Derivation, Characterization, and Differentiation of Human Embryonic Stem Cells

TLDR

Human embryonic stem cell derivation opens new avenues for studying human biology and medicine. The study presents the derivation and characterization of six human embryonic stem cell lines to meet growing demand. Using immunosurgery and pronase‑treated, hatched blastocysts, the inner cell mass was propagated to establish hES lines, which were then extensively characterized by marker expression, karyotyping, telomerase activity, and in vitro and in vivo pluripotency assays. Three lines displayed full pluripotency, one had trisomy 13 yet remained pluripotent, two (including a triploid line) showed limited in vivo pluripotency, and a clonally derived line could be maintained in an undifferentiated pluripotent state.

Abstract

The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.