International journal of stem cells最新文献

Human pluripotent stem cells (hPSCs) can be used to investigate hematopoietic development and have the potential to advance cell-based therapies and to facilitate developmental biology studies. However, efficient ex vivo differentiation into hematopoietic lineages, including red blood cells (RBCs) of the erythroid lineage and immune cells such as macrophages of the myeloid lineage, is hampered by the need for precise temporal regulation of cytokines and growth factors. In this study, we developed an optimized protocol for hematopoietic lineage specification from hPSCs by fine-tuning the temporal dynamics of cytokine and growth factor applications. Prolonged mesodermal specification in the absence of hemogenic cytokines significantly enhanced the generation of hematopoietic progenitors (CD34^＋CD45^＋) with robust functional potential. Early administration of interleukin (IL)-3 during hematopoietic specification promoted progenitor expansion and maturation. Supplementation of bone morphogenetic protein 4 at the hematopoietic maturation stage enhanced the differentiation efficiency and preferentially drove myeloid lineage commitment toward macrophages at the expense of erythroid differentiation. The timing of erythropoietin administration was important in erythroid lineage commitment, and delayed treatment (day 10) enhanced erythroblast expansion and RBC production. By contrast, the timing of IL-6, GM-CSF, and M-CSF exposure did not significantly affect macrophage differentiation efficiency, suggesting that myeloid lineage specification follows a default pathway under optimized differentiation conditions. These findings suggest a refined, time-controlled strategy for directing hematopoietic differentiation from hPSCs, and provide insight into therapeutic blood cell production, regenerative medicine, and ex vivo modeling of hematopoietic disorders.

Phenylketonuria (PKU), an autosomal recessive genetic disorder, has been documented to exhibit over 950 distinct mutations. This condition primarily affects the metabolism of phenylalanine, which is affected by a deficiency in the hepatic enzyme phenylalanine hydroxylase. The optimal treatment for PKU disease remains to be determined, necessitating further research. The severity of the disease and the most effective treatment method vary depending on the specific mutation, which necessitates the development of personalized treatment strategies. In this study, we successfully established induced pluripotent stem cell (iPSC) lines from the blood of a PKU patient with the R243Q mutation via Sendai virus-based reprogramming (R243Q-iPSCs). The established R243Q-iPSCs exhibited characteristics of pluripotency, as confirmed through quantitative reverse transcription polymerase chain reaction, western blot, immunocytochemistry, and karyotype analysis. Furthermore, these iPSCs not only successfully differentiated into hepatocytes but also exhibited a complete PKU disease phenotype. These results provide a valuable foundation for PKU disease research, including physiological studies of PKU, gene therapy, drug screening, and the development of platforms for novel cell therapy approaches.

Nanog is a key transcription factor that regulates the self-renewal and pluripotency of embryonic stem cells (ESCs). Although Kap1 has been demonstrated to regulate the stability of stemness factors, including Oct4 and Lin28A, its role in regulating Nanog protein stability in ESCs remains unexplored. In the present study, we examined the interaction between Kap1 and Nanog and its role in stabilizing the Nanog protein. Immunoprecipitation assays revealed that Nanog specifically interacted with the coiled-coil domain of Kap1. Kap1 overexpression increased the stability of the Nanog protein by inhibiting its ubiquitination and proteasomal degradation, whereas Kap1 silencing accelerated Nanog degradation. Furthermore, Kap1 overexpression inhibits Nanog degradation by interfering with the binding of Nanog to Fbxw8, an E3 ubiquitin ligase that promotes Nanog degradation via a proteasome-dependent process. These results indicate that Kap1 acts as a key regulator to preserve ESC properties by modulating the protein stability of stemness factors, including Oct4, Lin28A, and Nanog.

Transcription factor CP2-like protein 1 (Tfcp2l1), a naïve pluripotency transcription factor, is expressed in both early embryonic and adult tissues, where it enforces pluripotency of embryonic stem cells (ESCs) and stemness features of cancer cells, respectively. However, the detailed molecular pathways by which Tfcp2l1 is regulated in early embryonic development and cancer cells remain unknown. Here, we identified the pseudophosphatase dual specificity phosphatase 27 (Dusp27), also known as serine/threonine/tyrosine-interacting like-2, as a novel Tfcp2l1-interacting protein through a sterile alpha motif-like domain in the C-terminus of Tfcp2l1 in murine ESCs. The interaction between Dusp27 and Tfcp2l1 was dependent on the cell cycle status and increased during mitosis. Expression of Dusp27 was upregulated during naïve pluripotency and repressed during spontaneous differentiation of murine ESCs. Ectopic expression of Dusp27 enhanced the transcriptional activity of Tfcp2l1 and promoted features associated with the naïve pluripotent state, while suppressing meso-endodermal lineage differentiation. The present study demonstrates that Dusp27 is a novel positive regulator of Tfcp2l1 through a physical interaction and thereby fine-tunes the pluripotency status and meso-endodermal differentiation of murine ESCs.

Stem cell-derived embryo models (SCDEMs) create opportunities to investigate the morphological dynamics and underlying mechanisms of embryonic development, implantation, and post-implantation progression by recapitulating pre-and peri-implantation stages in vitro-an area that conventional in vivoapproaches struggle to investigate. This review provides a comprehensive overview of SCDEMs, detailing the methodologies used to generate synthetic embryos and the diverse types of stem cells employed. Furthermore, we describe how closely these models recapitulate key developmental processes pre- and post-implantation, thereby establishing their value as a platform for studying early mammalian embryogenesis. In addition, we suggest that synthetic embryos are valuable tools for studying environmental toxicity, yet ethical and technical constraints limit systematic in vivo investigations. We evaluate the strengths and limitations of these models in embryotoxicity studies and highlight future research strategies. SCDEMs are expected to significantly advance the broader field of early mammalian developmental biology, with impacts extending well beyond their use in embryotoxicology.

The advent of medical advances has resulted in the development of an array of treatments aimed at restoring damaged organs in humans. However, when chemical treatments, such as drug therapies, are constrained, organ transplantation may ultimately emerge as the sole viable solution. Nevertheless, despite the continually increasing demand for organ donations, the actual number of donated organs remains insufficient to meet this demand. Recently, a variety of organoids have been generated using stem cells and have been demonstrated to exhibit functionality comparable to that of native organs. This indicates that organoids may be a viable option for use in organ transplantation. However, while numerous recent publications have documented the regenerative effects of diverse organoid types when implanted into damaged regions, significant technical and ethical considerations must be addressed before organoids can be utilized as a replacement for human organs. This review presents an overview of experimental endeavors in regenerative therapies through organoid transplantation, while also addressing the challenges that must be overcome to enhance the feasibility of organoid use as a surrogate organ. As organoid technology continues to advance, organoids may eventually become a widely utilized surrogate source for organ replacement in clinical settings.

Alcohol dependence (AD) is one of the most prevalent neuropsychiatric disorders. Multiple polymorphisms in the Fyn tyrosine kinase gene (FYN) were found to be associated with AD. The function of AD-associated FYN variants remains largely unknown due to the absence of an appropriate model for studying them. Here, we generated human embryonic stem cell lines homozygous/heterozygous for rs706895 C/T alleles in 5' untranslated region (5' UTR) of FYN by CRISPR-Cas9 editing to explore the AD association. Transcriptome and reporter gene analyses demonstrated that induced neurons with the rs706895 C allele showed a significantly higher expression level of FYN under ethanol treatment. Our results suggest that FYN 5' UTR variant rs706895 may influence an individual's vulnerability to AD by altering FYN expression. Targeting AD-associated variants may provide a better understanding of disease mechanisms and a reliable basis for the personalized AD treatment.

Human pluripotent stem cell (hPSC)-derived brain organoids have emerged as innovative models for drug screening and cytotoxicity evaluation. However, their inherent cellular heterogeneity presents challenges in isolating targeted neuronal populations, such as upper motor neurons, which are crucial for motor cortex function. In this study, we developed motor cortex-like organoids enriched with excitatory glutamatergic and inhibitory GABAergic neurons to assess neurotoxicity in the upper motor neurons-a key component of voluntary motor control. By optimizing the differentiation protocols, we achieved robust expression of vGlut1 in excitatory neurons and GABA in inhibitory neurons by day 30 of the differentiation. The organoids were generated by co-culturing progenitor cells during the early differentiation phase, followed by lineage-specific maturation. Comparative analyses demonstrated that these organoids more accurately recapitulate the human cortical architecture than traditional neural cell line (SK-N-SH neuroblastoma cells). We observed that measures of cell viability and integrity-assessed via cleaved caspase-3 levels, growth-associated protein 43 (GAP43), and autophagy-related protein 5 (ATG5)-were significantly higher in 3D organoid cultures compared to conventional 2D systems. In toxicological assays, the motor cortex-like organoids exhibited a dose-dependent response to both toxic and non-toxic compounds, highlighting their potential as high-fidelity neurotoxicity screening models. Our findings suggest that hPSC-derived motor cortex-like organoids serve as a robust, physiologically relevant model that can replace animal models in toxicity assessments, offering enhanced accuracy in evaluating compounds that impact the motor cortex while reflecting better human brain physiology.

Brain organoids have emerged as transformative models for studying human neurodevelopment, neurological disorders, and personalized therapeutics. Central to their utility is the ability to monitor neural activity with high spatial and temporal resolution. Traditional electrophysiological tools-such as planar microelectrode arrays and patch-clamp techniques-offer limited access to the three-dimensional and dynamic nature of organoid neural networks. Recent technological advancements have led to the development of next-generation platforms including surface-embedded, flexible, and fully implantable electrodes. Moreover, multifunctional probes incorporating optical, chemical, and mechanical sensing open new avenues for multimodal interrogation of organoid physiology. This review summarizes the current state of electrophysiological technologies applied to brain organoids, highlighting innovations in recording fidelity, spatiotemporal resolution, and device-tissue integration. We also discuss key challenges such as maintaining organoid viability, achieving sufficient electrode density, and enabling non-disruptive, chronic interfacing throughout organoid development. Looking forward, future systems are expected to evolve toward ultra-dense, multimodal, and closed-loop interfaces capable of investigating organoid function throughout extended growth periods. These advances will not only deepen our understanding of brain-like activity in organoids but also support the design of more functionally accurate and translationally relevant neural models.