The potential use of stem cells derived from human amniotic fluid in renal diseases

Abstract

Amniotic fluid (AF) contains a variety of cell types derived from fetal tissues that can easily grow in culture. These cells can be obtained during amniocentesis for prenatal screening of fetal genetic diseases, usually performed during the second trimester of pregnancy. Of particular interest, some expanded sub-populations derived from AF cells are capable of extensive self-renewal and maintain prolonged undifferentiated proliferation, which are defining properties of stem cells. These human AF stem cells (hAFSCs) exhibit multilineage potential and can differentiate into the three germ layers. They have high proliferation rates and express mesenchymal and embryonic markers, but do not induce tumor formation. In this study, hAFSCs derived from amniocentesis performed at 16-20 weeks of pregnancy were isolated, grown in culture, and characterized by flow cytometry and by their potential ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-7 passages, 5 × 10⁵ hAFSCs were inoculated under the kidney capsule of Wistar rats that were subjected to an experimental model of chronic renal disease, the 5/6 nephrectomy model (Nx). After 30 days, Nx rats treated with hAFSCs displayed significant reductions in blood pressure, proteinuria, macrophages, and α-smooth muscle actin expression compared with Nx animals. These preliminary results suggest that hAFSCs isolated and expanded from AF obtained by routine amniocentesis can promote renoprotection in the Nx model. Considering that the AF cells not used for fetal karyotyping are usually discarded, and that their use does not raise ethical issues, they may represent an alternative source of stem cells for cell therapy and regenerative medicine.

Keywords: amniotic fluid stem cells; chronic kidney disease; mesenchymal stem cells.

Figure 1

The capacity of human amniotic fluid stem cells (hAFSCs) to differentiate into osteogenic, adipogenic, and chondrogenic lineages. (a) In culture, hAFSCs have fibroblast-like morphology. (b) Adipogenic differentiation was evaluated using Oil Red O staining. Cytoplasmic lipid droplets appear red ( × 400). (c, d) Osteogenic differentiation was confirmed by Alizarin red and alkaline phosphatase staining. Calcium deposits and alkaline phosphatase activity appear red ( × 100 and × 400, respectively). (e, f) Chondrogenic differentiation was confirmed by hematoxylin and eosin and immunofluorescence staining using specific monoclonal antibodies against type II collagen ( × 200 and × 400, respectively).

Figure 2

Surface antigen evaluation by flow cytometry. The human amniotic fluid stem cells (hAFSCs) were positive for CD29, CD44, CD90, and CD105; and negative for CD14, CD31, CD34, CD45, and CD117. IgG1-fluorescein isothiocyanate (FITC) and IgG2-phycoerythrin (PE) antibodies were used as isotype controls.