Stem Cell Reports最新文献

Cardiotoxicity is a significant challenge in cancer therapies, particularly with doxorubicin, a widely used anthracycline. More predictive in vitro models are needed to understand doxorubicin-induced cardiac damage and patient-specific responses. Here, human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs), cardiac fibroblasts (hPSC-cFBs), and endothelial cells (hPSC-ECs) were cultured in mono- or multi-cell-type formats and repeatedly treated with doxorubicin to mimic cumulative clinical exposure. A machine learning-based tool enabled continuous quantification of the early toxicity marker caspase-3/7 and accurately identified hPSC-CMs within mixed cultures. Notably, hPSC-ECs were more sensitive to doxorubicin than hPSC-CMs or hPSC-cFBs, with nitric oxide signaling contributing to the elevated cardiomyocyte toxicity observed in co-culture. These results question the conventional in vitro focus on cardiomyocytes regarding drug-induced cardiac damage, highlighting the interplay among different cardiac cell types in mediating the toxic effects of doxorubicin. Furthermore, the work demonstrates the potential of AI-based tools to provide scalable strategies for assessing drug-induced cardiotoxicity.

Two-cell (2C)-specific transcripts important for maternal zygotic transition, such as Zscan4 and endogenous retrovirus (MERVL), are sporadically expressed in approximately 1%-5% of an embryonic stem cell (ESC) population to maintain ESC and telomere homeostasis. However, the molecular mechanisms regulating the 2-cell-like state in ESCs are not fully understood. Here, we show that Sgo2a is an important suppressor of the 2-cell-like state and telomere length. Loss of Sgo2a reduces the enrichment of heterochromatic H3K9me3 on 2C genes, including Dux, Zscan4 gene clusters and downstream enhancers, resulting in activation of a 2-cell-like state. However, chromosome breakage was detected in Sgo2a-deficient ESCs. Mechanistically, SGO2A promotes the interaction between RIF1 and KAP1, regulates KAP1 ubiquitination, and maintains KAP1 stability at heterochromatin, repressing 2-cell genes. These results reveal a critical role for Sgo2a in suppressing the 2C-like state to maintain the homeostasis and genome stability of ESCs.

The mouse cortex is a canonical model for studying how functional neural networks emerge, yet it remains unclear which topological features arise from intrinsic cellular organization versus sensory input. Mouse forebrain organoids provide a powerful system to investigate these intrinsic mechanisms. We generated dorsal (DF) and ventral (VF) forebrain organoids from mouse pluripotent stem cells and tracked their development using longitudinal electrophysiology. DF organoids showed progressively stronger network-wide correlations, while VF organoids developed more refined activity patterns with enhanced small-world topology and increased modular organization. Both organoid types form small-world networks, but their topological organization differs. These differences emerge without extrinsic inputs and correlate with Pvalb⁺ interneuron enrichment in VF organoids. Our findings demonstrate how cellular composition influences neural circuit self-organization, establishing mouse forebrain organoids as a tractable platform to study cortical network architecture.

Male infertility often results from impaired interactions between germ cells and Sertoli cells. While in vitro fertilization and intracytoplasmic sperm injection are widely used, their success depends on the presence of haploid germ cells. Gene therapy remains challenging due to concerns about germline transmission. The mRNA offers a safer option, as its short-life reduces this risk. Here, we show that mRNA delivery into mouse testes restores fertility in a genetic model of infertility. Injected mRNA was specifically expressed in Sertoli cells; although it triggered an innate immune response, spermatogenesis resumed without major side effects. Delivery of naked Cldn11 mRNA into Cldn11-deficient mice, which have meiotic defects due to defective blood-testis barrier, allowed progression from spermatocytes to spermatids. Fertile offspring with normal imprinting were produced via microinsemination. These findings demonstrate the potential of mRNA-based therapy for treating male infertility by targeting testicular somatic cells, without introducing genetic material into the germline.

We used an embryonic stem cell line (H1) engineered for immune-evading properties to avoid rejection ("AlloAccept") and equipped with a "SafeCell" (SC) kill-switch to eliminate aberrantly proliferating cells. Utilizing a humanized immune system mouse model, we demonstrated the successful generation of allogeneic tissues from SafeCell-AlloAccept (SC-AlloAccept) cells in immunocompetent humanized mice in the immune-active subcutaneous region. These cells formed various tissue types, and their growth can be controlled with pro-drug ganciclovir to activate the kill switch, which eliminated proliferating cells and rendered the remaining tissue dormant. Strikingly, SC-AlloAccept-derived grafts survived for 5 months, underscoring their potential for long-term engraftment. Importantly, neither prior rejection of immunogenic parental H1 cells (sensitization) nor the presence of immune-evasive H1-derived tissue (potential immunocompromising) affected the immune response to a subsequent second transplant. This study validated the utility of SC-AlloAccept human cells in transplantation and enhanced the safety and efficacy of stem cell-based regenerative therapies.

Poor survival of transplanted midbrain dopaminergic neurons (mDANs) remains a key barrier for stem cell-based Parkinson's disease (PD) therapy. Ca_v1.3-containing L-type calcium channel blockers have been shown to prevent native mDAN loss in PD animal models and patients. Here, we aim to investigate whether they also enhance the post-transplant survival of stem cell-derived mDANs. We found that Ca_v1.3 is highly expressed in human embryonic stem cell-derived mDANs. Ca_v1.3 inhibitors including isradipine and cp-PYT significantly reduced apoptosis and improved mDAN survival in both interferon gamma (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-induced inflammatory models, as well as in the 6-OHDA-induced toxic model in vitro. Mechanistically, their protective effects are mediated through suppressing the CaMKII-p65-p53 signaling pathway. Moreover, we showed that cp-PYT treatment enhanced mDAN survival while reducing mature graft volume post-transplantation. Therefore, Ca_v1.3 inhibition may represent a clinically relevant strategy to enhance the survival of human pluripotent stem cell (hPSC)-derived mDANs in cell therapy for PD.

Human pluripotent stem cell (hPSC)-derived pancreatic beta cells provide an unlimited cell source for disease modeling and drug development. Generating highly purified beta cell populations from hPSCs remains challenging due to contamination by off-target and polyhormonal cells. Here, we present a robust cell-sorting-based purification strategy to enhance stem cell-derived beta (SC-beta) cell purity. Building on our previous work, we identified CD133 (PROM1) as a beta cell-enriched surface marker capable of distinguishing SC-beta cells from SC-alpha cells. Combining CD133 with the pan-endocrine marker CD49a (ITGA1) significantly increased beta cell enrichment while drastically reducing the fractions of alpha cells, polyhormonal cells, and ductal cells. This effect was consistent across multiple hPSC lines and differentiation protocols. Our approach yields SC-beta cell preparations with markedly improved purity, thereby advancing their application in disease modeling and drug development.

Biallelic pathogenic variants in STRADA (STE20-related adaptor alpha), an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study explores how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knockout human stem cell-derived brain organoids. In both dorsal and ventral forebrain STRADA knockout organoids, neurogenesis is delayed, with a predilection for progenitor renewal, increased proliferation and an expanded outer radial glia population. Ventrally, interneuron subtypes shift to an increase in neuropeptide Y-expressing cells. Inhibition of the mTOR pathway with rapamycin rescues most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest that mTOR inhibition during fetal brain development could be a potential therapeutic strategy in STRADA deficiency.

Direct somatic cell-to-neuronal fate reprogramming (induced neurons, iNs) is valuable for translational and basic research. N⁶-Methyladenosine (m⁶A), the most prevalent mRNA epitranscriptomic modification, is critical for neural biology, but its role in iN reprogramming remains elusive. Using our induced retinal ganglion cell-like neuron (iRGC) system, we found dynamic m⁶A epitranscriptomic adjustments during iRGC reprogramming. Mettl3, the core component of the m⁶A methyltransferase complex, promoted iRGC fate reprogramming and axon development. Integrated RNA-seq/MeRIP-seq analyses and gene function interrogations identified three m⁶A-modified genes (Prokr1, Rspo1, and Fmo2) as key mediators of Mettl3 effects. Collectively, our study elucidated the essential roles and molecular mechanisms of the m⁶A epitranscriptomic modification in neuronal fate reprogramming and axon development. These findings could aid future investigations designed to improve neuronal fate and axon regeneration outcomes for therapeutic purposes to treat neurodegenerative diseases.

The mechanism underlying retinal spheroid layer formation was investigated using Rax::GFP-positive retinal progenitor cells from human embryonic stem cell-derived retinal organoids. Single-cell RNA sequencing suggested the earlier cell-cycle exit in non-layered spheroids, while well-layered spheroids retained longer proliferative property with transiently activated canonical WNT2B-FZD7 signaling followed by temporary expression of non-canonical WNT5A. Despite structural differences in vitro, however, both non-layered and well-layered retinal spheroids on differentiation day 60 developed a layer of photoreceptors after transplantation in a retinal degeneration rat model, resulting in synaptic and functional integration. Additionally, part of the Rax::GFP-positive cells differentiated into non-retinal lineages, including ciliary marginal zone-like, retinal pigment epithelium, and spinal cord-like tissues in vitro, reflecting the heterogeneity of RAX-positive cells. These findings suggest that canonical and non-canonical WNT signaling pathways sequentially orchestrate early retinal morphogenesis, whereas environmental factors within the host retina strongly drive the alignment and functional integration of graft photoreceptors.