Multiple cell populations generate macrophage progenitors in the early yolk sac.

Yolk sac (YS) CSF1 receptor positive (CSF1R+) cells are thought to be the progenitors for tissue-resident macrophages present in various tissues. The YS progenitors for tissue-resident macrophages are referred to as erythroid-myeloid progenitors (EMPs). However, diverse types of hematopoietic progenitors are present in the early YS, thus it is not precisely known which type of hematopoietic cell gives rise to the CSF1R+ lineage. In this study, an analysis was conducted to determine when CSF1R+ progenitors appeared in the early YS. It showed that CSF1R+ cells appeared in the YS as early as embryonic day 9 (E9) and that the earliest hematopoietic progenitors that differentiate into CSF1R+ cells were found in E8. Since these progenitors possessed the capability to generate primitive erythroid cells, it was likely that primitive erythroid lineages shared progenitors with the CSF1R+ lineage. Mutual antagonism appears to work between PU.1 and GATA1 when CSF1R+ cells appear in the early YS. One day later (E9), multiple progenitors, including myeloid-restricted progenitors and multipotent progenitors, in the YS could immediately generate CSF1R+ cells. These results suggest that EMPs are not an exclusive source for the CSF1R+ lineage; rather, multiple hematopoietic cell populations give rise to CSF1R+ lineage in the early YS.

Isolation of CD35+ follicular dendritic cells and its role in the differentiation from B cells to IgA+GL7+ cells.

Follicular dendritic cells (FDCs) are non-hematopoietic cells that are localized in the germinal centers (GCs) of lymph nodes (LNs) and are involved in humoral immunity. FDCs are a rare population that are sensitive to mechanical and chemical stimuli, making their isolation for analysis difficult. In Peyer's Patches, which are the main IgA-inductive sites, FDCs have been reported to be activated by retinoic acid receptor (RAR) and toll-like receptor (TLR) signals to induce IgA production. However, little is known about FDCs in mesenteric LNs (MLNs), although MLNs are also an IgA-inductive site. In this study, we efficiently isolated FDCs as CD35+ cells using anti-CD35 antibodies (Abs) and magnetic bead sorting. We found that CD35+ FDCs facilitated differentiation from B220+ B cells into IgA+GL7+ GC B-like cells but not IgA+CD138+ plasma cells. Furthermore, using CD35+ FDCs from LPS-resistant C3H/HeJ mice, the generation of IgA+GL7+ GC B-like cells was not altered significantly between wild-type and LPS-resistant mice. Moreover, the addition of RAR antagonists and agonists revealed that differentiation into IgA+GL7+ GC B-like cells required the activation of RAR, especially RAR-ß, in FDCs. The differentiation of IgA+GL7+ cells was promoted by FDCs in peripheral LNs as well as MLNs in our in vitro assay. Taken together, these results indicate that magnetic bead sorting with anti-CD35 Abs enable the efficient isolation of FDCs. Our data suggested that CD35+ FDCs can support differentiation of B cells into IgA+GL7+ GC B-like cells in environments that are not limited to MLNs, which can be stimulated by retinoic acid.

Cellular Basis of Embryonic Hematopoiesis and Its Implications in Prenatal Erythropoiesis.

Primitive erythrocytes are the first hematopoietic cells observed during ontogeny and are produced specifically in the yolk sac. Primitive erythrocytes express distinct hemoglobins compared with adult erythrocytes and circulate in the blood in the nucleated form. Hematopoietic stem cells produce adult-type (so-called definitive) erythrocytes. However, hematopoietic stem cells do not appear until the late embryonic/early fetal stage. Recent studies have shown that diverse types of hematopoietic progenitors are present in the yolk sac as well as primitive erythroblasts. Multipotent hematopoietic progenitors that arose in the yolk sac before hematopoietic stem cells emerged likely fill the gap between primitive erythropoiesis and hematopoietic stem-cell-originated definitive erythropoiesis and hematopoiesis. In this review, we discuss the cellular origin of primitive erythropoiesis in the yolk sac and definitive hematopoiesis in the fetal liver. We also describe mechanisms for developmental switches that occur during embryonic and fetal erythropoiesis and hematopoiesis, particularly focusing on recent studies performed in mice.

Spatially Arranged Sparse Recurrent Neural Networks for Energy Efficient Associative Memory.

The development of hardware neural networks, including neuromorphic hardware, has been accelerated over the past few years. However, it is challenging to operate very large-scale neural networks with low-power hardware devices, partly due to signal transmissions through a massive number of interconnections. Our aim is to deal with the issue of communication cost from an algorithmic viewpoint and study learning algorithms for energy-efficient information processing. Here, we consider two approaches to finding spatially arranged sparse recurrent neural networks with the high cost-performance ratio for associative memory. In the first approach following classical methods, we focus on sparse modular network structures inspired by biological brain networks and examine their storage capacity under an iterative learning rule. We show that incorporating long-range intermodule connections into purely modular networks can enhance the cost-performance ratio. In the second approach, we formulate for the first time an optimization problem where the network sparsity is maximized under the constraints imposed by a pattern embedding condition. We show that there is a tradeoff between the interconnection cost and the computational performance in the optimized networks. We demonstrate that the optimized networks can achieve a better cost-performance ratio compared with those considered in the first approach. We show the effectiveness of the optimization approach mainly using binary patterns and apply it also to gray-scale image restoration. Our results suggest that the presented approaches are useful in seeking more sparse and less costly connectivity of neural networks for the enhancement of energy efficiency in hardware neural networks.

Recent advances in physical reservoir computing: A review.

Reservoir computing is a computational framework suited for temporal/sequential data processing. It is derived from several recurrent neural network models, including echo state networks and liquid state machines. A reservoir computing system consists of a reservoir for mapping inputs into a high-dimensional space and a readout for pattern analysis from the high-dimensional states in the reservoir. The reservoir is fixed and only the readout is trained with a simple method such as linear regression and classification. Thus, the major advantage of reservoir computing compared to other recurrent neural networks is fast learning, resulting in low training cost. Another advantage is that the reservoir without adaptive updating is amenable to hardware implementation using a variety of physical systems, substrates, and devices. In fact, such physical reservoir computing has attracted increasing attention in diverse fields of research. The purpose of this review is to provide an overview of recent advances in physical reservoir computing by classifying them according to the type of the reservoir. We discuss the current issues and perspectives related to physical reservoir computing, in order to further expand its practical applications and develop next-generation machine learning systems.

Mouse Yolk Sac Hematopoiesis.

The yolk sac is the first observed site of hematopoiesis during mouse ontogeny. Primitive erythroid cells are the most well-recognized cell lineages produced from this tissue. In addition to primitive erythroid cells, several types of hematopoietic cells are present, including multipotent hematopoietic progenitors. Yolk sac-derived blood cells constitute a transient wave of embryonic and fetal hematopoiesis. However, recent studies have demonstrated that some macrophage and B cell lineages derived from the early yolk sac may persist to adulthood. This review discusses the cellular basis of mouse yolk sac hematopoiesis and its contributions to embryonic and adult hematopoietic systems.

Subjectivity of the Anomalous Sense of Self Is Represented in Gray Matter Volume in the Brain.

The self includes complicated and heterogeneous functions. Researchers have divided the self into three distinct functions called "agency," "ownership," and "narrative self". These correspond to psychiatric symptoms, behavioral characteristics and neural responses, but their relationship with brain structure is unclear. This study examined the relationship between the subjectivity of self-related malfunctions and brain structure in terms of gray matter (GM) volume in 96 healthy people. They completed a recently developed self-reported questionnaire called the Embodied Sense of Self Scale (ESSS) that measures self-related malfunctions. The ESSS has three subscales reflecting the three distinct functions of the self. We also determined the participants' brain structures using magnetic resonance imaging (MRI) and voxel-based morphometry (VBM). Multiple regression analysis revealed a significant negative correlation between ownership malfunction and the insular cortex GM volume. A relationship with brain structure could thus only be confirmed for the ESSS "ownership" subscale. This finding suggests that distinct brain structures feel ownership and that the ESSS could partly screen for distinct brain structures.

A novel rat head gaze determination system based on optomotor responses.

The optomotor response of animals is commonly used to measure their visual performance, e.g., rats of different genetically altered strains or various drug tests. With the presentation of stimuli using computer screens or projectors, the common idea focuses on measuring the eye movement or head and/or body movement to characterize changes of the head gaze. However, traditional methods rely on either the invasive fixation of animals, or the judgment of a human observer who reports the stimulus-tracking movements. In this paper, we propose a novel head gaze determination system to automatically track the head movement of rats without artificial markers. The experiments were done to demonstrate the process of optimizing parameters in image processing. As a result, the head angle curve of the proposed method is consistent with that of ground-truth data annotated manually according to predefined rules. Hence, the proposed method provides a simple, convenient, and objective solution to automatically generate the head gaze orientations from massive amounts of recorded data for further visual performance analysis.

Depletion of Neural Crest-Derived Cells Leads to Reduction in Plasma Noradrenaline and Alters B Lymphopoiesis.

Hematopoietic stem cells and their lymphoid progenitors are supported by the bone marrow (BM) microenvironmental niches composed of various stromal cells and Schwann cells and sympathetic nerve fibers. Although neural crest (NC) cells contribute to the development of all the three, their function in BM is not well understood. In this study, NC-derived cells were ablated with diphtheria toxin in double-transgenic mice expressing NC-specific Cre and Cre-driven diphtheria toxin receptor with yellow fluorescent protein reporter. We found that yellow fluorescent protein-expressing, NC-derived nonhematopoietic cells in BM expressed hematopoietic factors Cxcl12 and stem cell factor The ablation of NC-derived cells led to a significant decrease in B cell progenitors but not in hematopoietic stem cells or myeloid lineage cells in BM. Interestingly, plasma noradrenaline was markedly decreased in these mice. The i.p. administration of 6-hydroxydopamine, a known neurotoxin for noradrenergic neurons, led to a similar phenotype, whereas the administration of a noradrenaline precursor in NC-ablated mice partially rescued this phenotype. Additionally, the continuous administration of adrenergic receptor ß antagonists partially decreased the number of B cell progenitors while preserving B lymphopoiesis in vitro. Taken together, our results indicate that NC-derived cell depletion leads to abnormal B lymphopoiesis partially through decreased plasma noradrenaline, suggesting this as a novel mechanism regulated by molecules released by the sympathetic neurons.

Repression of Primitive Erythroid Program Is Critical for the Initiation of Multi-Lineage Hematopoiesis in Mouse Development.

Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi-lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down-regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi-lineage hematopoiesis in the early yolk sac of developing embryos. J. Cell. Physiol. 232: 323-330, 2017. © 2016 Wiley Periodicals, Inc.

Common developmental pathway for primitive erythrocytes and multipotent hematopoietic progenitors in early mouse development.

Development of the hematopoietic system proceeds in a multistep manner. Primitive erythrocytes are the first hematopoietic cells to be observed that were produced transiently in developing embryos. Multilineage lymphohematopoiesis occurs after the primitive erythropoiesis. However, the lineage relationship of cells that comprise embryonic hematopoietic system is not well characterized. To clarify this process, careful analyses of the embryonic cells that differentiate into these cell lineages are necessary. We identified the common precursors of primitive erythrocytes and multipotent hematopoietic cells in mouse embryonic stem cell cultures and mouse embryos. A subset defined as CD45(-)CD41(+)AA4.1(-) cells showed bipotential capability to produce primitive erythrocytes and lymphomyeloid cells at the single-cell level. The cell population was present in vivo before hematopoietic stem cells (HSCs) appeared. Our results show that primitive erythrocytes and lymphomyeloid cells are not completely separate cell lineages, and these precursors comprise the embryonic hematopoietic system before HSC emergence.

Earliest hematopoietic progenitors at embryonic day 9 preferentially generate B-1 B cells rather than follicular B or marginal zone B cells.

The lymphoid potential of the hematopoietic system is observed as early as embryonic day 9 (E9) before transplantable hematopoietic stem cells (HSCs) appear at E11 in mice. However, it is largely unknown as to which cell fraction is responsible for the initial wave of lymphopoiesis and whether these earliest lymphocytes make any contributions to the adult lymphoid system. We previously isolated the earliest hematolymphoid progenitors at E9 that had CD45(+)c-Kit(+)AA4.1(+) phenotypes. In this study, the differentiation potency into B cell subsets of the E9 hematolymphoid progenitors was examined in detail. In culture, E9 hematolymphoid progenitors produced B220(-/low) B cell progenitors in striking contrast to adult BM c-Kit(+)Sca-1(+)Lin(-) cells. Upon in vivo transplantation, B cell progenitors derived from E9 hematolymphoid progenitors preferentially differentiated into the B-1 B lymphocyte subset, whereas their differentiation into B-2 B lymphocyte subsets [follicular B (FoB), marginal zone B (MZB) cells] was inefficient. Of note, these donor B lymphocytes permanently repopulated in host mice, even if adult mice were used as recipients. These results suggest that B cell progenitors produced from an initial wave of definitive hematopoiesis before authentic HSCs appear could be a permanent source for, at least, the B-1 B lymphocyte subset.

Origins and properties of dental, thymic, and bone marrow mesenchymal cells and their stem cells.

Mesenchymal cells arise from the neural crest (NC) or mesoderm. However, it is difficult to distinguish NC-derived cells from mesoderm-derived cells. Using double-transgenic mouse systems encoding P0-Cre, Wnt1-Cre, Mesp1-Cre, and Rosa26EYFP, which enabled us to trace NC-derived or mesoderm-derived cells as YFP-expressing cells, we demonstrated for the first time that both NC-derived (P0- or Wnt1-labeled) and mesoderm-derived (Mesp1-labeled) cells contribute to the development of dental, thymic, and bone marrow (BM) mesenchyme from the fetal stage to the adult stage. Irrespective of the tissues involved, NC-derived and mesoderm-derived cells contributed mainly to perivascular cells and endothelial cells, respectively. Dental and thymic mesenchyme were composed of either NC-derived or mesoderm-derived cells, whereas half of the BM mesenchyme was composed of cells that were not derived from the NC or mesoderm. However, a colony-forming unit-fibroblast (CFU-F) assay indicated that CFU-Fs in the dental pulp, thymus, and BM were composed of NC-derived and mesoderm-derived cells. Secondary CFU-F assays were used to estimate the self-renewal potential, which showed that CFU-Fs in the teeth, thymus, and BM were entirely NC-derived cells, entirely mesoderm-derived cells, and mostly NC-derived cells, respectively. Colony formation was inhibited drastically by the addition of anti-platelet-derived growth factor receptor-ß antibody, regardless of the tissue and its origin. Furthermore, dental mesenchyme expressed genes encoding critical hematopoietic factors, such as interleukin-7, stem cell factor, and cysteine-X-cysteine (CXC) chemokine ligand 12, which supports the differentiation of B lymphocytes and osteoclasts. Therefore, the mesenchymal stem cells found in these tissues had different origins, but similar properties in each organ.

Methods for investigation of osteoclastogenesis using mouse embryonic stem cells.

Investigation of osteoclastogenesis in vivo, especially in early development, has proven difficult because of the accessibility of these early embryonic stages. Our ability to culture embryonic stem cells (ESCs) in vitro has overcome this difficulty as these versatile cells can be expanded endlessly. Thus, the whole process of osteoclastogenesis can be monitored in these cultures through the microscope and with the help of molecular biology techniques. We have developed two methods to induce osteoclasts, the bone matrix remodeling cells, from murine ESCs. Surprisingly, one of these induction methods produces osteoclasts, osteoblasts, and also endothelial cells in the same culture dish. Hence, it is likely that ESCs in culture mimic the in vivo development of osteoclasts.

Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development.

The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.

Bmi-1-green fluorescent protein-knock-in mice reveal the dynamic regulation of bmi-1 expression in normal and leukemic hematopoietic cells.

The ability to self-renew is essential for all kinds of stem cells regardless of tissue type. One of the best candidate genes involved in conferring self-renewal capacity is Bmi-1, which has been proven to be essential for the maintenance of both normal adult hematopoietic and leukemia stem cells, as well as adult neural stem cells. To investigate the possible role of Bmi-1 in other cell types that also self-renew, we generated Bmi-1-green fluorescent protein (GFP)-knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of the Bmi-1 gene. Using these targeted reporter mice, we demonstrated that Bmi-1 is expressed in hematopoietic stem cells (HSCs) at its highest levels and downregulated upon commitment to differentiation. An in vivo reconstitution assay revealed that the frequency of HSCs was 1/16 in Bmi-1high c-kit+ lin -Sca-1+ bone marrow (BM) cells and 1/49 in Bmi-1 high lin- BM cells, suggesting that Bmi-1 may serve as a marker for normal HSCs. In murine leukemia models induced by P210BCR/ABL or TEL/PDGFbetaR + AML1/ETO, Bmi-1 was not overexpressed in leukemic HSCs, despite the increase in the HSC numbers. Bmi-1 was expressed at its highest levels in undifferentiated leukemia cells. Furthermore, in several other nonhematopoietic tissues, cells could be separated into distinct subpopulations with differential Bmi-1 expression. Thus, these mice allow for the isolation of viable Bmi-1-expressing cells and have the potential to become a useful tool for understanding the role of Bmi-1 in normal and cancer stem cells in multiple tissue types. Disclosure of potential conflicts of interest is found at the end of this article.

Myocardial restoration with embryonic stem cell bioartificial tissue transplantation.

BACKGROUND: The optimal cell-matrix combination for robust and sustained myocardial restoration has not been identified. The present study utilizes embryonic stem cells as the substrate of bioartificial myocardial tissue and evaluates engraftment in, and functional recovery of, the recipient heart. METHODS: Collagen type I was populated with undifferentiated green fluorescent protein (GFP)-positive mouse embryonic stem cells. An intramural left ventricular pouch was fashioned after ligation of the left anterior descending artery in an athymic nude rat heterotopic heart transplant model. The bioartificial mixture (0.125 ml) was implanted in the infarcted area within the pouch. Echocardiography was performed to assess fractional shortening in: Group I, infarcted rats that received cell-matrix implants; Group II, rats given matrix implant without cells; Group III, rats given no matrix or cells; and Group IV, rats receiving transplanted hearts without ligation (n = 5/group). Hearts were stained for GFP, cardiac markers (connexin-43, alpha-sarcomeric actin), hematoxylin-eosin (H&E) and trichrome. RESULTS: Embryonic stem cells formed stable intramyocardial grafts that were incorporated into the surrounding area without distorting myocardial geometry, thereby preventing ventricular wall thinning (anterior wall thickness was: Group I, 1.4 +/- 0.1 mm; Group II, 1.0 +/- 0.1 mm, Group III, 0.9 +/- 0.2 mm; and Group IV, 1.3 +/- 0.2 mm). The inoculated cells expressed connexin-43 and alpha-sarcomeric actin in vivo. Fractional shortening was better in embryonic stem cell-treated animals (Group I, 21.5 +/- 3.5%; Group II, 12.4 +/- 2.8%; Group III, 8.2 +/- 2.9%; Group IV, 23.2 +/- 4.2%). CONCLUSIONS: Embryonic stem cells are an efficient alternative substrate for myocardial tissue engineering and can prevent myocardial wall thinning and improve contractility after implantation into injured myocardium in a 3-dimensional matrix.

Stimulation of paracrine pathways with growth factors enhances embryonic stem cell engraftment and host-specific differentiation in the heart after ischemic myocardial injury.

BACKGROUND: Growth factors play an essential role in organogenesis. We examine the potential of growth factors to enhance cell engraftment and differentiation and to promote functional improvement after transfer of undifferentiated embryonic stem cells into the injured heart. METHODS AND RESULTS: Green fluorescent protein (GFP)-positive embryonic stem cells derived from 129sv mice were injected into the ischemic area after left anterior descending artery ligation in allogenic (BALB/c) mice. Fifty nanograms of recombinant mouse vascular endothelial growth factor, fibroblast growth factor (FGF), and transforming growth factor (TGF) was added to the cell suspension. Separate control groups were formed in which only the growth factors were given. Echocardiography was performed 2 weeks later to evaluate heart function (fractional shortening [FS]), end-diastolic diameter, and left ventricular wall thickness). Hearts were harvested for histology (connexin 43, alpha-sarcomeric actin, CD3, CD11c, major histocompatability complex class I, hematoxylin-eosin). Degree of restoration (GFP-positive graft/infarct area ratio), expression of cardiac markers, host response, and tumorigenicity were evaluated. Cell transfer resulted in improved cardiac function. TGF-beta led to better restorative effect and a stronger expression of connexin 43, alpha-sarcomeric actin, and major histocompatability complex class I. TGF-beta and FGF retained left ventricular diameter. FS was better in the TGF-beta, FGF, and embryonic stem cells-only group compared with left anterior descending artery-ligated controls. Growth factors with cells (TGF-beta, FGF) resulted in higher FS and smaller end-diastolic diameter than growth factors alone. CONCLUSIONS: Growth factors can promote in vivo organ-specific differentiation of early embryonic stem cells and improve myocardial function after cell transfer into an area of ischemic lesion. TGF-beta should be considered as an adjuvant for myocardial restoration with the use of embryonic stem cells.