HSC-independent definitive hematopoiesis persists into adult life.

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Engineered models for placental toxicology: Emerging approaches based on tissue decellularization.

Recent increases in prescriptions and illegal drug use as well as exposure to environmental contaminants during pregnancy have highlighted the critical importance of placental toxicology in understanding and identifying risks to both mother and fetus. Although advantageous for basic science, current in vitro models often fail to capture the complexity of placental response, likely due to their inability to recreate and monitor aspects of the microenvironment including physical properties, mechanical forces and stiffness, protein composition, cell-cell interactions, soluble and physicochemical factors, and other exogenous cues. Tissue engineering holds great promise in addressing these challenges and provides an avenue to better understand basic biology, effects of toxic compounds and potential therapeutics. The key to success lies in effectively recreating the microenvironment. One strategy to do this would be to recreate individual components and then combine them. However, this becomes challenging due to variables present according to conditions such as tissue location, age, health status and lifestyle. The extracellular matrix (ECM) is known to influence cellular fate by working as a storage of factors. Decellularized ECM (dECM) is a recent tool that allows usage of the original ECM in a refurbished form, providing a relatively reliable representation of the microenvironment. This review focuses on using dECM in modified forms such as whole organs, scaffold sheets, electrospun nanofibers, hydrogels, 3D printing, and combinations as building blocks to recreate aspects of the microenvironment to address general tissue engineering and toxicology challenges, thus illustrating their potential as tools for future placental toxicology studies.

The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells.

BACKGROUND: DNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. RESULTS: We determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs. CONCLUSIONS: GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

B1 lymphocytes develop independently of Notch signaling during mouse embryonic development.

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.

Hemogenic Endothelial Cells Can Transition to Hematopoietic Stem Cells through a B-1 Lymphocyte-Biased State during Maturation in the Mouse Embryo.

Precursors of hematopoietic stem cells (pre-HSCs) have been identified as intermediate precursors during the maturation process from hemogenic endothelial cells to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic day 10.5. Although pre-HSCs acquire an efficient adult-repopulating ability after ex vivo co-culture, their native hematopoietic capacity remains unknown. Here, we employed direct transplantation assays of CD45-VE-cadherin(VC)+KIT+(V+K+) cells (containing pre-HSCs) into immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that freshly isolated V+K+ cells exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multilineage repopulating capacity. Additionally, B cell colony-forming assays demonstrated the predominant B-1 progenitor colony-forming ability of these cells; however, increased B-2 progenitor colony-forming ability emerged after co-culture with Akt-expressing AGM endothelial cells, conditions that support pre-HSC maturation into HSCs. Our studies revealed an unexpected B-1 lymphocyte bias of the V+K+ population and acquisition of B-2 potential during commitment to the HSC fate.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

It is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

What do the lineage tracing studies tell us? Consideration for hematopoietic stem cell origin, dynamics, and leukemia-initiating cells.

The recent advance of technologies enables us to trace the cell fate in vivo by marking the cells that express the gene of interest or by barcoding them at a single cell level. Various tamoxifen-inducible Cre-recombinase mice combined with Rosa-floxed lines are utilized. In this review, with the results revealed by lineage tracing assays, we re-visit the long-standing debate for the origin of hematopoietic stem cells in the mouse embryo, and introduce the view of native hematopoiesis, and possible leukemic-initiating cells emerged during fetal stages.

Mechanism of hematopoiesis and vasculogenesis in mouse placenta.

The placenta plays a major role in the development of blood cells in the mouse and human embryo. Hematopoiesis and vasculogenesis in placenta are two closely interconnected processes. The mouse model has been widely used to study placental hematopoiesis. During mid-gestation, mouse placenta generate blood cells and support the proliferation, maturation and erythroid differentiation of hematopoietic cells within two major vascular niches in the labyrinth. Here, we review the vasculogenesis in chorioallantoic mouse placenta and the current knowledge on hematopoietic activity, niche and origin in mice placenta.

Localization of transient immature hematopoietic cells to two distinct, potential niches in the developing mouse placenta.

Previous studies have shown that human and mouse placentas have hematopoietic potential during mid-gestation. In this investigation, we used histological and immunohistological approaches to visualize hematopoietic cells in mouse placenta between 9.5 and 12.5 days of gestation (gd), identifying their topography and niche. Putative hematopoietic foci were present on 10.5 and 11.5 gd but not 9.5 or 12.5 gd and was restricted to the placental labyrinth. Two major niches each with distinctive hematopoietic cell clusters were present. One type of hematopoietic cell cluster involved the chorioallantoic vasculature and fetal vessels near the chorionic plate. These clusters resembled the hematopoietic stem cells produced by large embryonic arteries such as aorta that persist in postnatal marrow. The other type of hematopoietic cell cluster identified was at the opposite side of labyrinth next to the junctional zone and was composed of erythropoietic foci. Our results suggest that mouse placenta not only produces hematopoietic stem/progenitor cells but also a second wave of primitive erythrocytes that may support a rapid, mid-pregnancy, fetal growth trajectory. Our data also point to a close relationships in the origins of hematopoietic and endothelial cells within placenta.