic anomalies. We hypothesized that ADAMTS-9 was important during early embryogenesis. Human embryonic stem cell (hESC) was used as a model for studying ADAMTS9 expression and its role during early differentiation. Our results indicated that ADAMTS9 immunoreactivity was detected in cells located at the boundary of hESC colonies in undifferentiated state. Its mRNA and protein expression increased time-dependently in the first 24 days’ of embryoid body (EB) formation. The expression pattern was similar to that of mesoderm and endoderm markers. The positive correlation of ADAMTS9 with ESC differentiation was also found in the mouse system, in which Adamts9 expression was increased time-dependently during mouse EB formation and down-regulated during reprogramming from somatic cells into induced pluripotent cells. During hESC differentiation ADAMTS9 was co-localized with several specific mesoderm and endoderm markers. Transient ADAMTS9 knockdown by siRNA in hESC significantly decreased the expression level of mesoderm marker, REN. Induction of differentiation of hESCs towards the mesoderm lineages dramatically increased ADAMTS9 expression of the differentiated colonies. In the differentiated mesodermal cells, ADAMTS9 was co-expressed with vascular endothelial markers, VEGF and CD31, but not with pericyte markers, alpha muscle actin. Lentiviral vector encoding ADAMTS9 shRNA was used for long term knockdown of ADAMTS9. Knockdown of ADAMTS9 significantly reduced the expression of certain mesoderm markers, REN, PDGFRα and CD34. In conclusion, ADAMTS9 was induced during mesoderm differentiation and its knockdown led to down-regulation of mesoderm markers. The roles of ADAMTS9 during hESC differentiation and early embryo development warrant further investigation.