Isolation and initial characterization of nonhuman primate amniotic fluid-derived stem cells

Regenerative medicine promises to address the organ shortage by engineering replacement cells, tissues and organs using adult, progenitor, or human embryonic stem cells. However, embryonic stem cells can form teratomas and bone marrow-derived progenitor cells have limited expansion capabilities. We recently described a subpopulation of c-kit+ human amniotic fluid-derived stem (hAFS) cells that are highly expansive, do not form teratomas and can yield differentiated cell derivatives corresponding to the three germ layers. To this end, and because nonhuman primates (NHP) represent a critical step towards pre-clinical testing of the AFS technology, the goal of this study was to determine if AFS cells could be isolated from amniotic samples of monkeys. Expansive cell cultures were derived from 12/28 amniotic fluid samples of cynomolgus monkey. Cells were isolated and expanded in Chang’s media and c-kit+ cells were isolated, further expanded and clonal populations obtained. The putative AFS cells were characterized as to their karyotype, ability to expand, identification of cell-surface and nuclear markers (qPCR) and ability to differentiate to mesodermal lineages such as bone, muscle, fat, cartilage and endothelial cells. Highly expansive multipotent cells were isolated most readily from the second trimester of pregnancy (8/15 samples, or 53%), and less so from either the first (1/3, or 33%), or third (3/10, or 30%) trimester. Cell were diploid in all cases and were consistently expanded through at least 20 passages (10 passages for the 3rd trimester cells) without losing their proliferation potential, as confirmed by cell cycle analysis (G1, G2, S phase) and doubling times. The AFS cells had low-level expression of pluripotentiality markers (Oct4, Nanog, SOX2) compared to NHP embryonic stem cells, while expressing markers similar to that of adult mesodermal-lineage cells (CD44, CD90, CD73 and CD105). nhpAFS cells express the major, but not minor, histocompatability antigen and were consistently differentiated into adipogenic, osteogenic, myogenic, chondrocytic and endothelial lineages. Because of their robust expansion capabilities and ability to differentiate into several mesodermal lineages, it is concluded that nhpAFS cells, similar to hAFS cels, have the characteristics needed to bioengineer a wide-variety of mesodermal-cell containing organs and tissues.