Derivation of Three Clones from Human Embryonic Stem Cell Lines by FACS Sorting and Their Characterization

Here we describe the first report of three human embryonic stem cell (hESC) clones, hES 3.1, 3.2, and 3.3, derived from the parent line hES3 by sorting of single-cell preparations by flow cytometry. The viability of single-cell preparations before and after cell sorting remained >98%. The hESC were selected by size gating and forward-angle light scatter and were dispersed directly as singlecell/ well into 96-well plates containing human fetal fibroblasts as feeder layers. Single stem cell dispersion into 96-well plates was confirmed by using cells from a hES3 line that constitutively expressed green fluorescence protein (eGFP) under similar conditions of flow cytometry. Three clones were obtained from the parent line hES3—hES3.1, 3.2, and 3.3—and they have been in continuous culture for more than 1 year. The cloning efficiency was less than <0.5%. These hESC clones show normal stem cell characteristics, such as undifferentiated growth, high nucleocytoplasmic ratio, the same karyotype as that of the parent line (46 XX), stem cell surface markers (i.e., SSEA3, SSEA4, OCT4, TRA-1-60, and TRA-1-81), and gene expression for pluripotency (Oct-4 and nanog). They all formed embryoid bodies in suspension cultures, and after seeding in culture plates they showed pluripotency in vitro by forming cell lineages derived from all three germ layers as indicated by expression of the ectodermal marker nestin, the mesodermal marker renin, and the endodermal markersα-fetoprotein and GATA6. All clones showed normal expression of alkaline phosphatase activity, a marker of in vitro pluripotency. When hESC clones (1–2 × 10⁶ total) were injected into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice under the kidney capsule, all formed teratomas within 6–8 weeks. Analysis of the stem cell surface marker TRA-1-160 by flow cytometry showed nonsignificant (p < 0.05) differences between the clones and the parent line. The clones also differed in their expression of genes, with only one, hES 3.2, expressing the endodermal markers, i.e., α-fetoprotein and GATA6. The ability to produce clones from a parent hESC line rapidly by FACS sorting will help provide a homogeneous population of cells for achieving uniformed lineage specifications for future transplantation therapies and biomedical research.