Overview

The cells of the blastocyst inner cell mass only remain pluripotent for a short time. This state of pluripotency and self-renewal can be maintained in embryonic stem (ES) cell culture by adding specific chemicals or growth factors to ensure the cells can continue dividing and later differentiate into different cell types. In some cases, the cells are grown on a feeder layer of differentiated cells, which provides the growth factors and extracellular matrix components necessary for stem cell proliferation.

The ES cell state is regulated by the transcription factors, Oct4, Sox2, and Nanog. These factors activate the genes required for maintaining pluripotency and repress those involved in differentiation. Silencing any of these transcription factors results in lineage-specific differentiation. The factors operate along with many other transcription factors, including Klf4, Klf5, and Smad1, and transcriptional cofactors, such as p300, Mediator, and Nipb, help activate or repress genes without directly binding to DNA.

Chromatin regulators play a crucial role in maintaining the ES cell state. These are categorized as histone-modifying enzymes and ATP-dependent chromatin regulators. Histone modifying enzymes alter the DNA-histone interaction and transcriptionally activate specific genes. These enzymes also suppress development regulators such as polycomb group (PCG) protein complexes. ATP-dependent chromatin remodeling complexes use the energy released by ATP hydrolysis to either displace histones from DNA or enable the relocation of histones. This weakens the bonds between histones and DNA, providing access to transcription factors to bind to DNA.

Procedure

Embryonic stem cells in a culture medium must stay undifferentiated and divide indefinitely. This pluripotent state is maintained by several enzymes, transcription factors, and RNA molecules.

The enzyme telomerase is present at high levels in ES cells and adds telomeres, short DNA sequences at the end of chromosomes. This protects the chromosomes from damage and allows for cell renewal.

Chromatin regulators, such as histone-modifying enzymes, turn off genes that encode regulators of differentiation.

Several transcription factors are core regulators of the ES cell state–Oct4,  Sox2, and Nanog. These factors activate genes leading to pluripotency and self-renewal while suppressing those leading to differentiation.

The core regulators also activate genes encoding certain microRNAs. Some microRNAs allow cells to proliferate by regulating cell-cycle progression, and maintain the ES cell state.