Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells

The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture. Tümen Mansuroglu and József Dudás contributed equally to this work.     

BMP4/Smad5 dependent stress erythropoiesis is required for the expansion of erythroid progenitors during fetal development

The rapid growth of the embryo places severe demands on the ability of the cardiovascular system to deliver oxygen to cells. To meet this need, erythroid progenitors rapidly expand in the fetal liver microenvironment such that by E14.5, erythropoiesis predominates in the fetal liver. In this report we show that the BMP4/Smad5 dependent stress erythropoiesis pathway plays a key role in the expansion of erythroid progenitors in the fetal liver. These data show that the fetal liver contains two populations of erythroid progenitors. One population resembles the steady state erythroid progenitors found in the adult bone marrow. While the second population exhibits the properties of stress erythroid progenitors found in adult spleen. Here we demonstrate that defects in BMP4/Smad5 signaling preferentially affect the expansion of the stress erythroid progenitors in the fetal liver leading to fetal anemia. These data suggest that steady state erythropoiesis is unable to generate sufficient erythrocytes to maintain the rapid growth of the embryo leading to the induction of the BMP4 dependent stress erythropoiesis pathway. These observations underscore the similarities between fetal erythropoiesis and stress erythropoiesis.     

Retinoic acid, hypoxia, and GATA factors cooperatively control the onset of fetal liver erythropoietin expression and erythropoietic differentiation

The cytokine erythropoietin (Epo) is an essential factor promoting the survival, proliferation, and differentiation of erythroid progenitor cells. Epo expression and the initial phase of definitive erythropoietic differentiation in the fetal liver (E9-E12) are compromised in mouse embryos lacking the retinoic acid receptor RXRalpha. Our previous work demonstrated that the Epo gene is a direct target of retinoic acid action, via a retinoic acid receptor binding site in the Epo gene enhancer. However, Epo expression and erythropoietic differentiation become normalized in RXRalpha mutants from E12. In this study, we have investigated the molecular mechanisms underlying the transition in Epo gene regulation from RXRalpha-dependence to RXRalpha-independence. We find that three independent regulatory components are required for high level Epo expression in the early fetal liver: ligand-activated retinoic acid receptors, the hypoxia-regulated factor HIF1, and GATA factors. By E11.5, the fetal liver is no longer hypoxic, and retinoic acid signaling is no longer active; Epo expression from E11.5 onward is enhancer-independent, and is driven instead by basal promoter elements that provide a sufficient level of expression to support further erythropoietic differentiation.     

Hepatic lineages isolated from developing rat liver show different ways of maturation

Immunocytochemical analysis revealed that different hepatic cell types exist during liver development: (i). cells co-expressing the stem-cell marker Thy1 and the hepatic lineage marker CK-18 and (ii). cells only expressing CK-18 (hepatoblasts). In this study we separated the different hepatic cells and analyzed gene-expression and phenotype. Fetal rat livers were digested by collagenase solution. OX43- and OX44-positive hematopoietic cells were depleted and Thy1-positive cells were enriched using Magnetic cell sorting. The different cell compartments were analyzed by RT-PCR and immunocytochemistry for Thy1, CK-18, AFP, and albumin. Hepatoblasts expressed albumin at all times and AFP in the early stages. Thy1-enriched cells expressed CK-18 at all times, albumin in the early, and AFP in the late stages. Thy1-positive cells from fetal livers express liver specific genes. The data suggest that Thy1-positive hepatic cells develop towards hepatic stem cells, and hepatoblasts develop towards mature hepatocytes of the adult liver.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

Immunohistochemical analyses of cell–cell interactions during hepatic organoid formation from fetal mouse liver cells cultured in vitro

Cell–cell interactions among cell types constituting the fetal liver such as hepatoblasts, stellate cells and endothelial cells lead to functional lobule development. The present study was undertaken to investigate hepatic histogenesis in the primary culture of E12.5 mouse livers, including cell–cell and cell–matrix interactions. Fetal livers were dispersed with protease treatment and cultured for 5 days. Cellular adhesion of each hepatic cell type, gene expression and extracellular matrix deposition were analyzed by immunohistochemistry and immunoblotting. Immunohistochemical analysis demonstrated that the primary culture of fetal liver cells contained at least hepatoblasts, mesenchymal cells, endothelial cells, hemopoietic cells and Kupffer cells. Although hepatoblasts, mesenchymal cells, and endothelial cells aggregated separately in the initial step, they then formed a spheroid together, adhering to the glass slide, which led to the formation of flattened hepatic organoids. Hepatoblasts more preferentially adhered to mesenchymal cells than endothelial cells. Several extracellular matrix depositions were seen in aggregates consisting of at least hepatoblasts and mesenchymal cells within 12 h, but were poor in those lacking hepatoblasts. These data show that the primary culture of fetal liver cells contains most cell types constituting fetal livers, and may be useful for studying cell–cell interactions during liver development.     

alpha-Fetoprotein production and erythropoiesis in cell cultures of human fetal liver.

alpha-Fetoprotein and the synthesis of heme associated with hemoglobin were measured simultaneously in short-term cultures of human fetal liver cells to correlate the relationship of alpha-fetoprotein to erythroid cell function. Both synthetic processes decreased exponentially during the first 5 days of culture. The use of media supplemented with different batches of fetal calf serum and porcine portal vein serum indicated that the optimal conditions for the production of alpha-fetoprotein were different from those required for the synthesis of heme associated with hemoglobin. Moreover, the alpha-fetoprotein-producing cells could be separated from erythroid cells after velocity sedimentation in Ficoll gradients. Although it is well known that erythropoiesis and alpha-fetoprotein production occur simultaneously during ontogenesis, alpha-fetoprotein itself (0.01-100 micron g/ml) did not stimulate heme synthesis in liver erythroid cells. Erythropoietin did not stimulate alpha-fetoprotein production. It is concluded that there is no cause-effect relationship between alpha-fetoprotein production and erythroid cell fuction in human fetal liver cells and that the two processes occur independently in different cell types.     

The Wilms' tumor gene WT1 is a common marker of progenitor cells in fetal liver

It is well known that the Wilms' tumor gene WT1 plays an important role in cell proliferation and differentiation, and in organ development. In this study, to examine the role of the WT1 gene in lineage determination, fetal liver cells from LacZ-transgenic mice, in which WT1 expression was marked by the expression of the LacZ gene driven by WT1 promoter, were FACS-sorted according to LacZ expression of high (LacZ(++)) or undetectable (LacZ(-)) levels, which paralleled endogenous WT1 expression levels. LacZ(++) fetal liver cells were enriched by hepatocyte and endothelial progenitor cells. These results indicated that WT1 expression is a common marker of both hepatocyte and endothelial progenitors. These results also implied a role of the WT1 gene in lineage determination.     

Ultrastructural study of a cytoplasmic bridge connecting a pair of erythroblasts in mice

Summary A unique cytoplasmic connection between erythroblasts was studied by electron microscopy in mouse hemopoietic tissues (fetal liver, fetal and neonatal spleen and adult bone marrow). Many pairs of interphase erythroblasts were connected by a cytoplasmic bridge that was very thin and sometimes long in comparison with telophase bridges. The stage of maturation of the cells in a pair was similar. Small numbers of microtubules ran along the cytoplasmic bridge; a mid-body was not seen. The plasma membrane at approximately the middle of the bridge bulged to form a ring-shaped ridge filled with dense amorphous substances; this was called a bulging ring. Thus, the cytoplasmic bridge between erythroblasts did not morphologically correspond to the telophase bridge in the usual cytokinesis. Cytoplasmic bridges were observed in various differentiating stages of erythroblasts, whereas other cell types of the hemopoietic lineage did not have such a bridge. The cytoplasmic bridge is unique to erythroblasts and provides an evidence for the atypical cytokinesis of the erythroblastic lineage.     

胎肝滤液诱导大鼠胎盘间充质干细胞向肝细胞的分化

目的探讨PDMSCs向肝细胞增殖和分化的体外培养条件及方法。方法孕20 d的大鼠无菌条件下取胎盘,经胶原酶消化、密度离心、贴壁筛选法分离培养胎盘源间充质干细胞,并对其表面抗原进行鉴定。在体外培养体系中加入胎肝滤液,模拟体内肝脏微环境,诱导PDMSCs向肝细胞定向分化,以免疫细胞化学检测干细胞标志物;PAS检测糖原表达。结果在体外培养条件下,PDMSCs贴壁生长为成纤维样细胞,CD44表面标志物检测阳性;PDMSCs经胎肝滤液诱导14d时细胞呈现圆形、卵圆形的特征性改变,AFP、CK19表达阳性。结论胎肝滤液能够诱导PDMSCs定向分化为肝细胞样细胞。     

肝再生增强因子研究进展

肝再生增强因子是新近克隆的蛋白质因子,能特异地刺激肝源细胞的增殖,并对ＣＣl4所引起的急性肝衰竭有效治作用。本文综述了肝再生增强因子的发现、基因克隆及组织分布等。目前已开始了该因子的基因工程产品研制,它有望成为一种治疗肝病的新药。     

ABCC13, an unusual truncated ABC transporter,is highly expressed in fetal human liver

In the present study, we have cloned the cDNA of ABCC13, a novel ABC transporter, from the cDNA library of adult human placenta. The ABCC13 gene spans approximately 70kb on human chromosome 21q11.2 and consists of 14 exons. The open reading frame of the ABCC13 cDNA encodes a peptide consisting of 325 amino acid residues. The amino acid sequence corresponding to putative membrane-spanning domains was remarkably similar to ABCC1, ABCC2, ABCC3, and ABCC6. The ABCC13 gene was expressed in the fetal liver at the highest level among the organs studied. While ABCC13 was expressed in the bone marrow, its expression in peripheral blood leukocytes of adult humans was much lower and no detectable levels were observed in differentiated hematopoietic cells. The expression of ABCC13 in K562 cells decreased during cell differentiation induced by TPA. These results suggest that the expression of human ABCC13 is related with hematopoiesis.     

Effects on induction of tyrosine aminotransferase in fetal mouse liver in vitro of prednisolone,insulin and thyroxine

The hormonal requirements for formation of tyrosine aminotransferase (EC 2.6.1.5) in fetal mouse liver were investigated in organ culture using chemically defined medium. The hormones tested were insulin, thyroxine and prednisolone. Prednisolone alone resulted in a two-fold increase in tyrosine amino-transferase activity in explanted liver in hormone-free medium on day 6, and its effect was dose dependent, but neither insulin nor thyroxine alone induced the enzyme. Addition of prednisolone plus thyroxine and prednisolone plus insulin increased the enzyme activity 1.4- and 1.3-fold, respectively, over that of explants with prednisolone alone. These three hormones together had the greatest effect, causing induction of 1.5-fold more activity than that with prednisolone plus insulin or plus thyroxine. The three hormones were not all needed continuously during the culture period: prednisolone and insulin were required during the early part of cultivation and thyroxine during the later part. The effects of these hormones were blocked by actinomycin D or puromycin, suggesting that these hormones increase de novo synthesis of tyrosine aminotransferase. Phase-contrast microscopy showed that prednisolone stimulated liver epithelial cell outgrowth, probably acting with insulin.     

Haplotype mapping of a major quantitative-trait locus for fetal hemoglobin production, on chromosome 6q23.

Fetal hemoglobin (Hb F) and fetal cell (FC) levels in adults show considerable variation and are influenced by several genetic variants; the major determinants appear to be unlinked to the beta-globin gene cluster. Recently, a trans-acting locus controlling Hb F and FC production has been mapped to chromosome 6q23 in an Asian Indian kindred that includes individuals with heterocellular hereditary persistence of Hb F (HPFH) associated with beta thalassemia. We have extended the kindred by 57 members, bringing the total studied to 210, and have saturated the region with 26 additional markers. Linkage analysis showed tight linkage of the quantitative-trait locus (QTL) to the anonymous markers D6S976 (LOD score 11.3; recombination fraction .00) and D6S270 (LOD score 7.4; recombination fraction .00). Key recombination events now place this QTL within a 1-2-cM interval spanning approximately 1.5 Mb between D6S270 and D6S1626. Furthermore, haplotype analysis has led to a reevaluation of the genealogy and to the identification of additional relationships in the kindred.     

Positive effects of cryopreserved adult or fetal liver cell transplants on hypercholesterolemia and hepatic antioxidant defenses in cholesterol-fed rabbits

The liver plays a central role in lipid metabolism and the pathophysiology of many lipid disorders leads in turn to liver cell injury. Adult hepatocyte transplants provide well-recognized metabolic support, whilst hepatic stem cells may promote liver regeneration and repair, but in both cases, any clinical application would require low temperature banking of the cells. A model of dietary hypercholesterolemia was established in rabbits over 5 months, and transplants of cryopreserved adult hepatocytes (CH) and cryopreserved fetal liver cells (CFLC) were compared to Sham transplants. Cryopreservation was performed by a two-step freezing protocol using 1.5mol/l dimethyl sulfoxide (Me(2)SO). Serum contents of cholesterol lipid classes were measured during the subsequent 4 weeks, in addition to markers of serum and liver oxidative stress. Both CH and CFLC transplantation resulted in a decrease of serum lipids during the 1st week after transplantation. The effect of CH was limited to the 1st week, but CFLC provided a sustained lipid-lowering effect over the 4 weeks. The ultimate outcome of CFLC transplantation by the end of 4 weeks was more pronounced and statistically significant for both serum total cholesterol (0.15+/-0.05 versus 3.65+/-1.4mmol/l) and high-density lipoprotein-cholesterol (0.04+/-0.01 versus 0.56+/-0.06mmol/l) compared to Sham transplants (p<0.05 in both cases). CFLC transplantation also normalized hepatic tissue antioxidant defenses, namely an increase in reduced glutathione content, and enzyme activities for catalase and glutathione reductase (all significantly higher at p<0.05 than in Sham transplants) by 4 weeks.     

Phosphoenolpyruvate synthesis by fetal guinea-pig liver mitochondria

Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4–6 nmol/min per mg protein with 2 mM malate, succinate, and α-ketoglutarate as substrates. These rates were 90–110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.