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Human term amniotic fluid: a novel source of stem cells for regenerative medicine

      Objective

      Our objective was to characterize term amniotic fluid stem cells and compare them with previously described mid-trimester amniotic fluid stem cells. Stem cells are undifferentiated cells with the ability to self-replicate without differentiation. Mid-trimester amniotic fluid stem cells have been well-characterized as multipotent, but scant data exist for term amniotic fluid.
      • Loukogeorgakis S.P.
      • De Coppi P.
      Stem cells from amniotic fluid: potential for regenerative medicine.
      With the advent of noninvasive prenatal diagnosis, mid-trimester amniotic fluid will become less available from normal patients. In contrast, term amniotic fluid is available in large quantities from normal pregnancies and might be a resource for clinical transplantation.

      Study Design

      Institutional Review Board approval and informed consent were obtained before research began. Amniotic fluid was collected from uncomplicated, scheduled term cesarean deliveries with a small uterine incision and a soft catheter to collect fluid before the attempt to deliver the baby. Immediately after collection, cell viability from each fresh sample was assayed before culture with the use of Trypan Blue staining; cells were counted via hemocytometer. Cells were then cultured for expansion; aliquots were taken and frozen from each subsequent passage. Cells were seeded on gridded culture dishes and counted daily for 4 days to assess proliferation. Flow cytometry for surface markers and gene expression analysis by real-time polymerase chain reaction for genes that were associated with stem cells were performed at passage 3. Differentiation toward neural lineage was demonstrated by immunofluorescent microscopy with the use of monoclonal antibodies for nestin, B-tubulin III, and glial fibrillary acidic protein. Chondrocyte differentiation similarly was shown with monoclonal antibodies for aggrecan. Differentiation toward osteocyte lineage was assayed quantitatively for alkaline phosphatase activity and verified by alkaline phosphatase and Alizarin Red staining.
      • Chen Z.
      • Chan M.K.
      • Strelchenko N.
      • et al.
      Heterogeneity of stem cells in human amniotic fluid.

      Results

      Amniotic fluid was collected from 19 women with mean age of 33.9±2.7 years and mean gestation age of 39.0 ±0.7 weeks. Median volume of amniotic fluid that was obtained during collection was 61 mL (range, 20–118 mL). Samples from 8 patients were cultured successfully. The others were discarded because of meconium, blood, or slow growth. Mean cell viability was 80±7%, and cell counts were 1.3×105 to 1.5×106 per mL. The mean doubling time was 27 hours. Samples tolerated 10 passages without senescence and cryostorage for up to 18 months. All samples demonstrated cells with multiple surface markers and transcription genes that were associated with multipotent stem cells (Figure). Multipotency was demonstrated by differentiation into neural, osteocyte, and chondrocyte lineages.
      Figure thumbnail gr1
      FigureStem cell markers in term human amniotic fluid–derived stem cells
      A, The graph shows the results of flow cytometry studies that were performed on term human amniotic fluid cells that were harvested at passage 3 to determine the presence of surface markers associated with stem cells. For all samples, the average percent of cells that expressed each marker is shown. B, The graph shows the results of real-time polymerase chain reaction studies to determine the expression of genes that were associated with stem cells (OCT3/4, SOX2, NANOG, REX1, and PROM1) in term human amniotic fluid. The stem cell genes are compared to housekeeping genes normally expressed by all cells (HRPT1 and G6PDX) to compare gene expression levels.
      Dolin. Human term amniotic fluid. Am J Obstet Gynecol 2018.

      Conclusion

      Term and mid-trimester amniotic fluid stem cells demonstrate similar characteristics. We showed that term amniotic fluid stem cells were multipotent, differentiated into multiple cell lineages, and withstood cryostorage. Previous studies on term fluid have demonstrated only mesenchymal cells that required induction to pluripotent stem cells to achieve differentiation to neural lineage.
      • Moraghebi R.
      • Kirkeby A.
      • Chaves P.
      • et al.
      Term amniotic fluid: an unexploited reserve of mesenchymal stromal cells for reprogramming and potential cell therapy applications.
      Induced pluripotent cells are genomically unstable and tumorigenic; amniotic stem cells are not.
      • Rebuzzi P.
      • Zuccotti M.
      • Redi C.A.
      • Garagna S.
      Achilles’ heel of pluripotent stem cells: genetic, genomic and epigenetic variations during prolonged culture.
      Our study demonstrates for the first time that both ectodermal and mesenchymal differentiability are present in term amniotic fluid stem cells. Neural differentiation was achieved without the induction of pluripotency. Term amniotic fluid contains approximately 1 million cells per milliliter, which may represent a readily available, abundant, and noncontroversial source of stem cells with therapeutic potential.

      References

        • Loukogeorgakis S.P.
        • De Coppi P.
        Stem cells from amniotic fluid: potential for regenerative medicine.
        Best Pract Res Clin Obstet Gynaecol. 2016; 31: 45-57
        • Chen Z.
        • Chan M.K.
        • Strelchenko N.
        • et al.
        Heterogeneity of stem cells in human amniotic fluid.
        J Regen Med. 2014; 3
        • Moraghebi R.
        • Kirkeby A.
        • Chaves P.
        • et al.
        Term amniotic fluid: an unexploited reserve of mesenchymal stromal cells for reprogramming and potential cell therapy applications.
        Stem Cell Res Ther. 2017; 8: 190
        • Rebuzzi P.
        • Zuccotti M.
        • Redi C.A.
        • Garagna S.
        Achilles’ heel of pluripotent stem cells: genetic, genomic and epigenetic variations during prolonged culture.
        Cell Mol Life Sci. 2016; 73: 2453-2466