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Probing Stemness and Neural Commitment in Human Amniotic Fluid Cells

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Abstract

Recently, human amniotic fluid (AF) cells have attracted a great deal of attention as an alternative cell source for transplantation and tissue engineering. AF contains a variety of cell types derived from fetal tissues, of which a small percentage is believed to represent stem cell sub-population(s). In contrast to human embryonic stem (ES) cells, AF cells are not subject to extensive legal or ethical considerations; nor are they limited by lineage commitment characteristic of adult stem cells. However, to become therapeutically valuable, better protocols for the isolation of AF stem cell sub-populations need to be developed. This study was designed to examine the molecular components involved in self-renewal, neural commitment and differentiation of AF cells obtained at different gestational ages. Our results showed that, although morphologically heterogeneous, AF cells derived from early gestational periods ubiquitously expressed KERATIN 8 (K8), suggesting that the majority of these cells may have an epithelial origin. In addition, AF cells expressed various components of NOTCH signaling (ligands, receptors and target genes), a pathway involved in stem cell maintenance, determination and differentiation. A sub-population of K8 positive cells (<10%) co-expressed NESTIN, a marker detected in the neuroepithelium, neural stem cells and neural progenitors. Throughout the gestational periods, a much smaller AF cell sub-population (<1%) expressed pluripotency markers, OCT4a, NANOG and SOX2, from which SOX2 positive AF cells could be isolated through single cell cloning. The SOX2 expressing AF clones showed the capacity to give rise to a neuron-like phenotype in culture, expressing neuronal markers such as MAP2, NFL and NSE. Taken together, our findings demonstrated the presence of fetal cells with stem cell characteristics in the amniotic fluid, highlighting the need for further research on their biology and clinical applications.

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Acknowledgements

The authors are grateful to Dr. Fred Gage (Salk Institute, La Jolle, CA, USA) for providing the SOX2 promoter construct, and Sandhya Gangaraju and Brandon Smith for their technical assistance. AJ is funded by the CIHR - Frederick Banting and Charles Best Canada Graduate Scholarship.

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Authors and Affiliations

  1. Neurogenesis and Brain Repair, Neurobiology Program, Institute for Biological Sciences, National Research Council Canada, Building M-54, 1200 Montreal Road, Ottawa, Canada, K1A 0R6

    Anna Jezierski, Roger Tremblay, Dao Ly, Cathie Smith, Marianna Sikorska & Mahmud Bani-Yaghoub

  2. Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada

    Anna Jezierski, Andree Gruslin, Kursad Turksen & Mahmud Bani-Yaghoub

  3. Department of Obstetrics and Gynecology, Faculty of Medicine, University of Ottawa, Ottawa, Canada

    Andree Gruslin

  4. Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, Canada

    Kursad Turksen

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  1. Anna Jezierski
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Correspondence to Marianna Sikorska or Mahmud Bani-Yaghoub.

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Supplementary Figure 1

Expression of pluripotency stem cell markers in the C-KIT sorted AF cells. A–B. Immunofluorescence of dissociated AF cells stained with C-KIT (green) antibody prior to sorting for C-KIT positive cells (C-KIT+) by Fluorescence Activated Cell Sorting (FACS). B, merged immunofluorescence and phase contrast images. Scale bar: 50 μm. C. Flow cytometry gating for C-KIT+ AF cell population. D. qPCR analysis of mRNA harvested from C-KIT positive (C-KIT+) and negative (C-KIT−) AF cells directly following sorting discriminated the expression of SOX2, OCT4a/b and NANOG. Fold expression levels were normalized to β-ACTIN (ACTB). (GIF 43 kb)

High resolution (TIFF 7324 kb)

Supplementary Figure 2

Identification of SOX2 positive cells in AF cultures, using SOX2 promoter-EGFP retrovirus. A. The AP2 retroviral vector containing SOX2 promoter-EGFP was used to infect AF cell cultures. B–C. The SOX2 expressing AF cells were identified based on EGFP expression (green) prior (B) and after (C) differentiation into neurons. (GIF 31 kb)

High resolution (TIFF 3309 kb)

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Jezierski, A., Gruslin, A., Tremblay, R. et al. Probing Stemness and Neural Commitment in Human Amniotic Fluid Cells. Stem Cell Rev and Rep 6, 199–214 (2010). https://doi.org/10.1007/s12015-010-9116-7

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