Stem Cell Reports最新文献

OCT4 is a master regulator of pluripotency, with expression restricted to pluripotent and germ cells. Its expression is controlled by two cis-regulatory elements: the distal (DE) and proximal (PE) enhancers. Although widely used as markers for pluripotent stem cells (PSCs), their biological roles have remained incompletely defined. Here, we generated PSC lines and mouse models with targeted deletions of the Oct4 DE and PE. Our findings reveal that the DE is dispensable for sustaining the primed pluripotent state but required for the naive state, whereas the PE is necessary for the primed state but not for the naive state. Notably, PE-deficient naive mouse PSCs retained the capacity to differentiate into somatic lineages in vitro and to contribute chimeras. In contrast, deletion of either enhancer in vivo resulted in early embryonic lethality. These models offer powerful genetic tools to dissect the regulation of Oct4 expression during pluripotency and early development.

The master cell bank (MCB) system is essential for regenerative cell therapy. We have developed induced pluripotent stem cell (iPSC)-based immortalized megakaryocyte progenitor cell lines (imMKCLs) as an MCB for iPSC-derived platelet (iPSC-PLT) transfusion. However, imMKCLs exhibit both thrombopoietic and immune-skewed properties, with enhanced immune activity impairing platelet production. The link between immune properties and thrombopoietic efficiency remains unclear. Here, we demonstrate that proliferating imMKCLs in G1 and G2/M interphases contribute to platelet generation, while lysine acetyltransferase 7 (KAT7) suppresses immune-biased dominancy to maintain these interphases. KAT7 inhibition with WM3835 increases G0 cells, mimicking imMKCL aging, and induces cGAS-STING activation, chromatin instability, and the secretion of tumor necrosis factor (TNF)-α, interferon (IFN)-β, and other pro-inflammatory cytokines. Additionally, TNF-α treatment recapitulates the transition to G0 seen with KAT7 loss. These findings identify KAT7 as a key regulator of imMKCL proliferation by preventing immune-skewed properties, highlighting its potential as a quality control marker in iPSC-PLT manufacturing.

The human intestinal epithelial barrier is shaped by biological and biomechanical cues, including growth factor gradients and fluid flow. While these factors are known to affect adult stem cell (ASC)-derived intestinal epithelial cells in vitro, their impact on human induced pluripotent stem cell (hiPSC)-derived cells is largely unexplored. Here, we compare the cellular composition and gene expression profiles of hiPSC-derived intestinal epithelial cells exposed to various medium compositions and cultured as organoids, in Transwell and microfluidic intestine-on-chip systems. Modulating key signaling pathways (WNT, NOTCH, bone morphogenetic protein [BMP], and mitogen-activated protein kinase [MAPK]) influenced the presence of dividing, absorptive, and secretory epithelial lineages. Upon differentiation, intestinal epithelial cells expressed genes encoding digestive enzymes, nutrient transporters, and drug-metabolizing enzymes. Notably, these pathways were most enhanced in the intestine-on-chip system, along with an expression profile that suggests a more mature state. These findings highlight the potential of hiPSC-derived intestinal cells to model important intestinal functions and guide the selection of optimal culture conditions for specific applications.

Using anonymized, biobanked samples for induced pluripotent stem cells (iPSCs) creates new and unresolved ethical dilemmas. To elucidate the issues, we studied the perspectives of community members and bioethicists involved in the collection of the Yoruba Resident in Ibadan, Nigeria (YRI) HapMap samples. We found support for broad consent, commercial use of samples, more benefit sharing, sustained engagement of the community and local researchers, particularly for novel research, where a long time has elapsed between samples' collection and new research projects, and in the oversight of biobanked samples. Broad consent was durable when coupled with sustained community engagement, transparent governance, and practical mechanisms for reciprocal benefit.

Insulin-dependent diabetes could be treated by supplying patients with primary pancreatic islets or other types of insulin-secreting cells. Functional insulin-secreting cells can be induced in situ from the murine stomach using defined genetic factors, offering a promising method to directly produce autologous insulin-secreting cells. Here, we modeled whether such gastric insulin-secreting (GINS) cells could be generated in vivo from human stomach tissues. We produced human gastric organoids (hGOs) from human embryonic stem cells engineered with inducible expression of reprogramming factors. The hGOs were stably transplanted for 6 months and showed robust cytodifferentiation resembling the human stomach in structure and cellular composition. Upon hGO maturation in vivo, we activated the reprogramming factors and observed the formation of insulin⁺ cells, which secreted insulin into the circulation and ameliorated experimental diabetes. Our modeling indicates that GINS cells can be induced from human stomach tissues in vivo, warranting further therapeutic development for this technology.

Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem-cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple single guide RNA (sgRNA) delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.

Cell differentiation is regulated by transcription factors (TFs), but specific TFs needed for mammalian differentiation pathways are not fully understood. For example, during spinal motor neuron (MN) differentiation, 1,370 TFs are transcribed, yet only 55 have reported functional relevance. We developed a method combining pluripotent stem cell differentiation, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen and applied it to MN differentiation. The CRISPR screen identified 245 genes important for mouse MN differentiation, including 116 TFs. This screen uncovered important genes not showing differential transcription and identified a regulatory hub at the MN progenitor (pMN) stage. A secondary human screen of 69 selected candidates revealed a conservation between mouse pMN and human pMN and ventral pMN (vpMN) regulations. The validation of three hits required for efficient human MN differentiation supported the effectiveness of our approach. Collectively, our strategy offers a framework for identifying important TFs in various differentiation pathways.

This Forum article explores 4,481 YouTube videos about stem cells to map how medical knowledge is shaped online. By analyzing content and user metrics, the article identifies key mediators and influential creators, revealing a complex discourse dominated by celebrity influencers in the promotion and discussion of putative stem cell treatments.

RPGRIP1 encodes a connecting cilium (CC) protein essential for normal photoreceptor cell development and maintenance. Damaging variants in RPGRIP1 cause severe inherited retinal disease (IRD) and currently incurable vision loss, with mouse studies showing promising preclinical gene augmentation therapy results. Almost one-half of variants in RPGRIP1 in the ClinVar database are variants of uncertain significance (VUS), hindering genetic diagnosis for affected individuals and, hence, access to clinical trials of novel therapies and other management options. Here, we use human induced pluripotent stem cell (iPSC)-derived retinal organoids to model RPGRIP1-associated IRD, detecting biomarkers of disease including CC interactome dysfunction, stress response, and proteostasis abnormalities. In parallel, utilizing these novel disease biomarkers, we demonstrate the pathogenicity of a missense VUS, RPGRIP1 c.2108T>C p.(Ile703Thr). In addition, RPGRIP1 gene augmentation therapy rescued disease phenotypes, further supporting the utility of these biomarkers of RPGRIP1-IRD for reclassifying VUS and testing response to therapy.