Clusters of amniotic fluid cells and their associated early neuroepithelial markers in experimental myelomeningocele: Correlation with astrogliosis

Abstract

Myelomeningocele (MMC) is the most common and severe disabling type of spina bifida resulting in the exposure of vulnerable spinal cord to the hostile intrauterine environment. In this study, we sought to examine the cellular content of fetal amniotic fluid (AF) in MMC and explore a correlation between these cells and pathological development of MMC. MMC was induced in fetal rats by exposing pregnant mothers to all-trans retinoic acid and AF samples were collected before term. Cells were isolated from AF samples and morphologically and phenotypically characterized in short-term cultures. In addition, the spinal cord injury in MMC fetuses was assessed by immunohistochemical examination of astrogliosis. We identified a population of cells from the AF of MMC fetuses (MMC-AF) that formed adherent clusters of tightly packed cells, which were absent from the AF of normal control fetuses (norm-AF). MMC-AF clusters contained cells co-expressing adherens junction associated proteins (ZO-1), N-cadherin and F-actin at sites of cell-cell contacts. In addition, they expressed markers of early neuroepithelial cells such as SOX-1 and Pax-6 along with other stem/progenitor cell markers such as SOX-2 and nestin. Subpopulations of cells in MMC-AF clusters also expressed more advanced differentiation markers such as doublecortin and GFAP. We found that the appearance of cluster forming cells in cultures from MMC-AF correlated with activation of astrogliosis associated with the spinal cord injury in MMC fetuses. In summary, we identified a neuroepithelial cell population in the AF of MMC fetuses that formed adherent clusters in culture and we characterized cellular markers of these cells. Our data suggests that the phase of the disease is a crucial factor in the emergence of these cells into the AF and that these cells may provide a new and important platform for studying the progression of MMC and development of improved strategies for the repair and diagnosis of MMC prenatally.

Spinal Cord Injury in Myelomeningocele: Prospects for Therapy

“…Pathological specimens of human MMC do demonstrate signs of abrasion and loss of neural tissue almost exclusively in the dorsal spinal cord at the site of MMC defect, supporting the involvement of mechanical trauma in neural injury (Hutchins et al, 1996 ; Meuli et al, 1997 ; Sutton et al, 2001 ). Dorsal displacement of the spinal cord and the emergence of neural stem cells within the amniotic fluid during the development of rat MMC fetuses is also consistent with the contribution of mechanical trauma to neural damage (Danzer et al, 2011 ; Zieba et al, 2017 ). Our recent studies show that these pathological changes coincide with decreased levels of hyaluronic acid and lower amniotic fluid viscosity of late gestational age rat MMC fetuses (Zieba et al, 2019 ).…”

“…However, the actual concentration and activity of α-amylase in the MMC amniotic fluid were not established (Sasso et al, 2020 ). Interestingly, several recent studies have identified a multitude of live astrocytes and neural stem cells within the AF with amniotic fluid of MMC fetuses, indicating little, if any, neurotoxicity (Pennington et al, 2013 , 2015 ; Turner et al, 2013 ; Zieba et al, 2017 ). Also, studies using fetal lambs reported that MMC promoted mesenchymal stem/progenitor cell phenotypes and proliferation in the amniotic fluid (Ceccarelli et al, 2015 ), suggesting that amniotic fluid in MMC may be growth permissive.…”

Methotrexate and Valproic Acid Affect Early Neurogenesis of Human Amniotic Fluid Stem Cells from Myelomeningocele

“…Previous studies demonstrated that AFSCs derived from MMC Sprague-Dawley rat model showed significantly increased neural stem cell (NSC) markers, including SOX2 and NESTIN [ 32 , 33 ]. Moreover, in a recent study, MMC-AFSCs isolated from a rat model were characterized, showing high expression of neural progenitor markers SOX2 and NESTIN [ 34 ]. Similarly, in our study, human MMC-AFSCs significantly overexpressed early neural genes SOX2 and NESTIN and in addition PAX3 when compared to healthy AFSCs.…”

Hydrogel and neural progenitor cell delivery supports organotypic fetal spinal cord development in an ex vivo model of prenatal spina bifida repair

“…However, we should also acknowledge that the MMC spinal cord used in our experiments was harvested near term (E21), which does not take advantage of the likely enhanced regenerative capacity of the spinal cord earlier in gestation. 62,63 Some investigators have suggested that donor cells may be more likely to differentiate into neural lineages within an embryonic spinal cord niche (E16). 49 To date, most biomaterials described in animal models of MMC have comprised acellular, biodegradable sponges, sheets, or films whose primary aim is to provide tissue coverage over the spinal cord.…”