Mammalian Stem Cells Research Articles

The evolving hematopoietic niche during development.

Mammalian hematopoietic stem cells (HSCs) emerge from the hemogenic endothelium in the major embryonic arteries. HSCs undergo a complex journey first migrating to the fetal liver (FL) and from there to the fetal bone marrow (FBM), where they mostly remain during adult life. In this process, a pool of adult HSCs is produced, which sustains lifelong hematopoiesis. Multiple cellular components support HSC maturation and expansion and modulate their response to environmental and developmental cues. While the adult HSC niche has been extensively studied over the last two decades, the niches present in the major embryonic arteries, FL, FBM and perinatal bone marrow (BM) are poorly described. Recent investigations highlight important differences among FL, FBM and adult BM niches and emphasize the important role that inflammation, microbiota and hormonal factors play regulating HSCs and their niches. We provide a review on our current understanding of these important cellular microenvironments across ontogeny. We mainly focused on mice, as the most widely used research model, and, when possible, include relevant insights from other vertebrates including birds, zebrafish, and human. Developing a comprehensive picture on these processes is critical to understand the earliest origins of childhood leukemia and to achieve multiple goals in regenerative medicine, such as mimicking HSC development in vitro to produce HSCs for broad transplantation purposes in leukemia, following chemotherapy, bone marrow failure, and in HSC-based gene therapy.

Influence of Real and Simulated Microgravity on the Early Development of Mammalian Embryonic Stem Cells

Influence of Real and Simulated Microgravity on the Early Development of Mammalian Embryonic Stem Cells

Comparative Study of Single and Coaxial Electrospun Antimicrobial Cross-Linked Scaffolds Enriched with Aloe Vera: Characterization, Antimicrobial Activity, Drug Delivery, Cytotoxicity, and Cell Proliferation on Adipose Stem Cells and Human Skin Fibroblast.

The preparation of materials with application in the biomedical field needs to attend some characteristics such as biocompatibility, nontoxicity, adequate mechanical properties, and the ability to mimic the extracellular matrix. Scaffolds for use in cell culture were prepared based on gelatin, polylactic acid (PLA), aloe vera mucilage, and tetracycline. Fibers were prepared in single and coaxial configuration and then cross-linked with glutaraldehyde saturated vapor. The fibers were obtained with cylindric morphology and changed to ribbon morphology and porous membranes, similarly to the extracellular matrix, when cross-linked. Membranes prepared by coaxial electrospinning showed core-shell structures when observed by transversal images, which is beneficial for controlled drug release. Characterization techniques such as scanning electron microscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy demonstrated the cross-linking due to the increase in diameter, formation of imine groups, and improvement of thermal stability. Antibiotic release tests showed that the prevalent release mechanism is diffusion and can be controlled considering the encapsulation effect, when fibers are prepared with a coaxial configuration, increasing the drug release time, making it a suitable material for controlled release. The biological evaluation of the scaffolds was carried out in two cell lines: mammalian adipose stem cells (ASCs), used as a primary cell culture, and Detroit 548 human skin fibroblasts as a dermal cell model. Aloe vera enriched scaffolds showed better activity in contact with both cell lines, exhibiting cell viability values greater than 90% and favorable results in live-dead assays when no damaged cells were observed. Cell proliferation was evaluated using Detroit 548 human skin fibroblast on gelatin-based scaffolds by the staining of the adhered cells; the images showed good confluence and morphology of the cells on the aloe vera and antibiotic loaded membranes for both of the studied configurations. Antibiotic loaded membranes presented antimicrobial activity against S. aureus, and this behavior increased when aloe vera is included. According to the results, the scaffolds prepared on single configuration enriched with aloe vera and tetracycline could be used in dermal tissue engineering as burn dressings, diabetic foot apposite, and skin substitutes, and the scaffolds prepared with a coaxial configuration are recommended for controlled release systems of antibiotics as treatments for chronic wounds such as diabetic foot and burn healing.

A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock-in via CRISPR/Cas9 Technology.

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.

Autophagy accompanying the developmental process of male germline stem cells.

Germline stem cells are a crucial type of stem cell that can stably pass on genetic information to the next generation, providing the necessary foundation for the reproduction and survival of organisms. Male mammalian germline stem cells are unique cell types that include primordial germ cells and spermatogonial stem cells. They can differentiate into germ cells, such as sperm and eggs, thereby facilitating offspring reproduction. In addition, they continuously generate stem cells through self-renewal mechanisms to support the normal function of the reproductive system. Autophagy involves the use of lysosomes to degrade proteins and organelles that are regulated by relevant genes. This process plays an important role in maintaining the homeostasis of germline stem cells and the synthesis, degradation, and recycling of germline stem cell products. Recently, the developmental regulatory mechanism of germline stem cells has been further elucidated, and autophagy has been shown to be involved in the regulation of self-renewal and differentiation of germline stem cells. In this review, we introduce autophagy accompanying the development of germline stem cells, focusing on the autophagy process accompanying the development of male spermatogonial stem cells and the roles of related genes and proteins. We also briefly outline the effects of autophagy dysfunction on germline stem cells and reproduction.

Embryonic stem cells maintain high origin activity and slow forks to coordinate replication with cell cycle progression

Embryonic stem (ES) cells are pluripotent stem cells that can produce all cell types of an organism. ES cells proliferate rapidly and are thought to experience high levels of intrinsic replication stress. Here, by investigating replication fork dynamics in substages of S phase, we show that mammalian pluripotent stem cells maintain a slow fork speed and high active origin density throughout the S phase, with little sign of fork pausing. In contrast, the fork speed of non-pluripotent cells is slow at the beginning of S phase, accompanied by increased fork pausing, but thereafter fork pausing rates decline and fork speed rates accelerate in an ATR-dependent manner. Thus, replication fork dynamics within the S phase are distinct between ES and non-ES cells. Nucleoside addition can accelerate fork speed and reduce origin density. However, this causes miscoordination between the completion of DNA replication and cell cycle progression, leading to genome instability. Our study indicates that fork slowing in the pluripotent stem cells is an integral aspect of DNA replication.

Emergence and properties of adult mammalian epidermal stem cells

In this review we discuss how the mammalian interfollicular epidermis forms during development, maintains homeostasis, and is repaired following wounding. Recent studies have provided new insights into the relationship between the stem cell compartment and the differentiating cell layers; the ability of differentiated cells to dedifferentiate into stem cells; and the epigenetic memory of epidermal cells following wounding.

Hematopoietic Stem Cells and Their Niche in Bone Marrow.

Extensive research has explored the functional correlation between stem cells and progenitor cells, particularly in blood. Hematopoietic stem cells (HSCs) can self-renew and regenerate tissues within the bone marrow, while stromal cells regulate tissue function. Recent studies have validated the role of mammalian stem cells within specific environments, providing initial empirical proof of this functional phenomenon. The interaction between bone and blood has always been vital to the function of the human body. It was initially proposed that during evolution, mammalian stem cells formed a complex relationship with the surrounding microenvironment, known as the niche. Researchers are currently debating the significance of molecular-level data to identify individual stromal cell types due to incomplete stromal cell mapping. Obtaining these data can help determine the specific activities of HSCs in bone marrow. This review summarizes key topics from previous studies on HSCs and their environment, discussing current and developing concepts related to HSCs and their niche in the bone marrow.

Rejuvenating aged stem cells: therapeutic strategies to extend health and lifespan.

Aging is associated with a global decline in stem cell function. To date, several strategies have been proposed to rejuvenate aged stem cells: most of these result in functional improvement of the tissue where the stem cells reside, but the impact on the lifespan of the whole organism has been less clearly established. Here, we review some of the most recent work dealing with interventions that improve the regenerative capacity of aged somatic stem cells in mammals and that might have important translational possibilities. Overall, we underscore that somatic stem cell rejuvenation represents a strategy to improve tissue homeostasis upon aging and present some recent approaches with the potential to affect health span and lifespan of the whole organism.

Abstract 3526: GAIN-BRCA: A graphical explainable AI-net framework for breast cancer subtype classification using multiomics data

Abstract Higher standards for precise distinction and classification among the four breast cancer subtypes, i.e., Luminal A. Luminal B, HER-2-enriched, and Basal, are essential to delivering customized treatment in precision oncology. The effective integration of readily accessible multi-omics datasets from the same patient may improve the accuracy of subtype prediction algorithms and help decipher reliable molecular features for the prediction and prognosis of breast cancer. Previous models for predicting breast cancer subtypes relied mostly on concatenation and autoencoder methods but rarely utilized graph-based integration to leverage the biological associations among omics datatypes. To overcome these limitations, we developed a graph-based multi-omics integration method using features from mRNA, DNA methylation, and miRNA data and synthesized features from their interactions. GAIN-BRCA computes weightage scores from miRNA-mRNA and DNA methylation-mRNA interactions to derive a new transformed feature vector without compromising biological information. The neural network architecture was designed and trained on the transformed features to classify breast cancer subtypes. Neural network prediction accuracies were compared using concatenated, autoencoder, and GAIN-BRCA-based integrated datasets. GAIN-BRCA outperforms the existing similar models MOGONET (Acc: 0.72) and moBRCA-net (Acc: 0.86) with an accuracy of 0.91. These transformed features were then processed using SHAP, and the top 357 features were selected for functional characterization and pathway analysis. We identified specific pathways and genes related to each breast cancer subtype separately. The most enriched pathways found in each subtype are ribonucleotide reductase signaling and glucocorticoid signaling pathways in luminal A, PFKB4 signaling pathway in luminal B, adipogenesis and DNA methylation and transcriptional signaling in HER2+, and the role of OCT4 in mammalian embryonic stem cell pluripotency in basal. Similarly, the most discriminated gene features are BRAF, ESR1, KRAS, MAPK1, SMARCE1, PPP3CA, and TGFBR2 in luminal A, TGFB3 and NCOA3 in luminal B, HNF4A, SIN3A, and SOX9 in HER2+, and POU5F1 and FOXA2 in basal subtypes. The GAIN-BRCA framework, combined with SHAP, weighs independent omics as cross-disciplinary rather than multidisciplinary and presents a distinctive picture for each subtype. As a result, the framework presents the importance of features about the corresponding subtype, which might lead to a greater understanding of the molecular basis of breast cancer subtypes. Citation Format: Jai Chand Patel, Sushil Kumar Shakyawar, Sahil Sethi, Chittibabu Guda. GAIN-BRCA: A graphical explainable AI-net framework for breast cancer subtype classification using multiomics data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3526.

Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS_001, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS_001 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS_001-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0.021-0.94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS_001 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS_001 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS_001 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS_001 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS_001 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS_001 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS_001 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS_001 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS_001, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y.W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4701.

Phase separation of transcriptional proteins in mammalian stem cells studied by residue-resolution MD simulations

Phase separation of transcriptional proteins in mammalian stem cells studied by residue-resolution MD simulations

Signal-regulated Unmasking of Nuclear Localization Motif in the PAS Domain Regulates the Nuclear Translocation of PASK

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells through intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. This interaction serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting this association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the intramolecular interaction in PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

Roles and Regulation of H3K4 Methylation During Mammalian Early Embryogenesis and Embryonic Stem Cell Differentiation.

From generation of germ cells, fertilization, and throughout early mammalian embryonic development, the chromatin undergoes significant alterations to enable precise regulation of gene expression and genome use. Methylation of histone 3 lysine 4 (H3K4) correlates with active regions of the genome, and it has emerged as a dynamic mark throughout this timeline. The pattern and the level of H3K4 methylation are regulated by methyltransferases and demethylases. These enzymes, as well as their protein partners, play important roles in early embryonic development and show phenotypes in embryonic stem cell self-renewal and differentiation. The various roles of H3K4 methylation are interpreted by dedicated chromatin reader proteins, linking this modification to broader molecular and cellular phenotypes. In this review, we discuss the regulation of different levels of H3K4 methylation, their distinct accumulation pattern, and downstream molecular roles with an early embryogenesis perspective.

Role of ubiquitin-proteasome system in stem cell biology

Selective degradation of cellular proteins by the ubiquitin -proteasome system is one of the key regulatory mechanisms in eukaryotic cells. Accumulating data indicate that the ubiquitin - proteasome system is involved in the regulation of fundamental processes in mammalian stem cells, including proliferation, differentiation, cell migration, aging and programmed cell death. Regulation can be carried out either by proteolytic degradation of key transcription factors and signaling pathway proteins, or by posttranslational modifications of target proteins with ubiquitin or other ubiquitin-like modifiers. Studies of the molecular mechanisms of proteostasis maintenance in stem cells are of great importance for the development of new therapeutic approaches aimed at the treatment of autoimmune and neurodegenerative diseases, cancer and other socially significant pathologies. This review covers current data on the function of the ubiquitin-proteasome system in stem cells.

Live birth of chimeric monkey with high contribution from embryonic stem cells

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologouscells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditionsfor establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our resultshave major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

Salicylic acid and RNA interference mediate antiviral immunity of plant stem cells

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

Impact of Fiber Characteristics on the Interfacial Interaction of Mammalian Cells and Bacteria

An imperative requisite of tissue-engineered scaffolds is to promote host cell regeneration and concomitantly thwart microbial growth. Antibacterial agents are often added to prevent implant-related infections, which, however, aggravates the risk of bacterial resistance. For the first time, we report a fiber-based platform that selectively promotes the growth of mammalian cells and alleviates bacteria by varying fiber size, orientation, and material of polymeric yarns. The interactions of Gram-positive and -negative bacterial species with mammalian mesenchymal stem cells (MSC) were investigated on poly-€-caprolactone (PCL) yarns, polyethylene terephthalate (PET), poly-L-lactic acid (PLLA), and cotton. Various yarn configurations were studied by altering the fiber diameter (from nano- to microscale) and fiber orientations (aligned, twisted, and random) of PCL yarns. PCL nanofibrous yarn decreased the adhesion of S. aureus and E. coli, with a 2.7-fold and 1.5-fold reduction, respectively, compared to PCL microfibrous yarn. Among different fiber orientations, nanoaligned fibers resulted in an 8-fold and 30-fold reduction of S. aureus and E. coli adhesion compared to random fibers. Moreover, aligned orientation was superior in retarding the S. aureus adhesion by 14-fold compared to nanotwisted fibers. Our data demonstrate that polymeric yarns comprising fibers with nanoscale features and aligned orientation promote mammalian cell adhesion and spreading and concomitantly mitigate bacterial interaction. Moreover, we unveil the wicking of cells through polymeric yarns, facilitating early cell adhesion in fibrous scaffolds. Overall, this study provides insight to engineer scaffolds that couple superior interaction of mammalian cells with high-strength fibrous yarns for regenerative applications devoid of antibacterial agents or other surface modification strategies.

Micelle-like clusters in phase-separated Nanog condensates: A molecular simulation study.

The phase separation model for transcription suggests that transcription factors (TFs), coactivators, and RNA polymerases form biomolecular condensates around active gene loci and regulate transcription. However, the structural details of condensates remain elusive. In this study, for Nanog, a master TF in mammalian embryonic stem cells known to form protein condensates in vitro, we examined protein structures in the condensates using residue-level coarse-grained molecular simulations. Human Nanog formed micelle-like clusters in the condensate. In the micelle-like cluster, the C-terminal disordered domains, including the tryptophan repeat (WR) regions, interacted with each other near the cluster center primarily via hydrophobic interaction. In contrast, hydrophilic disordered N-terminal and DNA-binding domains were exposed on the surface of the clusters. Electrostatic attractions of these surface residues were responsible for bridging multiple micelle-like structures in the condensate. The micelle-like structure and condensate were dynamic and liquid-like. Mutation of tryptophan residues in the WR region which was implicated to be important for a Nanog function resulted in dissolution of the Nanog condensate. Finally, to examine the impact of Nanog cluster to DNA, we added DNA fragments to the Nanog condensate. Nanog DNA-binding domains exposed to the surface of the micelle-like cluster could recruit more than one DNA fragments, making DNA-DNA distance shorter.

Measuring Glutathione Regeneration Capacity in Stem Cells.

Glutathione (GSH) is a chief cellular antioxidant, affecting stem cell functions. The cellular GSH level is dynamically altered by the redox buffering system and transcription factors, including NRF2. Additionally, GSH is differentially regulated in each organelle. We previously reported a protocol for monitoring the real-time GSH levels in live stem cells using the reversible GSH sensor FreSHtracer. However, GSH-based stem cell analysis needs be comprehensive and organelle-specific. Hence, in this study, we demonstrate a detailed protocol to measure the GSH regeneration capacity (GRC) in living stem cells by measuring the intensities of the FreSHtracer and the mitochondrial GSH sensor MitoFreSHtracer using a high-content screening confocal microscope. This protocol typically analyses the GRC in approximately 4 h following the seeding of the cells onto plates. This protocol is simple and quantitative. With some minor modifications, it can be employed flexibly to measure the GRC for the whole-cell area or just the mitochondria in all adherent mammalian stem cells.