Fetal stem cell transplantation: Past,present, and future

Since 1928, human fetal tissues and stem cells have been used worldwide to treat various conditions. Although the transplantation of the fetal midbrain substantia nigra and dopaminergic neurons in patients suffering from Parkinson's disease is particularly noteworthy, the history of other types of grafts, such as those of the fetal liver, thymus, and pancreas, should be addressed as there are many lessons to be learnt for future stem cell transplantation. This report describes previous practices and complications that led to current clinical trials of isolated fetal stem cells and embryonic stem(ES) cells. Moreover, strategies for transplantation are considered, with a particular focus on donor cells, cell processing, and the therapeutic cell niche, in addition to ethical issues associated with fetal origin. With the advent of autologous induced pluripotent stem cells and ES cells, clinical dependence on fetal transplantation is expected to gradually decline due to lasting ethical controversies, despite landmark achievements.     

Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.     

Potential of embryonic and adult stem cells in vitro

Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.     

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.     

The placenta theory and the origin of infantile hemangioma

The pathogenesis of infantile hemangioma is unknown. In recent years, much of the focus has been placed at identifying the cell type(s) responsible for tumor initiation. New discoveries in infantile hemangioma suggest an involvement of progenitor cells in the pathogenesis of this vascular tumor. Both embryonic and extra-embryonic tissues have been postulated as potential sources for these progenitor cells. This review focuses on the placental theory, which proposes that a fetal placental progenitor is the cell type of origin for infantile hemangioma. Special emphasis will be placed on placental vasculogenesis and the presence and transit of placental progenitor cells during gestation.     

Fetal adnexa derived stem cells from domestic animal: progress and perspectives

The fetal adnexa such as umbilical cord, amnion and amniotic fluid have been proposed as ideal sources of different stem cell lineages. Use of adnexal tissue has many potential advantages, including the noninvasive nature of the isolation procedure, the large tissue mass from which cells can be harvested with high efficiency and the potential of these cells to differentiate. Moreover, particularly in human medicine, the harvesting of these tissues is more ethically acceptable making these sources of stem cells very attractive for regenerative therapies and biotechnological applications. The adnexal tissue cells preserve some of the characteristics of the primitive embryonic layers from which they originate. Indeed, many studies indicate that these stem cells exhibit some features of embryonic stem cells as expression of embryonic markers and proliferation capability, without showing immunogenicity. However, the differentiation potential of these cells, either in vivo or in vitro, is intermediate between the pluripotent embryonic stem cells and the multipotent adult stem cells. Non-embryonic extra-fetal derived stem cells have opened new perspectives for developmental biology and for regenerative medicine, not only in humans but also in animals. In this update, we report the state of the art of fetal adnexa-derived stem cells from domestic animals and analyze their applications and potential uses in veterinary medicine.     

Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells

In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.     

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

Background  

Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.     

Stem cells: their source,potency and use in regenerative therapies with focus on adipose-derived stem cells – a review

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called “stem cell-based cell-free therapy”. There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.     

Potential applications of germline cell-derived pluripotent stem cells in organ regeneration

Impressive progress has been made since the turn of the century in the field of stem cells. Different types of stem cells have now been isolated from different types of tissues. Pluripotent stem cells are the most promising cell source for organ regeneration. One such cell type is the germline cell-derived pluripotent cell, which is derived from adult spermatogonial stem cells. The germline cell-derived pluripotent stem cells have been obtained from both human and mouse and, importantly, are adult stem cells with embryonic stem cell-like properties that do not require specific manipulations for pluripotency acquisition, hence bypassing problems related to induced pluripotent stem cells and embryonic stem cells. The germline cell-derived pluripotent stem cells have been induced to differentiate into cells deriving from the three germ layers and shown to be functional in vitro. This review will discuss the plasticity of the germline cell-derived pluripotent stem cells and their potential applications in human organ regeneration, with special emphasis on liver regeneration. Potential problems related to their use are also highlighted.     

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm - early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm - were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.     

Subtractive hybridization reveals tissue-specific expression of ahnak during embryonic development

The gene product ahnak has been identified from extra-embryonic mesoderm cDNA enriched using a subtractive hybridization approach modified for using small amounts of starting material. Clones for cyclin D2 and H19 have also been isolated as being preferentially enriched in the extra-embryonic mesoderm compared with the embryo proper of embryonic day (E) 7.5 neural plate stage mouse embryos. The differential expression of these genes was confirmed at gastrulation stage using in situ hybridization. More detailed analysis of the human genomic ahnak sequence suggests that its highly repetitive structure was formed by unequal cross-over and gene conversion. During organogenesis, ahnak is expressed in a variety of tissues, including migratory mesenchyme. By E12.5, the major site of expression of ahnak is craniofacial mesenchyme. Immunohistochemical analysis has shown that ahnak protein is expressed mainly at the cell membrane of migratory mesenchymal cells, primarily in the nucleus of bone growth plate cells and mostly in the cytoplasm of differentiating nasal epithelia. The potential functions of ahnak are discussed in light of these results.     

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.     

Isolation of murine and porcine fetal stem cells from somatic tissue

Adult stem cells have been previously isolated from a variety of somatic tissues, including bone marrow and the central nervous system; however, contribution of these cells to the germ line has not been shown. Here we demonstrate that fetal somatic explants contain a subpopulation of somatic stem cells (FSSCs), which can be induced to display features of lineage-uncommitted stem cells. After injection into blastocysts, these cells give rise to a variety of cell types in the resultant chimeric fetuses, including those of the mesodermal lineage; they even migrate into the genital ridge. In vitro, FSSCs exhibit characteristics of embryonic stem cells, including extended self-renewal; expression of stem cell marker genes, such as Pou5f1 (Oct4), Stat3, and Akp2 (Tnap) and growth as multicellular aggregates. We report that fetal tissue contains somatic stem cells with greater potency than previously thought, which might form a new source of stem cells useful in somatic nuclear transfer and cell therapy.     

Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.

The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.     

CD117+ amniotic fluid stem cells

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

CD117+ amniotic fluid stem cells: State of the art and future perspectives

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

In vitro development of murine T cells from prethymic and preliver embryonic yolk sac hematopoietic stem cells.

Mature T cells are derived from prethymic stem cells, which arise at one or more extrathymic sites and enter and differentiate in the thymus. The nature of these prethymic stem cells is a critical factor for the formation of the T-cell repertoire. Although the bone marrow of adult mice can provide such stem cells, their origin during murine embryogenesis is still undetermined. Among potential sites for these progenitor cells are the fetal liver and the embryonic yolk sac. Our studies focus on the yolk sac, both because the yolk sac appears earlier than any other proposed site, and because the mammalian yolk sac is the first site of hematopoiesis. Although it has been shown that the yolk sac in midgestation contains stem cells that can enter the thymic rudiment and differentiate toward T-cell lineage, our aim was to analyze the developmental potential of cells in the yolk sac from earlier stages, prior to the formation of the liver and any other internal organ. We show here that the yolk sac from 8- and 9-day embryos (2-9 and 13-19 somites, respectively) can reconstitute alymphoid congenic fetal thymuses and acquire mature T-cell-specific characteristics. Specifically, thymocytes derived from the early embryonic yolk sac can progress to the expression of mature T lymphocyte markers including CD3/T-cell receptor (TCR), CD4 and CD8. In contrast, we have been unable to document the presence of stem cells within the embryo itself at these early stages. These results support the hypothesis that the stem cells capable of populating the thymic rudiment originate in the yolk sac, and that their presence as early as at the 2- to 9-somite stage may indicate that prethymic stem cells found elsewhere in the embryo at later times may have been derived by migration from this extra-embryonic site. Our experimental design does not exclude the possibility of multiple origins of prethymic stem cells of which the yolk sac may provide the first wave of stem cells in addition to other later waves of cells.