Abstract
Placental alkaline phosphatase (PLAP), biochemically and genetically distinct from other human tissue alkaline phosphatases, is expressed by trophoblast cells and increases throughout gestation. Recently, PLAP has been used extensively to isolate placenta-derived exosomes from biological specimens and quantitative differences of PLAP-positive exosomes have been associated with various adverse pregnancy outcomes. By examining exosomes from other uterine tissues and immunostaining of various feto-maternal tissues, we tested the placental specificity of PLAP. Exosomes from amnion, decidua, and myometrial cell cultures were isolated by differential centrifugation and characterized by size and exosome markers. PLAP expression in exosomes was tested by western blot using a PLAP-specific antibody (ab133602, Abcam). Additionally, amniochorion with decidua, and myometrium were collected from normal term pregnancies and PLAP localization was performed using immunohistochemistry (IHC) using the same antibody. Exosomes isolated from BeWo (trophoblast cell line) were used as positive control. Exosomes from various cell types were round with a size of 150nm and positive for exosomal proteins CD81, CD9 and CD63. Reactivity to PLAP was seen in exosomes isolated from all cell types (amnion, decidua and myometrium) (Figure 1A). As expected, BeWo cells were positive for PLAP. This was further confirmed by IHC were PLAP was localized in fetal (amnion, chorion, and cells of the extracellular matrix) and maternal uterine tissues (decidua and myometrium) confirming PLAP production by these cell types (Figure 1B). Chorion had the highest intensity staining and was weaker in decidua and myometrium. PLAP is produced by fetal membranes, uterine decidua and myometrial tissues and packaged in their respective exosomes. This led us to speculate that PLAP-containing exosomes isolated from maternal biological samples throughout pregnancy are not necessarily trophoblast in origin, but represent all gestational tissues. PLAP-specific exosome based biomarker studies should be cautious in interpreting their data as those exosomes are not likely indicators of placental function alone but overall gestational tissue functions.
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