Single cell analysis of lincRNA expression during human blastocyst differentiation identifies TERT(+) multi-lineage precursor cells

Abstract

SummaryChromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50-80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. Here, we carried out single cell expression profiling analyses of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of transposable element-derived lincRNAs highly expressed in human embryonic stem cells (hESCs) and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing 1,708 individual embryonic cells recovered from more than 100 human embryos and 259 mouse embryonic cells at different stages of preimplantation embryogenesis. Our experiments demonstrate that segregation of human blastocyst cells into distinct sub-populations based on single-cell expression profiling of just three HPATs (HPAT-21; -2; and -15) appears to inform key molecular and cellular events of naïve pluripotency induction and accurately captures a full spectrum of cellular diversity during human blastocyst differentiation. HPAT’s expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of viable blastocyst differentiation enabled identification of TERT(+) sub-populations, which are significantly enriched for cells expressing key naïve pluripotency regulatory genes and genetic markers of all three major lineages created during human blastocyst differentiation. Results of our analyses suggest that during early stages of preimplantation embryogenesis putative immortal multi-lineage precursor cells (iMPCs) are created, which then differentiate into trophectoderm, primitive endoderm and pluripotent epiblast lineages. We propose that cellular extinction events in cleavage-stage embryos are triggered by premature activation of HPAT lincRNAs reflecting failed iMPC’s creation attempts.HighlightsSingle cell analysis of 1,949 human & 259 mouse embryonic cellsIdentification of 5 most abundant HPAT lincRNAs in viable human blastocystsExpression profiling of just 3 lincRNAs captures cellular diversity of human blastocystsIdentification & characterization of TERT(+) multi-lineage precursor cellsMTTH/HPAT lincRNAs regulatory axis of naïve pluripotency induction in vivo