Stem Cells from Fetal Membranes – A Workshop Report
Article Outline
- Abstract
- 1. Introduction
- 2. Signalling pathways in TS cells
- 3. Towards a precise definition of trophoblast subtypes
- 4. Subtype-specific trophoblast cell lines
- 5. Decidua-mediated differentiation of hES cells to trophoblast
- 6. Identification of adult stem cells in human endometrium
- 7. Concluding remarks
- 8. Conflict of interest
- References
- Copyright
Abstract
Stem cells that can be derived from fetal membranes represent an exciting field of research that bears tremendous potential for developmental biology and regenerative medicine. In this report we summarize contributions to a workshop in which newest insights into the characteristics, subtypes and molecular determinants of stem cells from trophoblast and endometrial tissues were presented.
Keywords: Stem cells, Fetal membranes, Trophoblast, Endometrium
1. Introduction
Pluripotent embryonic stem cell (ES) lines that can differentiate into all three germ layers of the embryo were established over 25 years ago [1]. In contrast, stem cells from extraembryonic tissues have only come into the spotlight in more recent years. The derivation of trophoblast stem (TS) cells from the mouse was pioneered in 1998 [2]. TS cells can differentiate into all trophoblast cell types of the placenta, but are excluded from the embryo proper. Importantly, unlike ES cells that are the product of a derivation process in culture, TS cells are part of normal extraembryonic tissues and can be found in the polar trophectoderm of the blastocyst and in the extraembryonic ectoderm of the post-implantation mouse conceptus.
Compared to the lineage-determined stem cell types of the early mouse embryo, the situation in humans appears to be quite different. The derivation of embryonic stem cells succeeded in 1998 [3]. Further studies have shown that human ES cells can differentiate into trophoblast cells and can even give rise to trophoblast cells with stem cell potential [4]. Recent efforts are also focused on determining the stem cell potential of cell populations in fetal membranes such as the chorion and the amnion. Equally important for a successful outcome of pregnancy are investigations into the self-renewing and multipotent capacity of uterine endometrial cells. In this workshop, new insights were presented in the field of ‘stem cells derived from fetal membranes’.
2. Signalling pathways in TS cells
Dr. Yang presented insights into the signalling pathways that are required to maintain the stem cell character of TS cells. This work was prompted by the finding that mice deficient in the signalling molecule Shp2 are remarkably similar in phenotype to mutants in the TS cell marker Cdx2 [5]. Like for Cdx2 mutants, no TS cells can be derived from Shp2−/− blastocysts. Specification of the trophoblast lineage is initiated in the blastocyst, but the Shp2 mutation interferes with subsequent maintenance and proliferation of TS cells and instead leads to the induction of apoptosis. Detailed studies have elucidated that Shp2 is necessary for FGF receptor signalling in the trophoblast lineage to maintain the proliferative capacity and to suppress apoptosis. This signalling pathway leads to activation of the Ras/Raf/Erk2 MAP kinase pathway, which in turn phosphorylates the pro-apoptotic protein Bim. Erk2-mediated phosphorylation targets Bim for degradation, thereby suppressing apoptosis in trophoblast cells. However, parallel pathways downstream of Shp2 may also be involved since Bim degradation (∼80% knockdown by shRNA) accounts only for about 50–60% of TS cell survival. Further studies will have to show which signalling cascades contribute to TS cell maintenance in addition to Shp2/Erk2, with a good candidate being the Akt kinase signalling pathway.
3. Towards a precise definition of trophoblast subtypes
Dr. Natale's interest focuses on the molecular pathways that define specific trophoblast subtypes. Using a whole array of genes, he has analyzed the differentiation stage-specific expression of genes and the variability that is observed between different TS cell lines. Thus, trophoblast giant cell (TGC) subtypes of the mature placenta can be identified both in vivo and in TS cells in vitro by the specific expression of a combination of genes [6]. For example, placental lactogen 2 (Csh2) and cathepsin Q (Ctsq) co-expression identifies TGCs that line the maternal blood spaces within the labyrinthine layer of the placenta whereas expression of proliferin (Plf) exclusively identifies TGCs that are associated with spiral arteries in the decidua. Parietal TGCs lining the implantation site can be identified by expression of Csh1 [6]. Previous studies have identified genes such as Esx1, Dlx3 and Gcnf as general markers of labyrinthine trophoblast while Gcm1 has been identified as a marker of syncytiotrophoblast and more recently as a marker specifically of syncytiotrophoblast layer-II. Careful examination by in situ hybridization in the placenta revealed that these genes have somewhat unique expression patterns during development. This observation was confirmed in TS cells, further highlighting the presence of different trophoblast cell subtypes and the complexity of the cell culture model. Thus, while representing an extremely useful model system, these data highlight the need for careful analysis of gene expression in multiple TS cell lines and the use of different (and multiple) known molecular markers of differentiated trophoblast cell subtypes when assessing trophoblast differentiation in vitro.
4. Subtype-specific trophoblast cell lines
TS cells can be regarded as the most potent stem cells of the trophoblast lineage that can give rise to all trophoblast subtypes of the placenta, but the simultaneous differentiation of all three placental lineages may make the analysis difficult. However, it is likely that cells with more restricted potency persist in individual layers of the placenta. Dr. Brown presented studies on trophoblast subtype-specific cell lines with self-renewing capacities. The labyrinth-derived mouse SM10 and rat HRP-1 cell lines and the choriocarcinoma cell line Rcho-1 express genes characteristic of TS cells such as Cdx2 and/or Id2 and can be maintained continuously in cell culture. Upon TGFβ treatment of SM10 cells, the labyrinth-specific gene Gcm1 is upregulated, the cells aggregate, and they become multinucleate [7]. Although HRP-1 cells do express some lineage markers of labyrinthine trophoblasts, they do not become multinucleate or induce Gcm1 and thus should be used with this in mind. Spongiotrophoblast or trophoblast giant cell specific genes are never detected in these two cell lines. In contrast, Rcho-1 cells serve as a stem cell-like cell line committed to the giant cell lineage and can be identified by giant cell lineage expressed genes such as mSna and Hand1 [8]. Distinct steps in the differentiation process have been defined by down-regulation of Id2 expression, palladin production, stress fiber formation, and Csh1 induction. The data presented highlight the use of committed stem cell-like cells lines as important tools in determining the molecular regulation and function of specific differentiated trophoblast lineages.
5. Decidua-mediated differentiation of hES cells to trophoblast
Unlike mouse ES cells, human ES cells retain some capacity to differentiate into trophoblast cells. Dr. Dunk's work in collaboration with Dr. Jonathan Draper and Dr. Janet Rossant establishes that trophoblast differentiation is triggered by the environment of uterine decidual cells. When human embryonic bodies (hEB) are placed into contact with decidual explants and cultured at 3% oxygen, the hES cells differentiate into trophoblast cells that express trophoblast-specific markers such as cytokeratin 7 and 8 and exhibit invasive characteristics similar to extravillous cytotrophoblasts. The hEB implant into the decidual stroma and generate trophoblast that specifically targets the decidual arterioles, invades the vessel lumen and mediates vessel remodeling. Stable overexpression of the transcription factor Cdx2 in hES cells increases the differential capability and results in fused trophoblast-like epithelial sheets that stain positively for hCG. In the co-culture model clone CD10 generates large cytokeratin positive syncytial structures that invade the decidual stroma, while clone CD8 gives rise to large numbers of highly invasive individual trophoblasts that invade interstitially and disrupt the blood vessels in their path. This is the first demonstration that the human decidua can stimulate trophoblast differentiation from an undifferentiated hES cell line and may provide important insights into the mechanisms underlying implantation.
6. Identification of adult stem cells in human endometrium
Dr. Gargett presented pioneering work on mesenchymal stem cells that can be isolated from the endometrium, i.e. the maternal tissue that mediates implantation of the embryo. The human endometrium undergoes cycles of growth, differentiation and shedding during each menstrual cycle. It grows 4–7
mm within 7–10 days from the residual basalis layer every month; it also regenerates following parturition, extensive curettage and in post-menopausal women taking estrogen replacement therapy. It is likely that adult stem cells are responsible for this remarkable regenerative capacity. Initial studies identified rare epithelial and stromal cells with colony forming unit activity (CFU) suggesting two types of adult stem cells in the human endometrium [9]. Subsequent work using single cell suspensions of purified human endometrial stromal cells established that co-expression of CD146 and PDGFR-β enriches for human endometrial stromal CFU [10]. FACS sorted CD146+PDGFR-β+ stromal cells expanded in culture exhibit mesenchymal stem cell (MSC) properties as they undergo differentiation into myogenic, adipogenic, osteogenic and chondrogenic lineages. Their surface phenotype is similar to that of bone marrow MSC (CD29+, CD44+, CD73+, CD90+, CD105+, but Stro-1−) and negative for the haematopoietic markers CD34 and CD45. Cells co-localising CD146 and PDGFR-β are identified in a perivascular location around large and small blood vessels in the basalis and functional regions of the human endometrium. These studies demonstrate that the human endometrium contains a small population of MSC-like cells which may be responsible for its cyclical growth. It is also possible that dysfunctional endometrial MSC-like cells produce insufficient tissue in those women undergoing IVF treatment who are unable to produce an adequate endometrium for implantation of the developing embryo.
7. Concluding remarks
Stem cells from extraembryonic tissues represent an emerging area of research that bears tremendous potential for elucidating mechanisms underlying extraembryonic development. The analysis of TS cells and of stem cell-like cell lines with restricted differentiation potential is instrumental to identify factors that determine pluri-/multipotency and trophoblast subtype commitment. With our growing knowledge of the molecular frameworks that govern trophoblast differentiation, it is important to compare mouse and human stem cells to identify common and species-specific aspects of lineage determination and differentiation. In addition, stem cell-like character has been described in cells isolated from other extraembryonic tissues such as the chorionic and amniotic membranes, and the endometrium. These ‘adult’ stem cells are of enormous interest because of their general accessibility and potential to avoid ethical issues that are associated with early embryos. Overall, stem cells from fetal membranes represent an exciting field of research of outstanding importance with broad application to many different areas including normal and pathological development, assisted reproductive technology procedures and regenerative medicine.
8. Conflict of interest
The authors do not have any potential or actual personal, political, or financial interest in the material, information, or techniques described in this paper.
References
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PII: S0143-4004(07)00270-6
doi:10.1016/j.placenta.2007.11.006
© 2007 IFPA and Elsevier Ltd. All rights reserved.
