Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes

Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2‐Cre or VE‐cadherin‐Cre constructs to facilitate fate‐mapping of LSECs in liver regeneration. Some YFP‐positive LSECs were observed to convert into hepatocytes following a two‐thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non‐hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte‐like (iHep) cells may provide a new approach to tissue engineering.     

Generation of functional hemangioblasts from human embryonic stem cells

Recent evidence suggests the existence of progenitor cells in adult tissues that are capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Here we describe an efficient and reproducible method for generating large numbers of these bipotential progenitors-known as hemangioblasts-from human embryonic stem (hES) cells using an in vitro differentiation system. Blast cells expressed gene signatures characteristic of hemangioblasts, and could be expanded, cryopreserved and differentiated into multiple hematopoietic lineages as well as into endothelial cells. When we injected these cells into rats with diabetes or into mice with ischemia-reperfusion injury of the retina, they localized to the site of injury in the damaged vasculature and appeared to participate in repair. Injection of the cells also reduced the mortality rate after myocardial infarction and restored blood flow in hind limb ischemia in mouse models. Our data suggest that hES-derived blast cells (hES-BCs) could be important in vascular repair.     

Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein

Development of hematopoietic cells in the aorta-gonad-mesonephros (AGM) region in the midgestation mouse embryo involves a multistep process, sequentially changing from endothelial cell-like cells, including hemangioblasts, into hematopoietic stem cells, progenitors, and/or lineage-committed cells. An adaptor molecule, Lnk, is known to negatively control the production of pro- and pre-B cells and hematopoietic progenitor cells in adult bone marrow. Here we show a role of Lnk in hematopoietic development in the AGM region. Lnk was predominantly expressed in the endothelial cells lining the dorsal aorta at embryonic day 11.5 (E11.5). Overexpression of Lnk in the primary culture of the AGM region at E11.5 suppressed the emergence of CD45+ hematopoietic cells. Point mutation in the SH2 domain of Lnk, which abolishes the binding capability of Lnk to c-Kit upon stimulation with stem cell factor (SCF), led to loss of Lnk-dependent inhibition of hematopoietic cell development in AGM cultures, suggesting Lnk-mediated inhibition of the SCF/c-Kit signaling pathway. In cultured AGM cells from Lnk homozygous mutant mouse embryos, the number of emerged CD45+ cells was 2.5-fold larger than that from heterozygous littermates. Furthermore, aorta cells of E11.5 Lnk homozygous mutant mice also showed enhanced hematopoietic colony-forming activity. Thus, Lnk is a negative regulator of hematopoiesis in the AGM region.     

Using genetically engineered mice for radiation research

The laboratory mouse has been used for many decades as a model system for radiation research. Recent advances in genetic engineering now allow scientists to delete genes in specific cell types at different stages of development. The ability to manipulate genes in the mouse with spatial and temporal control opens new opportunities to investigate the role of genes in regulating the response of normal tissues and tumors to radiation. Currently, we are using the Cre-loxP system to delete genes, such as p53, in a cell-type specific manner in mice to study mechanisms of acute radiation injury and late effects of radiation. Our results demonstrate that p53 is required in the gastrointestinal (GI) epithelium to prevent radiation-induced GI syndrome and in endothelial and/or hematopoietic cells to prevent late effects of radiation. We have also used these genetic tools to generate primary tumors in mice to study tumor response to radiation therapy. These advances in genetic engineering provide a powerful model system to dissect both the mechanisms of normal tissue injury after irradiation and the mechanisms by which radiation cures cancer.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.     

Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization

Adults maintain a reservoir of hematopoietic stem cells that can enter the circulation to reach organs in need of regeneration. We developed a novel model of retinal neovascularization in adult mice to examine the role of hematopoietic stem cells in revascularizing ischemic retinas. Adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein. We performed serial long-term transplants, to ensure activity arose from self-renewing stem cells, and single hematopoietic stem-cell transplants to show clonality. After durable hematopoietic engraftment was established, retinal ischemia was induced to promote neovascularization. Our results indicate that self-renewing adult hematopoietic stem cells have functional hemangioblast activity, that is, they can clonally differentiate into all hematopoietic cell lineages as well as endothelial cells that revascularize adult retina. We also show that recruitment of endothelial precursors to sites of ischemic injury has a significant role in neovascularization.     

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ～ 84% of each lineage was EYFP⁺, and nearly all cells that come from Tie2‐expressing lineages are CD45⁺, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2⁺ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.     

Impaired endothelial proliferation and mesenchymal transition contribute to vascular rarefaction following acute kidney injury

Acute kidney injury induces the loss of renal microvessels, but the fate of endothelial cells and the mechanism of potential vascular endothelial growth factor (VEGF)-mediated protection is unknown. Cumulative cell proliferation was analyzed in the kidney of Sprague-Dawley rats following ischemia-reperfusion (I/R) injury by repetitive administration of BrdU (twice daily) and colocalization in endothelial cells with CD31 or cablin. Proliferating endothelial cells were undetectable for up to 2 days following I/R and accounted for only ～1% of BrdU-positive cells after 7 days. VEGF-121 preserved vascular loss following I/R but did not affect proliferation of endothelial, perivascular cells or tubular cells. Endothelial mesenchymal transition states were identified by localizing endothelial markers (CD31, cablin, or infused tomato lectin) with the fibroblast marker S100A4. Such structures were prominent within 6 h and sustained for at least 7 days following I/R. A Tie-2-cre transgenic crossed with a yellow fluorescent protein (YFP) reporter mouse was used to trace the fate of endothelial cells and demonstrated interstititial expansion of YFP-positive cells colocalizing with S100A4 and smooth muscle actin following I/R. The interstitial expansion of YFP cells was attenuated by VEGF-121. Multiphoton imaging of transgenic mice revealed the alteration of YFP-positive vascular cells associated with blood vessels characterized by limited perfusion in vivo. Taken together, these data indicate that vascular dropout post-AKI results from endothelial phenotypic transition combined with an impaired regenerative capacity, which may contribute to progressive chronic kidney disease.     

Syk and Slp-76 mutant mice reveal a cell-autonomous hematopoietic cell contribution to vascular development

Developmental studies support a common origin for blood and endothelial cells, while studies of adult angiogenic responses suggest that the hematopoietic system can be a source of endothelial cells later in life. Whether hematopoietic tissue is a source of endothelial cells during normal vascular development is unknown. Mouse embryos lacking the signaling proteins Syk and Slp-76 develop abnormal blood-lymphatic endothelial connections. Here we demonstrate that expression of GFPSlp-76 in a subset of hematopoietic cells rescues this phenotype, and that deficient cells confer focal vascular phenotypes in chimeric embryos consistent with a cell-autonomous mechanism. Endogenous Syk and Slp-76, as well as transgenic GFPSlp-76, are expressed in circulating cells previously proposed to be endothelial precursors, supporting a causal role for these cells. These studies provide genetic evidence for hematopoietic contribution to vascular development and suggest that hematopoietic tissue can provide a source of vascular endothelial progenitor cells throughout life.     

SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.     

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2^YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER⁺ and R26-SmoM2^YFP+ mice in this protocol); 2) inducing SmoM2^YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.     

Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice

Both LPL and HL are synthesized in parenchymal cells, are secreted, and bind to endothelial cells. To learn where endothelial lipase (EL) is synthesized in adult animals, the localization of EL in mouse and rat liver was studied by immunohistochemical analysis. Furthermore, to test whether EL could play a role in atherogenesis, the expression of EL in the aorta and liver of apolipoprotein E knockout (EKO) mice was determined. EL in both mouse and rat liver was colocalized with vascular endothelial cells but not with hepatocytes. In contrast, HL was present in both hepatocytes and endothelial cells. By in situ hybridization, EL mRNA was present only in endothelial cells in liver sections. EL was also present at low levels in aorta of normal mice. We fed EKO mice and wild-type mice a variety of diets and determined EL expression in liver and aorta. EKO mice showed significant expression of EL in aorta. EL expression was lower in the liver of EKO mice than in normal mice. Cholesterol feeding decreased EL in liver of both types of mice. In the aorta, EL was higher in EKO than in wild-type mice, and cholesterol feeding had no effect. Together, these data suggest that EL may be upregulated at the site of atherosclerotic lesions and thus could supply lipids to the area.     

Hematopoietic stem cells convert into liver cells within days without fusion

Both plasticity and cell fusion have been suggested to have a role in germ-layer switching. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs) in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2-7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.     

Selective role of a distinct tyrosine residue on Tie2 in heart development and early hematopoiesis

The development of the cardiovascular system and the development of the early hematopoietic systems are closely related, and both require signaling through the Tie2 receptor tyrosine kinase. Although endothelial cells and hematopoietic cells as well as their precursors share common gene expression patterns during development, it remains completely unknown how Tie2 signaling coordinately regulates cardiovascular development and early hematopoiesis in vivo. We show here that mice with a targeted mutation in tyrosine residue 1100 in the carboxyl-terminal tail of Tie2 display defective cardiac development and impaired hematopoietic and endothelial cell development in the paraaortic splanchnopleural mesoderm similar to that seen in Tie2-null mutant mice. Surprisingly, however, unlike Tie2-null mutant mice, mice deficient in signaling through this tyrosine residue show a normal association of perivascular cells with nascent blood vessels. These studies are the first to demonstrate the physiological importance of a single tyrosine residue in Tie2, and they suggest that multiple tyrosine residues in the receptor may coordinate cardiovascular development and early hematopoietic development.     

Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.     

Presence of mast cell precursors in fetal liver of mice

When liver cells obtained from 13- to 18-day embryos of beige (Chediak-Higashi syndrome) mice were transplanted into irradiated normal adult mice, tissue mast cells with giant granules showing beige mouse origin developed in the normal recipient mice. Mast cell precursors seem to have developed earlier in the liver of embryos than mast cells themselves since no mast cells were detectable in any tissues of 13- and 14-day embryos. This result suggests that tissue mast cells originate from hematopoietic tissues not only in adult mice but also in mouse embryos.     

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织的实验研究

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织,探索建立人-小鼠嵌合肝模型方法。15只SCID小鼠,以四氯化碳(CCL4)制备急性肝损伤模型,24h后行2/3肝切除,然后分为三个实验组细胞移植组(7只)、阴性对照组(3只)及空白对照组(5只);将人脐血单个核细胞悬液注入细胞移植组小鼠脾脏内,阴性对照组小鼠脾脏内注入等量磷酸盐缓冲液(PBS),空白对照组不注射细胞悬液和PBS。术后7d、14d及21d取小鼠肝组织观察病理变化、检测人白蛋白(ALB)及细胞角蛋白19(CK19),同时检测小鼠血清及肝组织匀浆中人ALB含量。全部小鼠表现出急性肝损伤组织学特征;细胞移植组小鼠术后7d、14d、21d肝组织内均见大量人ALB及CK19阳性表达细胞,血清及肝组织匀浆可检测出人ALB;阴性对照组小鼠肝组织未见人ALB及CK19阳性表达,血清及肝组织匀浆中未检测出人ALB。人脐血单个核细胞在部分肝切除的急性肝损伤小鼠肝组织内可大量分化为人肝细胞及胆管细胞,在建立模型方面已取得关键突破。     

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ～2 d of practical work.     

Enhanced regulatory T cell activity is an element of the host response to injury

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-beta1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.