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Regulation of hematopoiesis is dependent upon interactions between hematopoietic stem/progenitor cells and niche components, requiring a highly diverse array of different cell-cell interactions and cell signaling events. The overwhelming diversity of the components that can regulate hematopoiesis, especially when factoring in how the cell surface and intracellular protein expression profiles of hematopoietic stem/progenitor cells and niche components differ between homeostatic conditions and stressed conditions such as aging and irradiation, can make utilizing techniques like flow cytometry daunting, particularly while examining small cell populations such as hematopoietic stem cells (HSCs). Due to the complexity of the hematopoietic system, high-dimensional single-cell genomics and proteomics are constantly performed to understand the heterogeneity and expression profiles within this system. This chapter describes one such single-cell assay, which utilizes mass cytometry Time of Flight (CyTOF) technology to determine differences in expression profile within HSC, using changes in HSC populations due to gender and aging., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here, we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 18 h allows for tracking of cell-type-specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. By using live imaging in intact niches, we can better uncover the cellular and molecular mechanisms that regulate stem cell function in vivo., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In the context of plant regeneration, in vitro systems to produce embryos are frequently used. In many of these protocols, nonzygotic embryos are initiated that will produce shoot-like structures but may lack a primary root. By increasing the auxin-to-cytokinin ratio in the growth medium, roots are then regenerated in a second step. Therefore, in vitro systems might not or only partially execute a similar developmental program as employed during zygotic embryogenesis. There are, however, in vitro systems that can remarkably mimic zygotic embryogenesis such as Brassica microspore-derived embryos. In this case, the patterning process of these haploid embryos closely follows zygotic embryogenesis and all fundamental tissue types are generated in a rather similar manner. In this review, we discuss the most fundamental molecular events during early zygotic embryogenesis and hope that this brief summary can serve as a reference for studying and developing in vitro embryogenesis systems in the context of doubled haploid production., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Reactive oxygen species (ROS) may severely affect the biochemical viability of most cells. However, ROS may act also as key second messengers regulating important physiological functions in eukaryotic organisms. Of special interest is the potential role of ROS in the regulation of stem cell function and tissue homeostasis and regeneration in adult mammalian tissues. In this context, the hair follicle constitutes an excellent experimental model to study this aspect of ROS biology.Here we present a robust protocol to promote a sustained growth of ex vivo cultured human hair follicles based on the induction of a transient/modulable production of nonlethal endogenous ROS levels in the tissue through a protoporphyrin IX-dependent photodynamic procedure. The light-switchable ROS production activates hair follicle stem cell niches, induces cell proliferation, and maintains the growth/anagen phase for long time. This approach constitutes a complementary experimental tool to study the physiological roles of ROS in human tissues.

Plants are multicellular organism composed of different types of cells. These all kinds of cells are formed from pluripotent stem cells present at different positions in plant called stem cell niches. All these stem cell niches and their boundaries are maintained by complex regulatory mechanism at molecular level involving different genes, cofactors, and phytohormones. In this chapter, we discussed the regulatory mechanism and models of stem cell maintenance, specifying their boundaries at different stem cell niches.

Fluorescence lifetime imaging microscopy (FLIM), enabling live quantitative multiparametric analyses, is an emerging bioimaging approach in tissue engineering and regenerative medicine. When combined with stem cell-derived intestinal organoid models, FLIM allows for tracing stem cells and monitoring of their proliferation, metabolic fluxes, and oxygenation. It is compatible with the use of live Matrigel-grown intestinal organoids produced from primary adult stem cells, crypts, and transgenic Lgr5-GFP mice. In this chapter we summarize available experimental protocols, imaging platforms (one- and two-photon excited FLIM, phosphorescence lifetime imaging microscopy (PLIM)) and provide the anticipated data for FLIM imaging of the live intestinal organoids, focusing on labeling of cell proliferation, its colocalization with the stem cell niche, measured local oxygenation, autofluorescence, and some other parameters. The protocol is illustrated with examples of multiparameter imaging, employing spectral and "time domain"-based separation of dyes, probes, and assays.

The intestinal epithelium is known as one of the most regenerative tissues in our body. The lining of the intestine is composed of a single layer of epithelial cells generated by rapidly renewing stem cells residing at the crypt bottoms, resulting in a flow of cells to the villus tips. The stereotypical crypt-villus architecture makes the intestine an ideal model for stem cell research. Based on recent advances in research of stem cell niche signals in vivo, we have established an intestinal epithelial stem cell culture method. Under this culture condition, single Lgr5+ intestinal stem cells (ISCs) or isolated whole crypts efficiently expand into three-dimensional spherical structures recapitulating the intestinal crypt-villus organization. These organoids can be passaged weekly and maintained for years in culture. Moreover, they can be cryopreserved. As intestinal organoids recapitulate many aspects of the epithelial biology and are amenable to most, if not all, current experimental manipulations, they are widely used to study stem cell biology, cell fate determination, gene function, and disease mechanism.

Intestinal stem cells continuously self-renew throughout life to maintain gut homeostasis. With the advent of the organoid culture system, we are now able to indefinitely expand healthy and diseased tissue-derived human intestinal stem cells in vitro and use them for various applications. Nonetheless, investigating the behavior of human intestinal stem cells in vivo still remains challenging. We recently developed an orthotopic xenotransplantation system that realizes in vivo reconstruction of human intestinal epithelial tissue with preserved stem cell hierarchy by engrafting human normal colon organoids onto the mouse colon surface. We also introduced new growth factors, namely, insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2), into the culture condition for human intestinal organoids that significantly increase scalability and transfectability of the organoids. By integrating these recent advances, we organized a tissue-oriented platform encompassing derivation of patient-derived intestinal organoids and their orthotopic xenotransplantation. The research platform based on orthotopic xenotransplantation of human intestinal organoids provides a powerful tool for studying human intestinal stem cell biology in native tissue-relevant contexts as well as for establishing novel disease modeling systems.

It has been described that adult tissues contain mesenchymal stem cell populations. The specific areas where stem cells reside are known as niches. Crosstalk between cells and their niche is essential to maintain the correct functionality of stem cell. MSCs present a set of abilities such as migration, invasion, and angiogenic potentials, which make them ideal candidates for cell-based therapies. In order to test the regenerative capacity of these cells, we have described a methodology for the collection and for the evaluation of these mesenchymal precursors from different niches.

The niche plays critical roles in regulating functionality and determining regenerative outcomes of stem cells, for which establishment of favorable microenvironments is in demand in translational medicine. In recent years, the cell aggregate technology has shown immense potential to reconstruct a beneficial topical niche for stem cell-mediated regeneration, which has been recognized as a promising concept for high-density stem cell delivery with preservation of the self-produced, tissue-specific extracellular matrix microenvironments. Here, we describe the basic methodology of stem cell aggregate-based niche engineering and quality check indexes prior to application.

Adult skeletal muscle stem cells, termed satellite cells, are essential for regenerating muscle after tissue damage. Satellite cells are located in a specialized microenvironment between muscle fibers and their surrounding basal lamina. This local niche serves as a compartment to preserve satellite cell function and provides signals that facilitate the rapid response to injury. Visualization of this local niche enables the elucidation of such niche-derived signals. Here, we describe techniques for isolating single myofibers with their associated satellite cells for ex vivo visualization and analysis of an intact muscle stem cell niche.

The development, differentiation, and maturation of hematopoietic cells are regulated by the intrinsic and extrinsic regulation. Intrinsic activity is affected by cell autonomous gene expression and extrinsic factors originate from the so-called niche surrounding the hematopoietic cells. It remains unclear why the hematopoietic sites are shifted during embryogenesis. Flow cytometry and immunohistochemistry enable us to study embryonic regulation of hematopoietic niche in the mouse embryo.

The tracking of the hematopoietic potential of genetically manipulated fluorescent hematopoietic stem cells (HSC) in the bone marrow (BM) allows the assessment of regulatory processes involved in the re-establishment of hematopoiesis posttransplant. Herein, we describe the means to assess the consequence of expressing specific genes in HSC on their engraftment potential posttransplant.

The maintenance of adult stem cells in their normal quiescent state depends on intrinsic factors and extrinsic signals originating from their microenvironment (also known as the stem cell niche). In skeletal muscle, its stem cells (satellite cells) lose their regenerative potential with aging, and this has been attributed, at least in part, to both age-associated changes in the satellite cells as in the niche cells, which include resident fibro-adipogenic progenitors (FAPs), macrophages, and endothelial cells, among others. To understand the regenerative decline of skeletal muscle with aging, there is a need for methods to specifically isolate stem and niche cells from resting muscle. Here we describe a fluorescence-activated cell sorting (FACS) protocol to simultaneously isolate discrete populations of satellite cells and niche cells from skeletal muscle of aging mice.

The ability of cancer cells to compete with hematopoietic stem cells (HSCs) to target the bone marrow microenvironment, or the HSC niche, during the dissemination process is critical for the development of bone metastasis. Here, we describe the methods for testing the relative potential of cancer cells to compete with HSCs for occupancy of the HSC niche by measuring the peripheral blood level of engrafted HSCs by flow cytometry in mice after bone marrow transplantation and tandem cancer cell inoculation. This method is useful for determining the molecular mechanisms for the roles of HSCs in the regulation of bone metastases.

This chapter considers the techniques necessary and required for the reprogramming of exogenous stem/progenitor cell populations towards a mammary epithelial cell fate. The protocols describe how to isolate cells from alternate mouse organs such as testicles of male mice and mix them with mammary cells to generate chimeric glands comprised of male and female epithelial cells that are fully competent. During the reformation of mammary stem cell niches by dispersed epithelial cells, in the context of the intact epithelium-free mammary stroma, non-mammary cells are sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells. This therefore is a powerful technique for the redirection of cells from other organs/cancer cells to a normal mammary phenotype.

Terminal erythroid differentiation occurs in the bone marrow, within specialized niches termed erythroblastic islands. These functional units consist of a macrophage surrounded by differentiating erythroblasts and have been described more than five decades ago, but their function in the pathophysiology of erythropoiesis has remained unclear until recently. Here we propose that the central macrophage in the erythroblastic island contributes to the pathophysiology of anemia of inflammation. After introducing erythropoiesis and the interactions between the erythroblasts and the central macrophage within the erythroblastic islands, we will discuss the immunophenotypic characterization of this specific subpopulation of macrophages. We will then integrate these concepts into the currently known pathophysiological drivers of anemia of inflammation and address the role of the central macrophage in this disorder. Finally, as a means of furthering our understanding of the various concepts, we will discuss the differences between murine and rat models with regard to developmental and stress erythropoiesis in an attempt to define a model system representative of human pathophysiology.

Microgyria is associated with epilepsy and due to developmental disruption of neuronal migration. However, the role of endogenous subventricular zone-derived neural progenitors (SDNPs) in formation and hyperexcitability has not been fully elucidated. Here, we establish a neonatal cortex freeze-lesion (FL) model, which was considered as a model for focal microgyria, and simultaneously label SDNPs by CM-DiI. Morphological investigation showed that SDNPs migrated into FL and differentiated into neuronal and glia cell types, suggesting the involvement of endogenous SDNPs in the formation of FL-induced microgyria. Patch-clamp recordings in CM-DiI positive (CM-DiI(+)) pyramidal neurons within FL indicated an increase in frequency of spontaneous action potentials, while the resting membrane potential did not differ from the controls. We also found that spontaneous excitatory postsynaptic currents (EPSCs) increased in frequency but not in amplitude compared with controls. The evoked EPSCs showed a significant increase of 10-90% in rise time and decay time in the CM-DiI(+) neurons. Moreover, paired-pulse facilitation was dramatically larger in CM-DiI(+) pyramidal neurons. Western blotting data showed that AMPA and NMDA receptors were increased to some extent in the FL cortex compared with controls, and the NMDA/AMPA ratio of eEPSCs at CM-DiI(+) pyramidal neurons was significantly increased. Taken together, our findings provide novel evidence for the contribution of endogenous SDNPs in the formation and epileptogenicity of FL-induced focal microgyria.

Electromagnetic fields (EMFs) may affect the endogenous neural stem cells within the brain. The aim of this study was to assess the effects of EMFs on the process of toxin-induced demyelination and subsequent remyelination. Demyelination was induced using local injection of lysophosphatidylcholine within the corpus callosum of adult female Sprague-Dawley rats. EMFs (60 Hz; 0.7 mT) were applied for 2 h twice a day for 7, 14, or 28 days postlesion. BrdU labeling and immunostaining against nestin, myelin basic protein (MBP), and BrdU were used for assessing the amount of neural stem cells within the tissue, remyelination patterns, and tracing of proliferating cells, respectively. EMFs significantly reduced the extent of demyelinated area and increased the level of MBP staining within the lesion area on days 14 and 28 postlesion. EMFs also increased the number of BrdU- and nestin-positive cells within the area between SVZ and lesion as observed on days 7 and 14 postlesion. It seems that EMF potentiates proliferation and migration of neural stem cells and enhances the repair of myelin in the context of demyelinating conditions.

Hematopoietic stem and progenitor mobilization has revolutionized the field of hematopoietic transplantation. Currently, hematopoietic grafts acquired from the peripheral blood of patients or donors treated with granulocyte-colony stimulating factor (G-CSF) are the preferred source for transplantation. G-CSF mobilization regimens, however, are associated with known morbidities and a significant number of normal donors and patient populations fail to mobilize sufficient numbers of hematopoietic stem and progenitor cells for transplantation, necessitating the need for non-G-CSF mobilization strategies. Mechanistic studies evaluating hematopoietic bone marrow niche interactions have uncovered novel agents with the capacity for hematopoietic mobilization. This chapter provides a comprehensive overview of mobilizing agents, other than G-CSF, and experimental procedures and technical aspects important to evaluate and define their hematopoietic mobilizing activities alone and in combination.

Emerging evidence indicates that endothelial cells (ECs) not only form the passive building blocks of blood vessels that deliver oxygen and nutrients, but also instructively participate in organ regeneration and tumorigenesis by producing tissue-specific angiocrine factors. Due to a lack of unbiased, functional angiogenic models, the role of ECs in the homeostasis of tissue-specific stem cells and propagation of malignant cells is unknown. We established a means to maintain primary EC cultures by introducing phospho-ser473 Akt, enabling their survival for weeks under serum-/cytokine-free conditions. This lentiviral-based system maintains the angiogenic repertoire without immortalization and increased tumorigenic potential. Using our novel cytokine-/serum-free in vitro EC-based culture system, we have shown that ECs are endowed with the capacity to expand and maintain bona fide hematopoietic stem cells (HSCs) and survival of leukemic cells. This unbiased system described here can serve as a platform to identify EC-derived growth and to model treatment of a wide variety of hematological and malignant conditions.

The tracking of immunofluorescent labeled hematopoietic stem and progenitor cells (HSC/HPC) within the bone marrow (BM) cavity allows the assessment of the regulatory processes involved in transendothelial migration, trans-marrow migration, and finally lodgement into the HSC niche. This is of interest as the extracellular and cellular components involved in the regulation of HSC quiescence and differentiation are still not completely understood. Homing of transplanted HSC is the first critical step in the interaction between HSC and the microenvironment of the BM. As a consequence, murine models allowing the evaluation of the structural relationship between migrating HSC, the endosteal bone surface, and the vascular components of the BM enhance our understanding of hematopoietic regulation.

Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow in zones of residence specialized in supporting them which are referred to as niches. It is in such a specialized niche that normal HSPCs are maintained to perform their self-renewal and differentiation duties in a highly controlled manner. One challenge in dissecting the functional significance of the complex cellular and molecular interactions in the niche is to link the types and qualities of cell-cell contacts to the intracellular signaling components involved in cell regulation. Attempts to study the interactions of HSPC with their niche eventually have to be performed in their natural location in vivo, as isolation of the cells from bone -marrow will disrupt the HSPC-niche interactions and thus not reveal functionally critical cell-cell -contacts. Intravital imaging of individual cells in the bone marrow has just recently been introduced, almost exclusively focusing on imaging inside the marrow of the calvaria. However, calvarial marrow is functionally distinct from marrow of long bones, the major source of HSPC for both physiology and study. To overcome these limitations, we developed a novel method for multiphoton intravital imaging of HSPC in the marrow of long bones.

It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process.

Hematopoietic stem cells are at the foundation of the blood system. Their study is not only relevant to the understanding of the basic cellular mechanisms of self-renewal, lineage commitment, and differentiation, but they have also been the target of intense clinical research into the causes of leukemia and the exploitation of these cells for cell replacement therapies. The basic mechanisms of hematopoietic stem cell regulation become evident in the way these cells are first generated and expanded during development. Isolating and analyzing hematopoietic stem cells from the embryo is therefore of direct clinical importance.