Interaction network of human early embryonic transcription factors.

Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized.

The Emerging Role of B1 SINE in Pluripotent Reprogramming.

By screening a CRISPR knockout library for mouse pluripotent reprogramming roadblock genes, Kaemena et al. identify the KRAB-ZFP factor Zfp266 as a suppressor of efficient reprogramming. Furthermore, by analyzing DNA binding and chromatin openness, the authors found that ZFP266 has a role in suppressing reprogramming by targeting the B1 SINE sequences for silencing.

Comprehensive characterization of the embryonic factor LEUTX.

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions.

Transient DUX4 expression in human embryonic stem cells induces blastomere-like expression program that is marked by SLC34A2.

Embryonic genome activation (EGA) is critical for embryonic development. However, our understanding of the regulatory mechanisms of human EGA is still incomplete. Human embryonic stem cells (hESCs) are an established model for studying developmental processes, but they resemble epiblast and are sub-optimal for modeling EGA.

MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels.

MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells.

Polycomb repressive complex 2 shields naïve human pluripotent cells from trophectoderm differentiation.

The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages.

is a multifunctional factor priming human embryonic genome activation.

Double homeobox 4 () is expressed at the early pre-implantation stage in human embryos. Here we show that induced human expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes and .

Cisplatin overcomes radiotherapy resistance in OCT4-expressing head and neck squamous cell carcinoma.

Objectives: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patient-selective therapeutic utility.

CRISPR activation enables high-fidelity reprogramming into human pluripotent stem cells.

Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity.

Refined transcriptional blueprint of human preimplantation embryos.

Despite being a biologically fundamental question, the precise timing of lineage specification during human preimplantation development remains elusive. In this issue of Cell Stem Cell, Meistermann et al. (2021) refine our view through time-lapse embryo staging and single-cell sequencing and challenge the concept of a human inner cell mass.

Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators.

CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components.

Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter.

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site.

Human pluripotent reprogramming with CRISPR activators.

CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif).

Generation of a SOX2 reporter human induced pluripotent stem cell line using CRISPR/SaCas9.

SOX2 is an important transcription factor involved in pluripotency maintenance, pluripotent reprogramming and differentiation towards neural lineages. Here we engineered the previously described HEL24.3 hiPSC to generate a SOX2 reporter by knocking-in a T2A fused nuclear tdTomato reporter cassette before the STOP codon of the SOX2 gene coding sequence.

Generation of an OCT4 reporter human induced pluripotent stem cell line using CRISPR/SpCas9.

OCT4 is a crucial transcription factor in the pluripotent stem cell gene regulatory network and an essential factor for pluripotent reprogramming. We engineered the previously reported HEL24.3 hiPSC to generate an OCT4 reporter cell line by knocking-in a T2A nuclear EmGFP reporter cassette before the OCT4 gene STOP codon sequence.

p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming.

The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs.

Genetic Variability Overrides the Impact of Parental Cell Type and Determines iPSC Differentiation Potential.

Reports on the retention of somatic cell memory in induced pluripotent stem cells (iPSCs) have complicated the selection of the optimal cell type for the generation of iPSC biobanks. To address this issue we compared transcriptomic, epigenetic, and differentiation propensities of genetically matched human iPSCs derived from fibroblasts and blood, two tissues of the most practical relevance for biobanking. Our results show that iPSC lines derived from the same donor are highly similar to each other.

Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation.

CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming.

Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction.

Generation of iPSC line HEL47.2 from healthy human adult fibroblasts.

Human iPSC line HEL47.2 was generated from healthy 83-year old male dermal fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses.

Generation of iPSC line HEL24.3 from human neonatal foreskin fibroblasts.

Human iPSC line HEL24.3 was generated from healthy human foreskin fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses.

Selective microRNA-Offset RNA expression in human embryonic stem cells.

Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells.

Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids.