Mouse Fetal Liver Research Articles

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

Transcriptome of extracellular vesicles released by hepatocytes.

The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Induced maturation of hepatic progenitor cellsin vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Understanding the role of the microenvironment during definitive hemopoietic development

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Abstract 3818: TEL-AML1-induced STAT3 activation is essential for t(12;21)-associated childhood leukemia.

Abstract The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality in pediatric leukemia, present in up to 25% of children with pre-B cell Acute Lymphoblastic Leukemia (ALL). This translocation involves two transcription factors, TEL(ETV6) and AML(RUNX1), both of which have crucial roles in regulating hematopoiesis. Our lab has previously established a list of TEL-AML1 transcriptional target genes using Affymetrix GeneChip global gene expression analysis of mouse fetal liver hematopoietic progenitor cells (HPC) over-expressing the fusion cDNA. This analysis demonstrated deregulated expression of genes associated with STAT3 signaling, known to be one of the most important pathways required for proliferation and maintenance of multipotency in cancer stem cells. In this study, we analyze the importance of STAT3 activity in a mouse model of TEL-AML1 overexpression, in human TEL-AML1 positive leukemia cells and primary human leukemic samples. Our results show that activation STAT3, via RAC1 induction, is necessary for the survival, proliferation and self-renewal of TEL-AML1 positive leukemia through transcriptional regulation of MYC expression. In conclusion we show a novel signaling pathway important for the maintenance of TEL-AML1 leukemia that constitutes a promising novel therapeutic strategy for the treatment of TEL-AML1 leukemia. Citation Format: Maurizio Mangolini, Jasper de Boer, Vanessa Walf-Vorderwülbecke, Rob Pieters, Monique den Boer, Owen Williams. TEL-AML1-induced STAT3 activation is essential for t(12;21)-associated childhood leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3818. doi:10.1158/1538-7445.AM2013-3818

Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.

GATA-1 genome-wide occupancy associates with distinct epigenetic profiles in mouse fetal liver erythropoiesis

We report the genomic occupancy profiles of the key hematopoietic transcription factor GATA-1 in pro-erythroblasts and mature erythroid cells fractionated from day E12.5 mouse fetal liver cells. Integration of GATA-1 occupancy profiles with available genome-wide transcription factor and epigenetic profiles assayed in fetal liver cells enabled as to evaluate GATA-1 involvement in modulating local chromatin structure of target genes during erythroid differentiation. Our results suggest that GATA-1 associates preferentially with changes of specific epigenetic modifications, such as H4K16, H3K27 acetylation and H3K4 di-methylation. Furthermore, we used random forest (RF) non-linear regression to predict changes in the expression levels of GATA-1 target genes based on the genomic features available for pro-erythroblasts and mature fetal liver-derived erythroid cells. Remarkably, our prediction model explained a high proportion of 62% of variation in gene expression. Hierarchical clustering of the proximity values calculated by the RF model produced a clear separation of upregulated versus downregulated genes and a further separation of downregulated genes in two distinct groups. Thus, our study of GATA-1 genome-wide occupancy profiles in mouse primary erythroid cells and their integration with global epigenetic marks reveals three clusters of GATA-1 gene targets that are associated with specific epigenetic signatures and functional characteristics.

Gene Expression Profile and Functionality of ESC-Derived Lin-ckit+Sca-1+ Cells Are Distinct from Lin-ckit+Sca-1+ Cells Isolated from Fetal Liver or Bone Marrow

In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.

Transcription Factor Hes1 Functions As a Tumor Suppressor in MLL-Associated Acute Leukemia

AbstractAbstract 118 Background:The transcription factor Hairy enhancer of split1 (Hes1) is well characterised as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play bivalent roles in tumorigenesis, depending on the cell types. Notch/Hes1 pathway promotes leukemogenesis in T-cell acute lymphoblastic leukemia, and Hes1 also plays an oncogenic role in a mouse model of blastic transformation of chronic myelogenous leukemia. Recent studies, however, demonstrated that Notch signaling serves as a tumor suppressor for chronic myelomonocytic leukemia. Meanwhile, the role of Notch signaling in acute myeloid leukemia (AML) remains to be elucidated. To explore whether Hes1 could possibly play oncongenic or tumor suppressive roles in AML, we focused on a mouse leukemia model established by retroviral transduction with MLL-AF9. Methods:Common myeloid progenitors (CMPs), purified from Hes1−/− or Hes1+/+mouse fetal liver, were retrovirally transduced with MLL-AF9 cDNA. MLL-AF9-transduced cells were subjected to a colony-forming assay, with multiple rounds of replating. These cells were transplanted into lethally irradiated syngenic mice. CMPs from wild-type mouse bone marrow (BM-CMPs) were transduced with MLL-AF9 and dominant negative Hes1 (dnHes1), which lacks WRPW domain, and these cells were transplanted into irradiated mice. Secondary transplantation was performed by infusing leukemic bone marrow cells obtained from the primary recipients. Expression levels of downstream targets were evaluated by cDNA array and quantitative RT-PCR. Result:MLL-AF9-transduced Hes1−/− CMPs formed higher number of colonies at third replating compared to MLL-AF9-transduced Hes1+/+ CMPs, while colony numbers were not different in first and second plating. When infused to irradiated syngenic mice, MLL-AF9/Hes1−/− cells developed leukemia at shorter latencies than MLL-AF9/Hes1+/+ cells (MLL-AF9/Hes1−/−, 7–10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10–14 weeks, n=18; p<0.001). Both MLL-AF9/Hes1−/−- and MLL-AF9/Hes1+/+- derived leukemia cells prepared from bone marrow of the first recipients were transplantable and caused leukemia in the secondary recipients with shorter and similar latencies (MLL-AF9/Hes1−/−, 4 weeks, n=10 vs MLL-AF9/Hes1+/+, 4 weeks, n=5). Mice receiving BM-CMPs transduced with MLL-AF9 and dnHes1 also developed leukemia at shorter latencies than mice receiving BM-CMPs transduced with MLL-AF9 and mock (dnHes1/MLL-AF9 8–10 weeks, n=10 vs mock/MLL-AF9 14–20 weeks, n=10, p<0.01). Leukemia cells induced by both MLL-AF9 and dnHes1 were transplantable and caused leukemia in the secondary recipients with latencies shorter than those induced by MLL-AF9 alone (dnHes1/MLL-AF9 3–4 weeks, n=10 vs mock/MLL-AF9 4–5 weeks, n=5, p<0.05). MLL-AF9/Hes1−/− and MLL-AF9/Hes1+/+ leukemia cells were then compared for mRNA expression by cDNA microarray. Among the genes with different expression levels between MLL-AF9/Hes1−/− and MLL-AF9/Hes1+/+leukemia cells, Meis1 was expressed at significantly higher levels in MLL-AF9/Hes1−/− leukemia cells. Discussion:These results imply that Hes1 serves as a tumor suppressor in MLL-AF9-induced AML. In most of the MLL-associated leukemias, dysregulation of HOX genes is thought to be a mainstay of leukemogenesis. Meis1 is a key cooperative factor for the function of each HOX protein, and thus, presumed to play an important role in development of MLL-associated leukemias. Repression of Meis1 might be among mechanisms how Hes1 functions as a tumor suppressor in MLL-associated leukemia. Disclosures:No relevant conflicts of interest to declare.

Visualizing the Effects of Chemicals On Hematopoietic Stem Cell Engraftment in an Endogenous Niche

Abstract 506Hematopoietic stem and progenitor cells (HSPC) self-renew and give rise to all blood cell types throughout adulthood. Definitive HSPC arise from the hemogenic endothelium of the dorsal aorta, are released into circulation, and then seed an intermediate hematopoietic tissue before colonizing the adult marrow. In mammals this intermediate tissue is the fetal liver, and in the zebrafish it is the caudal hematopoietic tissue (CHT), a vascular plexus in the ventral tail of the embryo. We have generated the first highly specific zebrafish transgenic reporter of HSPC, using the previously described mouse Runx1 +23 kb intronic enhancer driving GFP (or mCherry) fluorescent protein. We have demonstrated that these Runx1+23 positive cells are capable of long-term engraftment and multi-lineage contribution. Using time-lapse live imaging in the embryo, we followed HSPC as they migrate to the CHT niche. Together with endothelial (kdrl(flk1):DsRed2) or stromal (cxcl12a(sdf-1a):DsRed2) reporter lines, we could visualize stem cell behavior directly in the endogenous niche. Upon arrival, HSPC underwent a number of distinct steps to engraftment, including: 1) adherence to the vessel wall; 2) extravasation; 3) migration to the abluminal space; 4) triggering of niche formation—endothelial cells actually remodel around a single HSPC to create a niche; 5) cell division decisions. To determine if endothelial niche formation is conserved in mammals during ontogeny, we performed live imaging of mouse fetal liver explants at embryonic day 11.5, the earliest stage of seeding by HSPC. We observed rare c-kit+/Ly6a(Sca1):GFP+ HSPC become centered in a rosette of CD31+/Lyve1+ sinusoidal endothelial cells. This dynamic remodeling of endothelial cells around an HSPC in the niche was strikingly similar to the cellular behaviors we observed in zebrafish. We hypothesized that chemical genetics could reveal the molecular mechanisms and signaling pathways that are associated with the distinct steps of HSPC engraftment. As proof-of-concept, we tested the CXCR4 antagonist AMD3100 because the CXCR4-CXCL12 receptors and ligands are expressed in the CHT, and found that it suppressed CHT hematopoiesis. Next, we performed a chemical genetic screen by applying ∼2400 individual compounds of known action to zebrafish embryos during colonization of the CHT. We found 40 compounds that increased and 107 compounds that decreased CHT hematopoiesis. Applying selected chemical hits in our live imaging assay we found that certain compounds actually modulated distinct steps during engraftment. We identified a role for sphingosine-1-phosphate signaling during extravasation. We observed that regulators of the transcription factor hypoxia inducible factor (HIF)-1α modulated migration into abluminal spaces. The HIF-1α stabilizer dimethyloxalylglycine (DMOG) promoted migration into hypoxic abluminal spaces, while conversely the HIF-1α inhibitor YC-1 promoted migration into normoxic luminal spaces. We found the plant alkaloid Lycorine promoted endothelial niche formation, creating more locations for HSPC and allowing longer residence times in the CHT. The transforming growth factor (TGF)-β receptor inhibitor SB-431542 increased the rate of HSPC division after they had arrived in the niche. Our studies provide the first genetic approach to understanding engraftment, and the chemicals found could be used therapeutically for patients receiving marrow transplantation. Disclosures:Tamplin:Boston Children’s Hospital: Employment, Patents & Royalties. Zon:Fate Therapeutics: Founder Other.

Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis

Abstract 3199Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development. Disclosures:No relevant conflicts of interest to declare.

Use of Surface Properties to Control the Growth and Differentiation of Mouse Fetal Liver Stem/Progenitor Cell Colonies

Multilayers of poly-l-lysine/poly-l-glutamic acid (PLL/PLGA) were constructed by layer-by-layer deposition on an end-tethered cationic PLL brush film serving as an initial layer. Increasing the number of coupling layers increased the thickness and the hydration of the films, and decreased the films' shear modulus and serum adsorption. These films were used to culture primary mouse fetal liver cells. Fetal liver stem/progenitor cells (FLSPCs) were isolated and maintained on the PLGA-terminal PLL/PLGA surfaces, forming colonies with clear boundaries that were partially attached to the surface, with cross-sectional areas of ~500 to ~2500 μm(2) after 2 days culture. Long-term studies showed that the cluster size of colonies slowly expanded and was correlated with the surface properties. For example, on the thicker films with shear modulus, G, less than 5 kPa, FLSPCs cluster size was constrained within a small distribution with less than 4000 μm(2) of projected area, whereas on the thinner films with G > 30 kPa, clusters were expanded and widely distributed, with projected areas over 4000 um(2). Immunostaining studies suggested that clusters with a small size maintained the self-renewal characteristics of stem cells, while the expanded clusters were clearly the results of spontaneous differentiation, exhibiting hepatocyte-like properties. On PLL-terminal t-(PLL/PLGA) films, which are less favorable for stem cell cultures than PLGA-terminal t-(PLL/PLGA) films, the cluster size distribution was also correlated with the film thickness, with more clusters of small size preserved on the thicker films. We observed that a soft, hydrated, serum-free surface could restrict the FLSPC expansion, resulting in self-maintenance of FLSPC colonies.

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

Immortalized mouse fetal liver stromal cells support growth and maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by co-culture with feeder cells. The feeder cells are important for the growth of hESCs. A novel spontaneously immortalizated mouse fetal liver stromal cell line, named KM3, was isolated from a13.5‑day mouse fetal liver. In this study, we examined whether KM3 cells could be used as feeders to support the growth of hESCs. hESCs cultured on KM3 cells showed a similar proliferation rate and characteristics to mouse embryonic fibroblasts(MEFs) after prolonged culture, including morphology, unlimited and undifferentiated proliferative ability, maintenance of normal karyotypes, formation of embryoid bodies invitro and typically immature teratomas invivo. Our results indicate that the immortalized KM3 cell line has the potential to support the growth and maintenance of hESCs. The cell line may be used for the large-scale expansion of hESCs in a low-cost and less labor-intensive manner.

Immunomagnetic exclusion of E-cadherin-positive hepatoblasts in fetal mouse liver cell cultures impairs morphogenesis and gene expression of sinusoidal endothelial cells

Previous studies have shown that various cell-cell interactions between hepatoblasts and nonparenchymal cells, including sinusoidal endothelial cells and stellate cells, are indispensable for the development of fetal murine hepatic architecture. The present study was undertaken to determine the effects of hepatoblasts on the sinusoidal structural formation using a culture system of fetal mouse livers. Primitive sinusoidal structures extensively developed in fetal livers, and were composed of LYVE-1- and PECAM-1-positive endothelial cells, desmin-positive stellate cells and F4/80-positive macrophages. When fetal liver cells at 12.5 days of gestation were cultured in vitro, hepatoblasts spread on glass slides and gave rise to hepatocytes on day 5. Desmin-positive stellate cells also spread on the glass slides. PECAM-1-positive endothelial cells became slender and developed into anastomosing capillary networks. When fetal liver cells were cultured without hepatoblasts, which were excluded by an immunomagnetic method using anti-E-cadherin antibodies, endothelial cells had impaired growth and capillary formation. These results demonstrated that capillary formation of endothelial cells was induced by the presence of hepatoblasts. VEGF and the conditioned medium containing humoral factors produced by hepatoblasts/hepatocytes did not induce capillary formation of endothelial cells in cultures of nonparenchymal cells, although they significantly increased the number of endothelial cells on the glass slides. The presence of hepatoblasts also significantly stimulated expression of CD32b mRNA, which is a sinusoidal endothelial marker. Hepatoblasts may work as a positive stimulator of sinusoid morphogenesis and maturation in liver development, in which a signal other than VEGF may play a decisive role, together with VEGF.

Hormonal and Chemical Regulation of Paraoxonases in Mice

In humans and rodents, paraoxonase (PON/Pon) 1 expression and activity in livers and serum are higher in females than in males, and some drugs increase paraoxonase's expression. However, the underlining mechanisms of gender-divergent expression and chemical regulation of Pon1 remain largely unknown. The present study determined the regulatory mechanisms contributing to gender-divergent and chemically altered Pon expression in mouse livers. Pon1 mRNA was much more abundant in the livers of mice than other tissues, with higher levels in female livers than male livers at mRNA and protein levels. Pon2 mRNA was ubiquitously expressed in mouse tissues, but minimally in mouse liver. Pon3 mRNA was most abundant in mouse lung and liver and less abundant in other tissues. Pon1 mRNA was lowest in fetal liver, markedly increased at parturition, and remained relatively constant thereafter. Pon2 and Pon3 mRNA are highly expressed in fetal liver and decreased after birth. Male-pattern growth hormone (GH) administration in hypophysectomized and lit/lit mice decreased Pon1 expression. Sex hormones and female-pattern GH administration had no effect on Pon1 expression, indicating the importance of male-pattern GH in regulating Pon1. Aryl hydrocarbon receptor, pregnane X receptor, and NF-E2-related factor activators had no effect on Pon1 mRNA. A constitutive androstane receptor (CAR) activator decreased Pon1 expression in wild-type but not CAR-null mice. In conclusion, Pon1 mRNA was most abundant in adult mouse livers, whereas Pon2 and Pon3 mRNAs were most abundant in fetal mouse livers. Female-predominant Pon1 expression in mouse livers is caused by the inhibitory effects of male-pattern GH secretion, and CAR activation decreases Pon1 expression.

The genomic and proteomic blueprint of mouse megakaryocytes derived from embryonic stem cells

Platelets are specialized cells, produced by megakaryocytes (MKs) in the bone marrow, which represent the first defense against hemorrhage. There are many diseases where platelet production or function is impaired, with severe consequences for patients. Therefore, new insights into the process of MK differentiation and platelet formation would have a major impact on both basic and clinical research. Embryonic stem (ES) cells represent a good in vitro model to study the differentiation of MKs, with the possibility of being genetically engineered and constituting an unlimited source of MKs. However, lack of knowledge about the molecular identity of ES-derived MKs (ES-MKs) may prevent any further development and application of this model. This paper presents the first comprehensive transcriptional and proteome profile analyses of mouse ES-MKs in comparison with MKs derived from mouse fetal liver progenitors (FL-MKs). In ES-MKs we found a down-regulation of cytoskeleton proteins, specific transcription factors and membrane receptors at both transcriptional and protein levels. At the phenotypic level, this molecular blueprint was displayed by ES-MKs' lower polyploidy, lower nuclear/cytoplasm ratio and reduced capacity to form proplatelets and releasing platelets. Overall our data demonstrate that ES-MKs represent a useful model to clarify many aspects of both MK physiology and pathological conditions where impaired MK functions are related to defective MK development, as in inherited thrombocytopenias and primary myelofibrosis.

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

Circadian Rhythms of Fetal Liver Transcription Persist in the Absence of Canonical Circadian Clock Gene Expression Rhythms In Vivo

The cellular circadian clock and systemic cues drive rhythmicity in the transcriptome of adult peripheral tissues. However, the oscillating status of the circadian clocks in fetal tissues, and their response to maternal cues, are less clear. Most clock genes do not cycle in fetal livers from mice and rats, although tissue level rhythms rapidly emerge when fetal mouse liver explants are cultured in vitro. Thus, in the fetal mouse liver, the circadian clock does not oscillate at the cellular level (but is induced to oscillate in culture). To gain a comprehensive overview of the clock status in the fetal liver during late gestation, we performed microarray analyses on fetal liver tissues. In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver. Nevertheless, JTK_CYCLE analysis identified some transcripts in the fetal liver that were rhythmically expressed, albeit at low amplitudes. Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes. These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver. These results raise important questions on the role of the circadian clock, or lack thereof, during ontogeny.

Transcriptome Comparison between Fetal and Adult Mouse Livers: Implications for Circadian Clock Mechanisms

Microarray transcriptome analyses of fetal mouse liver did not detect circadian expression rhythms of clock genes or clock-controlled genes, although some rhythmic transcripts that were likely not driven by endogenous cellular clocks were identified. This finding reveals a key distinction between the circadian oscillators in fetal and adult mouse livers. Thus, in this study, the transcriptomes of fetal and adult livers were systematically compared to identify differences in the gene expression profiles between these two developmental stages. Approximately 1000 transcripts were differentially enriched between the fetal and adult livers. These transcripts represent genes with cellular functions characteristic of distinct developmental stages. Clock genes were also differentially expressed between the fetal and adult livers. Developmental differences in liver gene expression might have contributed to the differences in oscillation status and functional states of the cellular circadian clock between fetal and adult livers.