Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is a rare, fatal, adult-onset neurodegenerative disease that is most often caused by mutations affecting the colony stimulating factor-1 receptor (CSF-1R). To understand how CSF-1R-mutation affects human microglia - the specialized brain-resident macrophages of the central nervous system - and the downstream consequences for neuronal cells, we used a macrophage and forebrain organoid co-culture system based on induced pluripotent stem cells generated from two patients with HDLS, with CSF-1R gene-corrected isogenic organoids as controls. Macrophages derived from iPSC (iMacs) of patients exhibited a metabolic shift toward the glycolytic pathway and reduced CSF-1 sensitivity, which was associated with higher levels of IL-1β production and an activated inflammatory phenotype. Bulk RNA sequencing revealed that iMacs adopt a reactive state that leads to impaired regulation of neuronal cell populations in organoid cultures, thereby identifying microglial dysregulation and specifically IL-1β production as key contributors to the degenerative neuro-environment in HDLS.

Cellular heterogeneity within cancer tissues determines cancer progression and treatment response. Single-cell RNA sequencing (scRNA-seq) has provided a powerful approach for investigating the cellular heterogeneity of both cancer cells and stroma cells in the tumor microenvironment. However, the common practice to characterize cell identity based on the similarity of their gene expression profiles may not really indicate distinct cellular populations with unique roles. Generally, the cell identity and function are orchestrated by the expression of given specific genes tightly regulated by transcription factors (TFs). Therefore, deciphering TF activity is essential for gaining a better understanding of the uniqueness and functionality of each cell type. Herein, metaTF, a computational framework designed to infer TF activity in scRNA-seq data, is introduced and existing methods are outperformed for estimating TF activity. It presents the improved effectiveness in characterizing cell identity during mouse hematopoietic stem cell development. Furthermore, metaTF provides a superior characterization of the functional identity of breast cancer epithelial cells, and identifies a novel subset of neural-regulated T cells within the tumor immune microenvironment, which potentially activates BCL6 in response to neural-related signals. Overall, metaTF enables robust TF activity analysis from scRNA-seq data, significantly enhancing the characterization of cell identity and function.

Due to the inaccessibility of early human embryos for large, robust studies, many questions regarding the mechanisms of early embryogenesis remain. To address these questions, multiple research groups have developed human stem cell-based models of the pre-implantation blastocyst stage. These models, known as blastoids, mimic several key processes that natural blastocysts undergo as they prepare to implant into the uterine wall. One of the main advantages of blastoids is their scalability, making them suitable for both screenings and molecular studies. To leverage this advantage, we established a protocol for the parallel formation and culture of blastoids in thermoformed microwell arrays. Thin-walled thermoformed microwell platforms allow for uniform, large-scale generation of blastoids and enable in situ high-resolution imaging through both widefield and confocal microscopy. Here we present a step-by-step protocol for the culture of blastoids in thermoformed microwell platforms.

Hematopoietic stem and progenitor cells (HSPCs) can migrate and reside within the bone marrow in distinct microenvironments or niches. The niches organize around specific stromal cells, such as endothelial cells at the capillary or sinusoid walls, and osteoblasts along the bone matrix. Within each niche, a specific combination of external cues, including secreted and diffusible factors, cell-matrix, and cell-cell interactions, controls HSPCs behavior and fate. Deciphering the interplay between HSPCs and stromal cells of the niches is challenging: in vivo, it is hindered by the opacity of the bone matrix; in vitro, classical co-culture models only poorly recapitulate essential features of the physiological niches. The difficulty is moreover amplified by the exceptional migration capacity of HSPCs.In this chapter, we present a method to overcome these limitations by producing arrays of microwells designed to mimic bone marrow niches in a functional manner. These "microniches" promote a long-term interaction between the HSPC and a stromal cell of interest. We describe their microfabrication based on a maskless photolithography method allowing the production of arrays of microwells with reproducible volume and geometry, and the iterative improvement of the geometric design of the wells. We describe the loading and culture of stromal cells with HSPCs. We discuss the potentiality of microwells, in basic and applied research, as a platform to investigate molecular mechanisms involved in direct cell-cell interactions and local effects of diffusible factors, for any adherent and non-adherent cells of interest.

Recent advances in three-dimensional (3D) modeling of post-implantation human embryos using human pluripotent stem cells (hPSCs) have revolutionized our ability to investigate this crucial yet enigmatic stage of development. Here we detail the generation of the human extra-embryoid (hEE), a 3D stem cell-based embryo model that uniquely captures key spatiotemporal events of peri-gastrulation development through the formation and co-development of post-implantation embryonic and extra-embryonic lineages, with high efficiency and robustness across genetic backgrounds. This chapter provides a detailed protocol for generating hEEs in vitro, including guidance on hPSC maintenance, expected cell morphology, troubleshooting strategies, and key culture techniques.

Protocols for human pluripotent stem cell differentiation commonly yield a heterogeneous mix of cell types. To understand the source of heterogeneity at the single-cell level, it may be necessary to link final cell state to the cell's history and initial state, for example to determine gene expression or morphogen signaling over the course of differentiation. Here we present methods to quantify and track single cells in time-lapse fluorescence microscopy during stem cell differentiation and link single-cell dynamics to the final resulting state in the same cells.

Implantation triggers critical morphological transformations in the embryo, where the epiblast transitions from a cluster of unpolarized cells into a highly organized, polarized epithelium characterized by a central lumen. Human pluripotent stem cells (hPSCs) are valuable models for studying this process, but conventional matrices like Matrigel have significant limitations, including variability and poor control over mechanical properties. To overcome these challenges, we developed a synthetic polyethylene glycol (PEG) hydrogel system with tunable mechanical stiffness to model peri-implantation epiblast morphogenesis.Our platform enables hPSCs to form unpolarized 3D aggregates that undergo stiffness-dependent transformation into lumen-forming, apicobasal-polarized structures resembling epiblast morphogenesis during peri-implantation. Unlike natural ECMs, PEG hydrogels maintain hPSC pluripotency for extended periods and support trilineage differentiation upon induction. The modular hydrogel design facilitates targeted mechanistic studies on the biophysical and biochemical regulation of cell morphogenesis. We present a comprehensive protocol for fabricating PEG hydrogels, encapsulating hPSCs, and assessing cell polarity, lumen formation, and pluripotency using immunostaining and RT-PCR. This platform provides a robust, cost-effective, and versatile tool for advancing developmental biology and regenerative medicine.

Stem cell-based embryo models (SEMs) have the potential to transform our understanding of early human embryogenesis. A critical step in engineering SEMs is the generation of the major cell types that compose preimplantation embryos including two primary extraembryonic lineages: (i) trophoblast cells, which are crucial for implantation and the establishment of maternal-fetal exchange, and (ii) hypoblast cells, which contribute to yolk sac formation. In addition, both cell types provide key signaling cues necessary for embryonic development. CRISPR-based epigenome editors are programmable devices that allow for efficient and precise activation (CRISPRa) or repression (CRISPRi) of cell fate-determining factors by modulating endogenous regulatory elements. Here, we present a step-by-step method to implement CRISPRa for controlling cell fate in embryonic stem cells based on our work in generation of CRISPR-programmed mouse embryo models.

The bone marrow (BM) niche comprises of diverse cell types, each playing a distinct role in hematopoiesis and disease. Single-cell RNA sequencing enables investigation of various cell populations within heterogeneous tissues. In this chapter, we describe the method to isolate BM niche cells from femurs and tibias of mice by fluorescence-activated cell sorting for single-cell RNA sequencing. This method ensures isolation of high-quality BM niche cells with high viability and purity.

Chondrocyte-based strategies are widely used for cartilage repair and show promising results in the treatment of articular cartilage damage and osteoarthritis. In cartilage repair approaches that involve the implantation of autologous chondrocytes or tissue-engineered scaffolds, it is essential to obtain significant amounts of viable cells. Nasal chondrocytes serve as a clinically valuable source of cells for cartilage regeneration, and their isolation is a critical step in cartilage tissue engineering. This chapter outlines an optimized protocol for obtaining nasal chondrocytes from neonatal rats using a Leica VT1200 vibratome to obtain nasal cartilage fragments. Enzymatic isolation of nasal chondrocytes and their subsequent in vitro culture highlight the importance of the stem and progenitor cell niche in supporting the further culture and differentiation of these cells for effective cartilage regeneration.

Managing sacroiliac joint (SIJ) pain is challenging and unpredictable. There are no internationally accepted recommendations. In light of the lack of global consensus and guidelines and the ongoing advancements in management options, a widely accepted treatment algorithm remains absent. This systematic review updates and evaluates the existing evidence on strategies for managing SIJ pain.

Ethically challenging areas of research raise the question of how science can proceed in an ethically reflective and careful way and what role ethical deliberative bodies at the local level play in this. Important lessons can be drawn from the experience of Embryonic Stem Cell Research Oversight Committees (ESCROs). ESCROs have evolved as reflective spaces in response to changes in the science, including research on "humanization" of experimental rodents, embryogenesis, and developing human germ cells. Institutional efforts can contribute to broader goals of open and democratic deliberation.

Despite the growing interest in the use of probiotics in broilers as feed additives, studies conducted to investigate the effect of probiotic administration on acute phase responses and the impact on muscle growth parameters in broilers are still limited. In this study, we investigated the effect of Bacillus amyloliquefaciens CECT 5940 on relative gene expression for growth factors involved in muscle development (insulin-like growth factors, myogenic factor 5, paired-box transcription factor), percentage of proliferating antigen cell nuclei in breast muscle, and secretion of acute phase proteins (serum amyloid A, haptoglobin, alpha1-acid glycoprotein) in the peripheral blood of broilers. Sixty one-day-old chicks from the experimental group were sprayed with a probiotic containing B. amyloliquefaciens at a dose of 1 × 1010 CFU/g directly after hatching and received the probiotic in drinking water (50 × 1010 CFU/1000 L) for 5 consecutive days of life. Sampling was performed on the 5th, 8th, and 12th day of life of the chicks. From the obtained results, we can conclude that B. amyloliquefaciens modulated the gene expression of selected growth parameters in the pectoral muscle, thereby increasing the number of satellite cells, however, their uptake into muscle fibers and thus increased hypertrophic growth was not proven at the time of the last sampling. At the same time, it demonstrated a potentiating effect on PCNA expression in chicken breast muscles in the early intensive growth phase of broiler chickens and modulated the production of acute phase proteins, which may contribute to improving adaptive processes during the growth and development of broilers.

In the human brain, the nigrostriatal pathway regulates motor functions, and its selective deterioration leads to the onset of Parkinson's disease (PD), a neurodegenerative disorder characterized by motor dysfunction and significant disability. The A9 neurons, a subgroup of ventral mesencephalic dopaminergic (DA) neurons, form the nigrostriatal pathway that emerges from the nigral region and innervates into the striatum. These DA neurons exhibit extensive and arborized axonal terminals projecting into the dorsal striatum. This review examines the distinct molecular determinants underlying the development, projection pattern, survival, maintenance, and vulnerability of A9 neurons, distinguishing them from other ventral midbrain DA subgroups such as A8 and A10. Key transcription factors (e.g., Lmx1a/b, FoxA2, Pitx3), signaling cascade pathways (e.g., Sonic Hedgehog, Wnt/β-catenin), and molecular markers (e.g., Aldh1a1, GIRK2, ANT2) are discussed in detail. A comparative assessment of the electrophysiology, cytoarchitecture, energy demand, and antioxidant reserves of A9 DA neurons versus the neighboring ventral mesencephalic DA subgroups elucidates the role of intrinsic determinants in susceptibility and selective degeneration in PD. The unique susceptibility of A9 cells to redox imbalance, neuronal inflammation, and mitochondrial dysfunction is also explored. Furthermore, recent advancements in stem cell-based approaches for generating A9-like neurons and their application in cell transplantation therapies for PD are discussed. Current challenges, including integration and long-term survival of transplanted neurons, are highlighted alongside prospects of cell replacement therapy. By evaluating the molecular biology of A9 neurons, this review aims to understand PD pathology and develop strategies for novel therapeutic approaches.

Exosome roles in cellular cross-talking within tumor microenvironment (TME) is a critical event in tumorigenesis. Type 2 macrophages (M2), cancer-associated fibroblasts (CAFs) and cancer stem cells (CSCs) are the three most important cells in cancer progression and metastasis, and targeting their connectome route can be an effective anti-cancer strategy. Exosomes mediate bidirectional cross-talking between the three cell types in which exosomes secreted from CSCs promote polarization of M2 macrophages and CAFs, and that M2- and CAF-derived exosomes promote cancer stemness through activation of epithelial-mesenchymal transition (EMT)-related signaling including transforming growth factor (TGF)-β, WNT/β-catenin and epidermal growth factor (EGF). CSC-derived exosomal TGF-β is a key driver of CAF and M2 macrophage polarization, with the latter mediated through activation of signal transducer and activator of transcription 3 (STAT3). β-catenin activity also seems to take important role in exosomal cross-talk between CAFs and stemness state of cancer. Incubation of exosomes with inhibitors of signaling inter-connecting CSCs, M2 and CAFs is a key anti-cancer strategy and a promising supplementary to the routine immunotherapeutic approaches in cancer therapy.

Slow skeletal troponin T (ssTnT, TNNT1) is the tropomyosin-binding subunit of the troponin complex in the slow-twitch fibers of skeletal muscle. Exon 5 of TNNT1 is alternatively spliced, and retention of the 3' region of intron 11 (exon 12') has also been described. Variants in TNNT1 are known to cause nemaline myopathy (NM).

Neuropathic pain is a multifaceted syndrome posing significant challenges to patient quality of life and healthcare systems. Conventional treatments primarily focus on general pain modulation, which fail to address specific underlying mechanisms, leading to limited efficacy and infinite side effects. Calcitonin gene-related peptide (CGRP) has played a pivotal role in neuro-immune repair, contributing to vasodilation, nociception, and immune modulation following tissue injury. Herein, a bupivacaine-loaded cerium-based metal-organic framework (CUB) is designed to integrate sustained release of analgesia with immunomodulatory and antioxidant capabilities. In vivo models of chronic constriction injury (CCI) have demonstrated that CUB significantly reduced neuroinflammation, promoted M2 microglial polarization, and enhanced myelin regeneration for the prolonged analgesia. Deep mechanism analysis revealed that the designed CUB can significantly elevate TSP-1 expression to activate CGRP signal in modulating the neuro-immune interaction, contributing to the repair process. Notably, the CUB outperformed standalone bupivacaine or cerium nanoparticles in terms of pain relief, motor function recovery, and neuroglial regulation. The findings highlight the potential of CUB as a multifactorial therapeutic for treating neuropathic pain, offering new perspectives on the integration of nanotechnology in chronic pain management through neuro-immune pathways.

Porcine epidemic diarrhea virus (PEDV) causes severe intestinal damage and high mortality in neonatal piglets. The continuous emergence of new strains has brought new challenges to prevention and control. In this study, we isolated and characterized a prevalent PEDV virulent strain and analyzed 19,612 jejunal cells from PEDV-infected and control piglets using single-cell sequencing, revealing significant changes in cellular composition, gene expression, and intercellular communication. In response to PEDV infection, epithelial repair was enhanced through increased proliferation and differentiation of stem cells, transit-amplifying (TA) cells, and intestinal progenitor cells into enterocytes. Additionally, PEDV disrupted intercellular communication, compromising epithelial functionality while triggering immune responses, with IFN-γ and IL-10 signaling activation acting as critical regulators of immune balance and tissue homeostasis. Beyond enterocytes, viral genes were detected in various other cell types. Further experiments confirmed that PEDV could initiate replication in B and T lymphocytes but was unable to produce infectious progeny, with T cells additionally undergoing virus-induced apoptosis. These findings provide new insights into PEDV tropism, immune evasion, and epithelial repair, revealing complex host-pathogen interactions that shape disease progression and tissue regeneration, thereby contributing to a better understanding of enteric coronavirus pathogenesis.IMPORTANCEThe persistent circulation of porcine epidemic diarrhea virus (PEDV) poses a major threat to the swine industry, with emerging strains complicating prevention and control efforts. Currently, no effective measures completely prevent virus transmission, highlighting the need to understand PEDV-host interactions. In this study, we isolated a prevalent virulent strain and used single-cell sequencing to identify new PEDV-infected cell types and explore the complex interplay between the host and PEDV. These findings provide essential insights into viral pathogenesis and facilitate the design of targeted antiviral interventions.

The TATA box-binding protein (TBP) is an evolutionarily conserved basal transcription factor common in the pre-initiation complex of all three eukaryotic RNA polymerases (RNA Pols). Despite their high conservation, homologous TBPs exhibit species- and tissue-specific functions that may contribute to the increasingly complex gene expression regulation across evolutionary time. To determine the molecular mechanisms of species- and tissue-specificity for homologous TBPs, we examined the ability of yeast TBP and murine TBP paralogs to replace the endogenous TBP in mouse embryonic stem cells (mESCs). We show that, despite the high conservation in the DNA-binding domain among the homologs, they cannot fully rescue the lethality of TBP depletion in mESCs, which correlates with their inability to support RNA Pol III transcription. Furthermore, we show that the homologs differentially support stress-induced transcription reprogramming, with the divergent N-terminal domain playing a role in modulating changes in transcriptional response. Lastly, we show that the homologs have vastly different DNA binding dynamics, suggesting a potential mechanism for the distinct functional behavior observed among the homologs. Taken together, these data show a remarkable balance between flexibility and essentiality for the different functions of homologous TBP in eukaryotic transcription.

Skeletal ciliopathies result from defects in primary cilia, which are crucial for embryonic development through transduction of extracellular signals, including Hedgehog. Selective transport of ciliary proteins is mediated by the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes and the kinesin-2 and dynein-2 motors. Biallelic loss-of-function variants in genes encoding dynein-2-specific subunits, including DYNC2LI1, cause skeletal ciliopathies. As mesenchymal stem cells (MSCs) differentiate into osteoblasts, we investigated the effects of pathogenic variants of DYNC2LI1 on osteogenic differentiation of the MSC-like line C3H10T1/2. Dync2li1-knockout cells expressing disease-causing DYNC2LI1 variants demonstrated defects in the retrograde ciliary protein trafficking, including Hedgehog pathway GPCRs, Smoothened and GPR161. Furthermore, Dync2li1-knockout cells expressing the pathogenic variants demonstrated impaired Hedgehog signaling, in particular, a reduced ratio of the GLI3 repressor form to total GLI3, resulting in impaired osteogenic differentiation of MSCs. By contrast, osteogenic differentiation via BMP signaling was derepressed in Dync2li1-knockout cells. This suggests that skeletal ciliopathies caused by DYNC2LI1 variants could be attributable in part to impaired osteogenic differentiation due to defects in Hedgehog signaling, resulting from defects in retrograde ciliary protein trafficking.