Zhuan Li

Mouse Embryonic Head as a Site for Hematopoietic Stem Cell Development

In the mouse embryo, the aorta-gonad-mesonephros (AGM) region is considered to be the sole location for intraembryonic emergence of hematopoietic stem cells (HSCs). Here we report that, in parallel to the AGM region, the E10.5-E11.5 mouse head harbors bona fide HSCs, as defined by long-term, high-level, multilineage reconstitution and self-renewal capacity in adult recipients, before HSCs enter the circulation. The presence of hemogenesis in the midgestation head is indicated by the appearance of intravascular cluster cells and the blood-forming capacity of a sorted endothelial cell population. In addition, lineage tracing via an inducible VE-cadherin-Cre transgene demonstrates the hemogenic capacity of head endothelium. Most importantly, a spatially restricted lineage labeling system reveals the physiological contribution of cerebrovascular endothelium to postnatal HSCs and multilineage hematopoiesis. We conclude that the mouse embryonic head is a previously unappreciated site for HSC emergence within the developing embryo.

Tracing haematopoietic stem cell formation at single-cell resolution

Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45− and CD45+ pre-HSCs in the aorta–gonad–mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications.

ATF4 plays a pivotal role in the development of functional hematopoietic stem cells in mouse fetal liver

The fetal liver (FL) serves as a predominant site for expansion of functional hematopoietic stem cells (HSCs) during mouse embryogenesis. However, the mechanisms for HSC development in FL remain poorly understood. In this study, we demonstrate that deletion of activating transcription factor 4 (ATF4) significantly impaired hematopoietic development and reduced HSC self-renewal in FL. In contrast, generation of the first HSC population in the aorta-gonad-mesonephros region was not affected. The migration activity of ATF4(-/-) HSCs was moderately reduced. Interestingly, the HSC-supporting ability of both endothelial and stromal cells in FL was significantly compromised in the absence of ATF4. Gene profiling using RNA-seq revealed downregulated expression of a panel of cytokines in ATF4(-/-) stromal cells, including angiopoietin-like protein 3 (Angptl3) and vascular endothelial growth factor A (VEGFA). Addition of Angptl3, but not VEGFA, partially rescued the repopulating defect of ATF4(-/-) HSCs in the culture. Furthermore, chromatin immunoprecipitation assay in conjunction with silencing RNA-mediated silencing and complementary DNA overexpression showed transcriptional control of Angptl3 by ATF4. To summarize, ATF4 plays a pivotal role in functional expansion and repopulating efficiency of HSCs in developing FL, and it acts through upregulating transcription of cytokines such as Angptl3 in the microenvironment.

Tissue damage negatively regulates LPS-induced macrophage necroptosis

Infection is a common clinical complication following tissue damage resulting from surgery and severe trauma. Studies have suggested that cell pre-activation by antecedent trauma/tissue damage profoundly impacts the response of innate immune cells to a secondary infectious stimulus. Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that control cell release of inflammatory mediators from important innate immune executive cells such as macrophages (Mφ), which critically regulate the progress of inflammation. In this study, we investigated the mechanism and role of trauma/tissue damage in the regulation of LPS-induced Mφ necroptosis using a mouse model simulating long-bone fracture. We demonstrate that LPS acting through Toll-like receptor (TLR) 4 promotes Mφ necroptosis. However, necroptosis is ameliorated by high-mobility group box 1 (HMGB1) release from damaged tissue. We show that HMGB1 acting through cell surface receptor for advanced glycation end products (RAGE) upregulates caveolin-1 expression, which in turn induces caveolae-mediated TLR4 internalization and desensitization to decrease Mφ necroptosis. We further show that RAGE-MyD88 activation of Cdc42 and subsequent activation of transcription factor Sp1 serves as a mechanism underlying caveolin-1 transcriptional upregulation. These results reveal a previous unidentified protective role of damage-associated molecular pattern (DAMP) molecules in restricting inflammation in response to exogenous pathogen-associated molecular pattern molecules.

Interleukin-3 promotes hemangioblast development in mouse aorta-gonad-mesonephros region

Background The hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0–7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined. Design and Methods The BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel- or OP9-based methods were employed to evaluate endothelial progenitor activity. Results Functionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19+ B lymphocytes, and CD3+TCRβ+ T lymphocytes. Meanwhile, the BL-CFC-derived adherent cells generated CD31+ tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31+, endomucin+ and CD105+ subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis. Conclusions The hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.

Endothelial Smad4 restrains the transition to hematopoietic progenitors via suppression of ERK activation

In mouse mid-gestational embryos, definitive hematopoietic stem progenitor cells are derived directly from a very small proportion of the arterial endothelium. However, the physiological mechanisms restraining excessive endothelial-hematopoietic transition remain elusive. We show here that genetic deletion of Smad4 from the endothelium stage (using Tie2-Cre), but not from embryonic hematopoietic cells (using Vav-Cre), leads to a strikingly augmented emergence of intra-arterial hematopoietic clusters and an enhanced in vitro generation of hematopoietic progenitors, with no increase in the proliferation and survival of hematopoietic cluster cells. This finding indicates a temporally restricted negative effect of Smad4 on the endothelial to hematopoietic progenitor transition. Furthermore, the absence of endothelial Smad4 causes an increased expression of subaortic bone morphogenetic protein 4 and an activation of aortic extracellular signal-regulated kinase, thereby resulting in the excessive generation of blood cells. Collectively, our data for the first time identify a physiological suppressor that functions specifically during the transition of endothelial cells to hematopoietic progenitors and further suggest that endothelial Smad4 is a crucial modulator of the subaortic microenvironment that controls the hematopoietic fate of the aortic endothelium.

Subregional localization and characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head

Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Genetic and live imaging data have supported this. Recently, the embryonic head has been shown to contain fully functional hematopoietic stem cells (HSC). By lineage tracing, cerebrovascular specific endothelial cells were shown to contribute to the postnatal mouse hematopoietic system. Since Ly6aGFP is a marker of all HSCs, some hematopoietic cluster cells and hemogenic endothelial cells in the midgestation mouse aorta, we examine here whether embryonic head HSCs and vascular endothelial cells are positive for this marker. Whereas some head vasculature, single hematopoietic cells and all HSCs are Ly6aGFP expressing, we do not find clusters of hematopoietic cells emerging from the cerebrovasculature that are characteristic of endothelial-to-hematopoietic transition.

Migration of dorsal aorta mesenchymal stem cells induced by mouse embryonic circulation

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid‐gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45−CD31−Flk‐1−CD44+CD29+), expressed perivascular markers (α‐SMA+NG2+PDGFRβ+PDGFRα+), and had tri‐lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5–E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA‐MSCs through platelet‐derived growth factor (PDGF) receptor‐, transforming growth factor‐beta receptor‐, but not basic fibroblast growth factor receptor‐mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood‐ or PDGF‐mediated migration of DA‐derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis. Developmental Dynamics, 2011.

Characterization of hemangioblast in umbilical arteries of mid-gestation mouse embryos

Hemangioblasts are the common precursors of hematopoietic and vascular cells, and are characterized as blast colony-forming cells (BL-CFCs) in vitro. We previously identified BL-CFCs in the mouse aorta–gonads–mesonephros (AGM) region, but not yolk sac, placenta, circulation, or fetal liver. Here, we aim to determine whether BL-CFCs develop in the umbilical arteries (UA) that link the dorsal aorta (sub-region of AGM) and placenta. We find that the UA cells of E11.5 mouse embryos were capable of generating typical blast colonies. On replating, these colonies produced erythroid/myeloid progenitors and B220+ B lymphocytes in vitro, corroborating their definitive hematopoietic nature. They also generated CD31+ or endomucin+ tube-like structures on OP9 stromal cells, showing their endothelial potential. The proximal and distal regions of UA had equal numbers of BL-CFCs. To evaluate whether BL-CFCs can be autonomously maintained or expanded in UA or AGM, in vitro organ culture was performed. Interestingly, the BL-CFC pool in the AGM was significantly amplified, in striking contrast to a decrease in the UA. Taken together, our findings indicate that in addition to the AGM the UA serves as an important, but less supportive, niche for hemangioblast development.