Isolation and Initial Characterization of Nonhuman Primate Amniotic Fluid-Derived Stem CellsJ. Koudy Williams, Silmi Mariya, Anthony Atala, Shay Soker
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-kitr human 3.I1’lI1l0lllC ﬂuid-derived stem (hAF S) 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 AF S technology, the goal of this study was to determine if AF S 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-kitr cells were isolated, further expanded and clonal populations obtained. The putative AF S 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 leagt 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). nhpAF S cells express the major, but not minor, histocompatability antigen and were onsistently 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 nhpAF S cells, similar to hAF S cels, have the characteristics needed to bioengineer a wide-variety of mesodermal-cell containing organs and tissues.