Faculty Bibliography

Amniotic fluid contains multipotent cells and could be a source of stem cells for clinical use. Amniotic fluid cells (AFC) are made up of a heterogeneous population of fetal cells that can be retrieved during pregnancy without ethical concernsas it is a standard clinical procedure-. Studies of proliferation, multipotent marker expression, differentiation, and gene expression were performed after culturing with dexamethasone, valproic acid and magnesium sulfate. There were correlations between known drug effects on the human fetus and changes seen in human AFC in culture, as well as previously undescribed observations in neural and chondrogenic inducibility. Gene expression profiles confirmed these observations. AFC culture may provide a novel method to evaluate pharmacological agents before clinical use in pregnancy.

Amniotic fluid cells (AFC) from 2nd trimester amniocentesis have been found to be a source of multipotent stem cells which might overcome the limitations of expansion, histocompatibility, tumorigenesis, and ethical issues associated with using human embryonic cells, umbilical cord, cord blood, bone marrow, and induced pluripotent cells. Previous work by our group and others demonstrated multipotency and the ability to grow well in culture. However, all these studies were done in media containing fetal calf serum. We sought to observe the properties of AFC grown in serum-free media as that would be required for clinical transplantation in humans. Fresh samples were obtained from three patients, and each sample divided into a culture whose cells were not exposed to fetal calf serum, and the other half into a standard culture medium containing fetal calf serum. Doubling time and stem cell marker expression by flow cytometry were assessed. Differentiation to neural, osteoid, and chondrogenic lineages was induced using appropriate media and confirmed by fluorescent microscopy, histology, and immunohistochemistry. There were no statistically significant differences between cells grown serum-free and in standard media in any of these parameters. The data supports the possibility of clinical use of AFC in stem cell transplantation.

INTRODUCTION: Trisomy is a common chromosomal abnormality associated with fetal loss and multi-systemic complications of survival in humans. Animal models cannot inform developmental pathophysiology in trisomies. Previous studies have shown slow cell kinetics and reliable differentiation into hematopoietic and neural lineages of trisomic human amniotic fluid cells (AFC). We investigated the behavior of trisomic AFCs compared with euploid AFCs in culture to guide better understanding of abnormal development. METHODS: Amniotic fluid was collected from women identified at risk for fetal abnormality at second trimester amniocentesis. Three samples were identified as trisomic (2x Trisomy21, 1x Trisomy18). AFCs were isolated and cultured to observe morphology and multiplication potential. Fluorescence activated cell sorting (FACS) was performed to evaluate cell surface markers of pluripotency, as euploid AFCs are known to be pluripotent. Cells were grown as 3D spheroids for differentiation into osteogenic, chondrogenic and neural lineages. Immunohistochemistry staining and fluorescent imaging was performed for analysis of lineage markers. Trisomic cultures were compared to matched euploid controls. RESULTS: Fetal Trisomy 18 and Trisomy 21 AFCs were morphologically indistinguishable from euploid samples. Trisomic AFCs exhibited rapid division for a 96 hr period, exceeding euploid pace. FACS analysis using antibodies for stem cell pluripotency markers SSEA4, TRA-1-60 and CD90 revealed expression patterns similar to euploid samples. Trisomy18 spheroids differentiated similarly to euploid samples into osteogenic, chondrogenic and neural lineages. Trisomy21 samples uniformly induced either more weakly or did not induce at all into all three lineages. CONCLUSIONS: We have shown rapid growth kinetics in Trisomy 21 and Trisomy 18 samples. Presence of stem-like pluripotency markers and inducibility characteristics are novel findings. Of interest, Trisomy21 showed reduced differentiation to neural lineage, which is tempting to relate to the clinical condition. AFC cultures may be useful for the study of developmental differences in aneuploid fetuses. (Figure Presented)

Stem cells are undifferentiated cells with the capacity for differentiation. Amniotic fluid cells (AFC) have only recently emerged as a possible source of stem cells for clinical purposes. There are no ethical or sampling constraints using amniocentesis as a standard clinical procedure for obtaining an abundant supply of AFC. AFC of human origin proliferate rapidly and are multipotent with the potential for expansion in vitro to multiple cell lines. Tissue engineering technologies using AFC are being explored. AFC may be of clinical benefit for fetal therapies, degenerative disease and regenerative medicine applications. We present a comprehensive review of the evolution of human amniotic fluid cells as a possible modality for therapeutic use.