Table 1.

Enrichment of genes comprising top 100 lineage-specific genetic markers of each of three major embryonic lineages of human preimplantation embryos among genes that are significantly up-regulated in the MLME cells.

Classification category	Number of genes	Number of up-regulated genes in the MLME cells	Percent	P value*	Observed/expected ratio**
Human genome	26,178	9,430	36.0
Genetic markers of the human Epiblast (EPI)	100	91	91.0	1.186E−30	2.53
Genetic markers of the human Primitive Endoderm (PE)	88	41	46.6	0.0107581	1.29
Genetic markers of the human Trophectoderm (TE)	100	81	81.0	2.799E−20	2.25

*p values were estimate using the hypergeometric distribution test; **, expected values were estimated based on the number of all analyzed genes (26,178) and the number of genes significantly up-regulated in the human embryonic MLME (9,430); MLME, multi-lineage markers expressing cells; A total of 819 telomerase-positive (TERTpos) individual human embryonic cells were analyzed and each single cell was identified as the putative immortal MLME cell if it expressed genetic markers of each of the three major lineages (epiblast, EPI; throphectoderm, TE; and primitive endoderm, PE) and all three (NANOG; POU5F1; SOX2) pluripotent state master regulators. The MLME phenotype was assigned to individual telomerase-positive cells that co-expressed at least six genetic markers of the EPI lineage; seven genetic markers of the TE lineage; and four genetic markers of the PE lineage; and three main master pluripotency transcription factors. The expression levels of 58 genetic markers of human embryonic lineages were considered individually in a particular single cell by comparing the expression values of the markers in a given cell and the median expression value of the marker in the population of single cells of human embryos as previously reported (18, 140). The marker was considered expressed when the expression value in a cell exceeded the median expression value. The set of 58 genetic markers of human embryonic lineages analyzed in these experiments during the selection a total of 135 MLME cells from 839 TERTpos human embryonic cells is listed in the Supplemental Table S4 (18) and was originally reported elsewhere (141). Independent sets of lineage-specific markers comprising of top 100 individual genetic markers for each embryonic lineage were utilized for validation of the MLME phenotype and were reported elsewhere (140).