Recent advances in the derivation of germ cells from the embryonic stem cells

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.     

Differential expression of the embryo/cancer gene ECSA(DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development

In this paper, we examine the expression profiles of two new putative pluripotent stem cell genes, the embryo/cancer sequence A gene (ECSA) and the cancer/testis gene Brother Of the Regulator of Imprinted Sites (BORIS), in human oocytes, preimplantation embryos, primordial germ cells (PGCs) and embryo stem (ES) cells. Their expression profiles are compared with that of the well-known pluripotency gene, OCT4, using a primer design that avoids amplification of the multiple OCT4 pseudogenes. As expected, OCT4 is high in human oocytes, down-regulated in early cleavage stages and then expressed de novo in human blastocysts and PGCs. BORIS and ECSA show distinct profiles of expression in that BORIS is predominantly expressed in the early stages of preimplantation development, in oocytes and 4-cell embryos, whereas ECSA is predominantly expressed in the later stages, blastocysts and PGCs. BORIS is not detected in blastocysts, PGCs or other fetal and adult somatic tissue tested. Thus, BORIS and ECSA may be involved in two different aspects of reprogramming in development, viz., in late gametogenesis, and at the time of formation of the ES cells (inner cell mass (ICM) and PGC), respectively. However, in human ES cells, where a deprogrammed stem cell state is stably established in culture, an immunofluoresence study shows that all three genes are co-expressed at the protein level. Thus, following their derivation from ICM cells, ES cells may undergo further transformation in culture to express a number of embryo and germ line stem cell functions, which, in normal development, show different temporal and spatial specificity of expression.     

Cloned human embryonic stem cells for tissue repair and transplantation

One approach to overcome transplant rejection of human embryonic stem (ES) cells is to derive ES cells from nuclear transfer of the patient’s own cells. Because an efficient protocol for human somatic cell nuclear transfer (SCNT) has not been reported, several critical factors need to be determined and optimized. Our experience with domestic animals indicate that reprogramming time (the period of time between cell fusion and oocyte activation), activation method and in vitro culture conditions each play a critical role in chromatin remodeling and the developmental competence of SCNT embryos. In this review, we describe the optimization of human SCNT and derivation of human cloned ES cells. In our study, about approx 25% of human reconstructed embryos developed into blastocysts when we allowed 2 h for reprogramming to support proper embryonic development. Since sperm-mediated activation is absent in SCNT, an artificial stimulus is needed to initiate embryo development. Incubation with 10 μM calcium ionophore for 5 min followed by incubation with 2.0 μM 6-dimethyl amino purine was found to be the most efficient chemical activation protocol for SCNT using human oocytes. In order to overcome inefficiencies in embryo culture, we prepared human modified synthetic oviductal fluid with amino acids (hmSOFaa) by supplementing mSOFaa with human serum albumin and fructose instead of bovine serum albumin and glucose, respectively. Culturing human SCNT-derived embryos in G1.2 medium for the first 48 h followed by hmSOFaa medium produced more blastocysts than culturing in G1.2 medium for the first 48 h followed by culture in G2.2 medium or culturing continuously in hmSOFaa medium. The protocol described here produced cloned blastocysts at rates of 19–29%, which is comparable with the rates in cattle (approx 25%) and pigs (approx 26%) using established SCNT methods. A total of 30 SCNT-derived blastocysts were cultured, 20 inner cell masses (ICMs) were isolated by immunosurgical removal of the trophoblast, and one human cloned ES cell line (SCNT-hES1) with typical ES cell morphology and pluripotency was derived. Our approach opens the door for the use of autologous cells derived from nuclear transfer ES (ntES)-derived cells in transplantation medicine.     

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

BACKGROUND: Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. METHODS: Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. RESULTS: Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. CONCLUSIONS: This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.     

Expression of the CD46 antigen, and absence of class I MHC antigen, on the human oocyte and preimplantation blastocyst.

Expression of CD46 and class I major histocompatibility complex (MHC) antigens by human oocytes and 6-8-day unhatched expanded preimplantation blastocysts has been studied by immunocytochemistry. The CD46 antigen, a cell surface complement regulatory protein, was expressed by unfertilized oocytes as well as strongly by both the inner cell mass and trophectoderm of preimplantation blastocysts. In contrast, class I MHC antigens were not usually expressed by either oocytes or blastocysts. These data support the concept that gametes and embryonic cells involved in fertilization and early implantation events, respectively, may be protected from immunological recognition or attack both by the lack of class I MHC antigens and by expression of the CD46 complement regulatory protein.     

Immunogenicity of human embryonic stem cells: can we achieve tolerance?

Human embryonic stem cells are unique in their capacity to propagate without losing pluripotency, and at the same time may readily differentiate to various cell types of the three embryonic germ layers. It is widely accepted today that differentiated human embryonic stem cells may in the future enable repair of vital tissues of the body. Detailed differentiation protocols need to be developed and safety issues associated with cellular therapeutics must be examined. One of the greatest hurdles facing transplantation is the development of immune rejection processes towards non-autologous cells. Profiling of histocompatibility antigens expressed on the cells reveals that they might be subjected to immune response. Here we describe the routes of immune recognition that can identify these antigens and the proposed ways for overcoming the rejection of human embryonic stem cell derivatives.     

How to cross immunogenetic hurdles to human embryonic stem cell transplantation

Implantation of human embryonic stem cells (hES), derived progenitors or mature cells derived from hES has great therapeutic potential for many diseases. If hES would come from genetically unrelated individuals, it would be probably rejected by the immune system of the recipient. Blood groups, MHC and minor antigens are the immunogenetic hurdles that have to be crossed for successful transplantation. Autologous transplantation with adult stem cells would be the best approach but several elements argue against this option. Classical immunosuppression, depleting antibody, induction of tolerance and stem cell banking are alternative methods that could be proposed to limit the risk of rejection.     

Pluripotent stem cell lines

The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while maintaining the ability to differentiate into advanced derivatives of all three germ layers, features very useful for understanding the differentiation and function of human tissues, for drug screen and toxicity testing, and for cellular transplantation therapies. Here we review the family of pluripotent cell lines derived from early embryos and from germ cells, and compare them with the more recently described induced pluripotent stem cells.     

Interspecies somatic cell nuclear transfer and preliminary data for horse-cow/mouse iSCNT

Nuclear transfer (NT) experiments in mammals have demonstrated that adult cells are genetically equivalent to early embryonic cells and the reversal of the differentiated state of a cell to another that has characteristics of the undifferentiated embryonic state can be defined as nuclear reprogramming. The feasibility of interspecies somatic cell NT (iSCNT) has been demonstrated by blastocyst formation and the production of offspring in a number of studies. Embryo and oocyte availability is a major limiting factor in conducting NT to obtain, blastocysts for both reproductive NT studies in genetically endangered animals and in embryonic stem cell derivation for species such as the horse and human. One approach to generate new embryonic stem cells in human as disease models, or in species where embryos and oocytes are not widely available, is to use oocytes from another species. Utilization of oocytes for recipient cytoplasts from other species that are accessible and abundant, such as the cow and rabbit, would greatly benefit ongoing research on reprogramming and stem cell sciences. The use of iSCNT is an exciting possibility for species with limited availability of oocytes as well as for endangered or exotic species where assisted reproduction is needed. However, the mechanisms involved in nuclear reprogramming by the oocyte are still unknown and the extent of the "universality" of ooplasmic reprogramming of development remains under investigation.     

Overexpression of Nodal promotes differentiation of mouse embryonic stem cells into mesoderm and endoderm at the expense of neuroectoderm formation

Understanding how to direct the fate of embryonic stem (ES) cells upon differentiation is critical to their eventual use in therapeutic applications. Clues for controlling ES cell differentiation may be found in the early embryo because mouse ES cells form derivatives of all three embryonic germ layers upon injection into blastocysts. One promising candidate for influencing the differentiation of ES cells into the embryonic germ layers is the transforming growth factor-beta (TGF-beta) growth factor, Nodal. Nodal null mouse mutants lack mesoderm, and injection of Nodal mRNA into nonmammalian embryos induces mesodermal and endodermal tissues. We find that overexpression of Nodal in mouse ES cells leads not only to up-regulation of mesodermal and endodermal cell markers but also to downregulation of neuroectodermal markers. These findings demonstrate the importance of Nodal's influence on the differentiation of pluripotent cells to all three of the primary germ layers. Accordingly, altering expression of factors responsible for cell differentiation in the intact embryo provides an approach for directing ES cell fates in vitro toward therapeutically useful cell types.     

Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation

BACKGROUND: This study was designed to establish human embryonic stem cell (hESC) lines, to identify the differences when maintained in serum-containing versus serum-free medium and to test their potential of in vitro differentiation. METHODS: Procedures including immunosurgery were performed on 11 donated human blastocysts to establish hESC lines. The cell lines were characterized and maintained using either serum-free or serum-containing media to compare their morphology, Oct-4 expression, apoptosis and growth speed. Differentiation of these lines was evaluated by the morphology and the expression of genes belonging to the three embryonic germ layers and the germ cell lineage. RESULTS: Three hESC lines were established, and they grew at similar speed in both media (serum-containing or serum-free), but hESC cultured in serum-containing medium yielded significantly higher percentages of morphologically good colonies and cells expressing Oct-4. These cell lines differentiated spontaneously in vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. CONCLUSIONS: Three hESC lines with Taiwanese ancestry have been established, and they retain the in vitro differentiation potential with or without embryoid body (EB) formation. The data support that hESC may be capable of differentiation into germ cells although further confirmation is needed. It is also suggested that strategies such as stepwise adaptation will be needed before implementing a serum-free culture condition for hESC lines that have previously been derived in a medium containing serum.     

Transplantation of Mammalian Embryonic Stem Cells and Their Derivatives to Avian Embryos

Xenografting of normal and transformed mammalian tissues and cells to chick embryos has been performed for almost 100 years. Embryonic stem cells, derived more than 25 years ago from murine, and more than 10 years ago from human blastocysts, have transformed many fields of biological research. There is a growing body of studies combining these two widely-used experimental systems. This review surveys those reports in which murine or human embryonic stem cells, or differentiated derivatives of these pluripotent stem cells, were transplanted to embryonated chick eggs. Many of these studies have utilized the unique characteristics of both experimental models to obtain answers to developmental questions that are difficult or impossible to approach with xenografting to adult rodents or tissue culture-only techniques.     

人胚胎干细胞原代克隆生长及其传代的研究

目的 评价人胚胎干细胞建系与囊胚质量、原代克隆生长的关系。方法 D3废弃胚胎成组共培养获得不同质量的囊胚，免疫刀去除囊胚滋养外胚层细胞后，将内细胞团(ICM)接种到饲养细胞层上生长、传代。结果 从质量好的囊胚得到的人胚胎干细胞传代的代数更多；原代克隆生长快的人胚胎干细胞传代效率更高。结论 人胚胎干细胞建系与囊胚质量、原代克隆生长情况密切相关。     

In vitro differentiation of human parthenogenetic stem cells into neural lineages

Human parthenogenetic stem cells are derived from the inner cell mass of blastocysts obtained from unfertilized oocytes that have been stimulated to develop without any participation of male gamete. As parthenogenesis does not involve the destruction of a viable human embryo, the derivation and use of human parthenogenetic stem cells does not raise the same ethical concerns as conventional embryonic stem cells. Human parthenogenetic stem cells are similar to embryonic stem cells in their proliferation and multilineage in vitro differentiation capacity. The aim of this study is to derive multipotent neural stem cells from human parthenogenetic stem cells that are stable to passaging and cryopreservation, and have the ability to further differentiate into functional neurons. Immunocytochemistry, quantitative real-time PCR, or FACS were used to confirm that the derived neural stem cells express neural markers such as NES, SOX2 and MS1. The derived neural stem cells keep uniform morphology for at least 30 passages and can be spontaneously differentiated into cells with neuron morphology that express TUBB3 and MAP2, and fire action potentials. These results suggest that parthenogenetic stem cells are a very promising and potentially unlimited source for the derivation of multipotent neural stem cells that can be used for therapeutic applications.     

Analysis of Esg1 expression in pluripotent cells and the germline reveals similarities with Oct4 and Sox2 and differences between human pluripotent cell lines

Establishment of pluripotent epiblast cells is a critical event during early mammalian development because all somatic lineages and the primordial germ cells (PGCs) are derived from them. The epiblast and PGCs are in turn the precursors of pluripotent embryonic stem cells and embryonic germ cells, respectively. Although PGCs are specialized cells, they express several key pluripotency-related genes, such as Oct4 and Sox2. We have analyzed Esg1 expression in mouse and human cells and shown that in the mouse the gene is specifically expressed in preimplantation embryos, stem cells, and the germline. Moreover, Esg1 coexpresses with Oct4 and Sox2, confirming its identity as a marker of the pluripotent cycle. Esg1 is also expressed with Oct4 and Sox2 by human embryonic stem cells and in germ cell carcinoma tissue but not by all human embryonal carcinoma cell lines. These data suggest that together with Oct4 and Sox2, Esg1 plays a conserved role in the pluripotent pathway of mouse and human stem and germ cells.     

Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders

BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).     

Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines

BACKGROUND: Serum-containing and serum-free media were used to derive human embryonic stem (HES) cells from donated oocytes and embryos. METHODS and RESULTS: Inner cell masses (ICM) were isolated by immunosurgery. The HES cells were found to be easily obtained and expanded in a serum-free medium. The efficacy in establishing human embryonic stem cell lines improved in a serum-free medium compared with that in serum-containing media. Four HES cell lines were derived from 13 isolated ICM on mouse embryonic fibroblast feeder layers. All four cell lines possess the same characteristics and differentiating potency: normal 46, XX or 46, XY karyotype; and expressing a series of surface markers such as APase, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1. They can form embryoid bodies in suspension culture and develop teratomas comprising derivatives of three embryonic germ layers when injected into severe combined immunodeficient mice. CONCLUSION: These preliminary results suggest that serum-free cultivation may be superior to serum-containing cultivation for deriving human embryonic stem cells.     

The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores

BACKGROUND: Immune rejection can lead to the failure of human embryonic stem cell (hES cell) transplantation. One approach to address the problem is to establish hES cell line banks. Due to the limited source of human embryos and to ethical reasons, the hES cell lines are not readily available. This study was undertaken to determine whether discarded day 3 embryos with low morphological scores could develop into blastocysts and produce hES cell lines. METHODS: A total of 130 day 3 embryos with low morphological scores were cultured to blastocyst stage, and inner cell masses (ICM) were isolated by immunosurgery. Colonies derived from the ICM were passaged every 4-7 days and evaluated for cell surface markers, differentiation potentials and karyotypes. RESULTS: A total of 19 blastocysts were obtained from 130 embryos (quality score <16), which resulted in the formation of 10 ICM, and two cell lines. Both cell lines satisfied the criteria that characterize pluripotent hES cells. CONCLUSION: Our results suggest that a subset with poor quality day 3 embryos judged on the basis of morphological assessment can form blastocysts and give rise to hES cell lines.     

Embryonic Stem Cell Marker Expression Pattern in Human Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, Heart and Dermis

Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn’s disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.