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

Date Published: March 30, 2017

Publisher: Public Library of Science

Author(s): Jolanta Zieba, Amanda Miller, Oleg Gordiienko, George M. Smith, Barbara Krynska, Irina Kerkis.

http://doi.org/10.1371/journal.pone.0174625

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.

Partial Text

Myelomeningocele (MMC), the most common and severe form of spina bifida, is a devastating congenital defect. [1,2]. It is characterized by protrusion of the meninges and spinal cord through the overlying vertebral defect and wound opening in the skin [3]. Children affected by MMC face significant and life-long physical disabilities including leg paralysis, sensory loss, bowel and bladder dysfunctions, skeletal deformations, and Arnold-Chiari II malformation with secondary hydrocephalus often requiring lifelong support and institutional care [4–6]. The etiology in most cases of MMC is multifactorial involving teratogenic, genetic and nutritional factors [7–9]. In particular, folic acid deficiency has been implicated in increased risk of neural tube defects, including MMC [10,11]. However, despite mandatory folate supplementation and routine treatment of women with folic acid before or during early pregnancy, neural tube defects remain among the most common congenital abnormalities in humans. Treatment and management of patients with these defects continues to have a huge economic burden on the health care system [12,13].

All animal studies in this project were performed under protocols approved by Temple University’s Institutional Animal Care and Use Committee (IACUC). The Temple University IACUC functions to ensure compliance with all federal and state regulations on the humane care and use of animals in research, including the provisions of the Animal Welfare Act and the PHS Policy on Humane Care and Use of Laboratory Animals. All methods of euthanasia were consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA).

In this study of amniotic fluid cells from MMC-AF samples, we identified clusters of adherent cells in short-term cultures and characterized their associated phenotypic markers. These cell clusters are phenotypically reminiscent of the early neuroepithelium and they are absent in cultures from norm-AF samples. Phenotypic characterization of cells in MMC-AF clusters showed that a subset of tightly packed cells co-expressed adherens junction associated proteins ZO-1 and N-cadherin, which contribute to cell-cell adhesion characteristic of early neuroepithelial cells [36]. As previously reported, N-cadherin-based adherens junctions contribute to the strong adhesion between neuroepithelial cells during early embryonic development to maintain the structure of the neuroepithelia [40–42]. The majority of cells in MMC-AF clusters were positive for SOX-1 and Pax-6 markers of early neuroepithelial cells. Likewise, the neural stem/progenitor cell markers SOX-2 and nestin were also present in these clusters. Nestin, an intermediate filament, is transiently expressed in neuroepithelial stem/progenitor cells of the developing CNS. It is recognized as a marker for developing glial cells and neural progenitor cells and its downregulation correlates with differentiation of these cells [43]. Nestin expressing GFAP positive astrocytes were distributed throughout MMC-AF cell clusters, indicating their immature phenotype. In addition, we also found a number of mature astrocytes expressing GFAP in the absence of nestin. These findings are in line with earlier studies, which have reported the presence of GFAP positive astrocytes in a fraction of rapidly adhering cells in cultures from AF of fetuses with neural tube defects [25,26,28]. Likewise, following epigenetic selection, a sub-population of SOX-2 and nestin positive neural stem cells was isolated in culture from AF of experimental neural tube defects [29]. Our data confirm and extend on these observations, in which we show, for the first time, presence of the earliest population of neuroepithelial cells expressing ZO-1 and N-cadherin as well as SOX-1 and Pax-6 isolated from AF of MMC fetuses. We also showed that these cells exist along with SOX-2 and nestin positive cells, GFAP positive astrocytes, and doublecortin positive neurons. Thus, the data presented here combined with previous reports [29–31] shows neural cells isolated from the AF in the setting of neural tube defects contain various populations of neuroepithelial cells.

 

Source:

http://doi.org/10.1371/journal.pone.0174625

 

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