Generation of an NKX2-1-EGFP reporter iPSC line with inducible Cas9 for lung progenitor cell tracing.

CRISPR/Cas9-mediated generation of a homozygous MT4 knockout mouse embryonic stem cell line.

The CRISPR/Cas9 system is utilized to generate a homozygous Mt4 knockout (Mt4-/-) mouse embryonic stem cell (mESC) line that maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers.

Generation of GABAergic Neurons from Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible ASCL1/DLX2 Expression at AAVS1.

Human induced pluripotent stem cells (hiPSCs) offer an unprecedented opportunity to model human neurological diseases in vitro. Reliable generation of defined neuronal populations, such as gamma-aminobutyric acid (GABAergic) neurons, is essential for these studies. This protocol details a robust method for differentiating hiPSCs into GABAergic neurons using a doxycycline-inducible system. The approach relies on the stable integration of transgenes encoding the transcription factors ASCL1 and DLX2 into the safe-harbor adeno-associated virus integration site 1 (AAVS1.) locus via CRISPR/Cas9-mediated knock-in. This targeted integration avoids the transgene silencing often observed with random lentiviral integration. The protocol covers the maintenance of hiPSCs, the nucleofection process for transgene integration, puromycin selection to enrich successfully engineered cells, and the step-by-step differentiation procedure. Upon induction with doxycycline, the engineered hiPSCs rapidly exit the cell cycle, acquire neuronal morphology, and express GABAergic lineage markers within 21 days. Key steps include neuro-induction and subsequent maturation using specific growth factors, as well as potential approaches to improve cellular adhesion during maturation. This standardized method yields enriched populations of induced neurons expressing GABAergic lineage markers, providing a valuable tool for neuroscience research and cellular modeling.

Netrin-1 Acts as a Guardian of Naive Pluripotency in Human Embryonic Stem Cells

We investigated the role of Netrin-1 (NTN1) in human naïve pluripotency using complementary loss- and gain-of-function approaches. In primate embryos and human embryonic stem cells (hESCs), Netrin-1 expression is associated with the naïve pluripotent state. Disruption of NTN1 had no detectable effect on hESCs maintained on murine embryonic fibroblasts. However, under sub-optimal culture conditions, NTN1-knockout cells exhibited compromised naïve pluripotency, which was rescued with feeder cells overexpressing Netrin-1. Netrin-1 overexpression in hESCs accelerated acquisition of the naïve state and markedly increased resistance to differentiation. These effects were accompanied by extensive epigenetic remodeling, including H3K27ac and H2K27me3. Proteomic and phospho-proteomic analyses further revealed rapid Netrin-1-dependent alterations in pathways controlling cell adhesion, signaling, and chromatin regulation. Together, these findings extend the role of Netrin-1 beyond its established functions and identify it as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naïve pluripotency.

Modeling myeloid cell development in health and disease using induced pluripotent stem cells

Key applications of iPSC technology in hemato-oncology are summarized, its major advantages and current limitations are discussed, and emerging directions are highlighted, including scalable iPSC-derived blood cell therapies for inherited and acquired bone marrow failure syndromes and leukemia.

CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage.

Intervertebral disc (IVD) degeneration, a leading cause of chronic lower back pain, is associated with loss of vacuolated notochordal cells (NCs) and fibrotic remodeling of the nucleus pulposus. Emerging therapies increasingly focus on NCs, which are rare but therapeutically relevant cells for regenerating degenerated IVDs. In this study, we used CRISPR-based transactivation (CRISPRa) to direct the differentiation of human induced pluripotent stem cells (iPSCs) into the NC lineage. We tested CRISPRa-mediated activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9, coupled with single-cell sequencing of Aggrecan-2A-mScarlet reporter iPSCs. This approach identified the SOX5/6/9 combination (SOX-trio) as critical for promoting NC lineage commitment. The SOX-trio yielded the largest cell population expressing a range of genes previously associated with NC identity, including SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, and KRT18. Our study demonstrates the integration of CRISPRa with single-cell technologies as a powerful platform for investigating and enriching iPSC-derived NCs, supporting future regenerative strategies across various fields.

A fast 2C-induction method reveals a barrier role of SP2 for totipotency