Derivation, Characterization, and Differentiation of Human Embryonic Stem Cells

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 pronasetreated 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. Stem

Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines

“…Approximately 20 lines from the 78 derivations undertaken before August 9, 2001, are available in sufficient quantities for general research use (National Institutes of Health [NIH] stem cell registry, http://stemcells.nih.gov/research/registry). Of these, only a small subset of lines is available for detailed characterization [1][2][3][4][5][6][7][8]. As expected, various hESC lines have a number of similarities.…”

“…As expected, various hESC lines have a number of similarities. For example, undifferentiated hESCs are similar in expressing surface antigens and markers characteristic of the undifferentiated ESC state, including Oct4 (POU5F1), Nanog, UTF1, DPPA5, TERT, gap junction proteins, and SSEA and TRA antigens [1][2][3][4][5][6][7][8]. hESCs are also similar in their ability to proliferate and differentiate into cell types of the three germ layers in vitro and in vivo [9 -16].…”

Low O 2 tensions and the prevention of differentiation of hES cells

“…hES cells are generally derived from the inner cell mass of blastocysts produced by in vitro fertilization techniques (1,3,(10)(11)(12)(13). It is not widely appreciated that the environment of the mammalian reproductive tract, to which naturally conceived embryos are exposed, is 1.5-5.3% O 2 (14).…”

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells

“…The unique proliferative properties of human ES cells may be related to a naïve transcriptome which is dedicated to self-propagation through mitotic cell division and sustenance of a pluripotent uncommitted state (Thomson et al, 1998;Amit et al, 2000;Pera et al, 2000;Bhattacharya et al, 2004;Brandenberger et al, 2004;Miura et al, 2004;Rao et al, 2004;Boyer et al, 2005;Wei et al, 2005). Defining the molecular basis of pluripotency and the capability of human ES cells for self renewal is critical for the rational design of biomedical applications of human ES cells as a cellular source for tissue replacement (Carpenter et al, 2003;Sato et al, 2003;Heins et al, 2004;Stojkovic et al, 2004;Zwaka and Thomson, 2005).…”