Debiao Zhao

Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development In Vivo

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three‐stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore, we have provided evidence that the hESC‐derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen, and generation and secretion of plasma proteins. More importantly, the hESC‐derived hepatocytes express several members of cytochrome P450 isozymes, and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes, which may be useful as an in vitro system for toxicity screening in drug discovery.

Stably Transfected Human Embryonic Stem Cell Clones Express OCT4‐Specific Green Fluorescent Protein and Maintain Self‐Renewal and Pluripotency

Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos; they can be cultured indefinitely and differentiated into many cell types in vitro. These cells therefore have the ability to provide insights into human disease and provide a potential unlimited supply of cells for cell‐based therapy. Little is known about the factors that are important for maintaining undifferentiated hESCs in vitro, however. As a tool to investigate these factors, transfected hES clonal cell lines were generated; these lines are able to express the enhanced green fluorescent protein (EGFP) reporter gene under control of the OCT4 promoter. OCT4 is an important marker of the undifferentiated state and a central regulator of pluripotency in ES cells. These OCT4‐EGFP clonal cell lines exhibit features similar to parental hESCs, are pluripotent, and are able to produce all three embryonic germ layer cells. Expression of OCT4‐EGFP is colocalized with endogenous OCT4, as well as the hESC surface antigens SSEA4 and Tra‐1‐60. In addition, the expression is retained in culture for an extensive period of time. Differentiation of these cells toward the neural lineage and targeted knockdown of endogenous OCT4 expression by RNA interference downregulated the EGFP expression in these cell lines, and this correlates closely with the reduction of endogenous OCT4 expression. Therefore, these cell lines provide an easy and noninvasive method to monitor expression of OCT4 in hESCs, and they will be invaluable for studying not only OCT4 function in hESC self‐renewal and differentiation but also the factors required for maintenance of undifferentiated hESCs in culture.

Somatic sex identity is cell autonomous in the chicken.

In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

Evidence for avian cell autonomous sex identity (CASI) and implications for the sex-determination process?

For the majority of animals, males and females are obviously different in terms of appearance, behaviour and physiology, and until recently, these differences were considered to be the result of hormone actions. However, there is now considerable evidence that the development of some sexually dimorphic structures/behaviours is a function of properties inherent to male and female cells (hormone independent). The relative contribution of hormones and cellular identity to the development of the phenotype is not clear and is likely to vary from species to species. The study of gynandromorph birds and chimeric embryos has greatly assisted efforts to distinguish between the effects of hormones and inherent cellular factors on phenotype. It is now clear that in birds, male/female differences are not primarily the result of hormone action and that male and female somatic cells possess a cell autonomous sex identity (CASI). Here, we review evidence for CASI in birds and discuss the implications for the process of sex determination.

Gonadal Asymmetry and Sex Determination in Birds

Although vertebrates display a superficial bilateral symmetry, most internal organs develop and locate with a consistent left:right asymmetry. There is still considerable debate as to when this process actually begins, but it seems that, at least for some species, the initial steps occur at a very early stage of development. In recent years, a number of model systems, including the chick embryo, have been utilised to increase our understanding of the molecular basis of this complex developmental process. While the basic elements of asymmetry are clearly conserved in chick development, the chick embryo also exhibits an additional unusual asymmetry in terms of the development of the gonads. In the female chick embryo, only 1 gonad and accessory structures fully develop, with the result that the adult hen has only 1 ovary and a single oviduct - both on the left side. With a small number of exceptions, this is a consistent feature of avian development. Here, we describe the morphological development and molecular basis of this unusual asymmetry, consider the implications for avian sex determination, and discuss the possible biological reasons why many birds have adopted a single-ovary system.

Cell-Autonomous Sex Differences in Gene Expression in Chicken Bone Marrow–Derived Macrophages

We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome–specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN–responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes.

Real-Time Sexing of Chicken Embryos and Compatibility with in ovo Protocols

The chicken embryo is an established model system for studying early vertebrate development. One of the major advantages of this model is the facility to perform manipulations in ovo and then continue incubation and observe the effects on embryonic development. However, in common with other vertebrate models, there is a tendency to disregard the sex of the experimental chicken embryos, and this can lead to erroneous conclusions, a lack of reproducibility, and wasted efforts. That this neglect is untenable is emphasised by the recent demonstration that avian cells and tissues have an inherent sex identity and that male and female tissues respond differently to the same stimulus. These sexually dimorphic characteristics dictate that analyses and manipulations involving chicken embryos should always be performed using tissues/embryos of known sex. Current sexing protocols are unsuitable in many instances because of the time constraints imposed by most in ovo procedures. To address this lack, we have developed a real-time chicken sexing assay that is compatible with in ovo manipulations, reduces the number of embryos required, and conserves resources.

Sex Determination and Gonadal Development in Birds

The mechanisms regulating sex determination and gonadal development in birds and mammals share many common morphological and molecular features. However, these mechanisms have evolved independently and there are elements of these processes that are absent in placental mammals but are present in most avian species. These include, a left-right gonadal asymmetry, the direct involvement of estrogen in primary sex determination, a strong cellular sex identity in non-reproductive tissues, and the type of dosage compensation mechanism employed. In terms of genetic control, the avian testis-determining pathway is thought to be regulated by expression of the Z-linked gene DMRT1 (sex chromosomes are ZZ in males and ZW in females), while the autosomal FOXL2 gene product is thought to play a major role in ovarian development. As our understanding of this process improves, it is likely that genetic and hormonal elements will merge into one interacting network that regulates sex determination and the sexual phenotype in birds.

01-P012 The role of micro-RNAs in the development of the chick gonads

causative gene. We have identified a 187 kb microdeletion on chromosome 15q11.2 which narrows down the candidate region to a cluster of non-coding small nucleolar RNAs (snoRNAs), the SNORD116. The deletion results in a loss of expression of the SNORD116 cluster in the patient. The snoRNAs in the adjacent SNORD115 cluster, and the neighbouring genes UBE3A, MAGEL and NECDIN are expressed. Knockout mouse models of Snord116 show features of PWS, thus supporting a role of SNORD116 in the aetiology of PWS. However, nothing is known about the physiological role of the SNORD116 snoRNA cluster and how their lack of expression could cause the multiple symptoms of PWS. SnoRNAs are small non-coding RNAs that are found in the nucleolus,which guide modification of ribosomal RNA via sequence homology to the rRNA target. However, the SNORD116 snoRNAs do not show any sequence homology to any known rRNA or mRNA target. They are so-called ‘orphan’ snoRNAs. To date, it is unknown with which proteins or other RNAs SNORD116 interacts. To identity binding partners of SNORD116, we performed a StreptoTag binding assay.