INTRODUCTION: Recent publications have suggested that a highly pluripotent stem cell population may be derived from the cellular fraction of amniotic fluid. During the surgical correction of twin to twin transfusion syndrome by fetoscopic laser ablation, large amounts of amniotic fluid are harvested, the cellular component of which could serve as a potentially rich source of amniotic fluid-derived pluripotent stem cells. To explore this hypothesis, we collected large amounts of amniotic fluid following fetoscopic laser ablation procedures and sought to generate and characterize amniotic fluid-derived stem cells (AFS) as described previously.1
METHODS: Amniotic fluid was collected following fetoscopic laser ablation with IRB approval. The total cellular component was cultured in α-MEM (Hyclone) supplemented with 15% ES FBS (Invitrogen), 100 U/ml penicillin G/streptomycin, 2 mM L-glutamine, 18% Chang Basal Medium, and 2% Chang C Medium (both from Irvine Scientific). Adherent cells were expanded and selected with CD117 microbeads (Miltenyi Biotec). Cells were characterized by flow cytometry, and hematologic pluripotence was assessed by culture in 100 ng/ml GM-CSF, co-culture with OP-9 cells, or co-culture with primary human bone-marrow stroma. Proliferative capacity was assessed and doubling times were determined for all serial passages.
RESULTS: Amniotic fluid derived cells were typically CD90posCD105posHLA-DRneg with minority populations that were HLA-DRlow and CD133pos. This phenotype is consistent with that of bone marrow-derived MSCs. Cellular morphology was also consistent with that of bone marrow-derived MSCs. We did not detect any expression of CD34, CD45, or CD38 either pre- or post-CD117 selection. Some samples were fully devoid of CD117pos cells; however, others did exhibit CD117-expressing cells which could be selected. Attempts at transdifferentiation of either CD117pos or CD117neg cells by ES cell methods were unsuccessful; the AFS cell phenotype remained consistent throughout the addition of exogenous cytokines or co-culture with bone marrow stroma. CD117neg AFS cell lines began to divide more slowly, exhibiting doubling times of 90 hours by post-selection passage 10. In contrast, CD117pos AFS cell lines maintained a doubling time of 25–30 hours through post-selection passage 10.
CONCLUSIONS: The AFS cells derived from fetoscopic laser ablation procedures exhibited an MSC phenotype and did not appear to have pluripotent plasticity. We hypothesize that AFS cells with greater plasticity might exist at the time of diagnostic amniocentesis (week 14–16) but appear to be absent in the fluid samples that we obtained on weeks 24–26. The AFS MSCs had a proliferative potential greater than that exhibited by bone marrow MSCs, and the presence of surface CD117 (SCF receptor) seemed to be predictive of an enduring proliferative ability. Further study will be necessary to determine the validity and significance of this correlation.
Disclosure: No relevant conflicts of interest to declare.