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The handle http://hdl.handle.net/1887/62812 holds various files of this Leiden University dissertation.
Author: Moerkamp, A.T.
Title: The building blocks for cardiac repair : isolation and differentiation of progenitor cells from the human heart
Issue Date: 2018-06-12
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E XTRAEMBRYONIC EN DODERM (XEN) CELLS AS A MODEL OF ENDODERM DEVELOPMENT
Moerkamp, A.T.
1, Paca, A.
2, Goumans, M.J.
1, Kunath, T.
2, Kruithof, B.P.T.
1, Kruithof- de Julio, M.
11. Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands.
2. MRC Centre for Regenerative Medicine, University of Edinburgh, United King- dom.
Published in Development, Growth and Differentiation (2013)
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Abstract
When provided with the correct signals cardiac progenitor cells (CPCs) are able to dif- ferentiate into the different cardiac cell types, including cardiomyocytes, which is of great therapeutic value. In order to obtain a homogenous population of cardiomy- ocytes for cardiac repair, it is of importance to understand the signaling networks that direct CPC differentiation. During development, the visceral endoderm, inter- acts with the nascent mesoderm to induce the cardiac fate. Extraembryonic endo- derm (XEN) cells could be used to recapitulate this developmental process in vitro.
These multipotent XEN cells are derived from the primitive endoderm, which will
give rise to the visceral and parietal endoderm and later on form the yolk sac. As such
XEN cells could be used to support in vitro human CPC to cardiomyocyte differen-
tiation; as an alternative differentiation strategy, to accelerate the current available
cardiomyocyte differentiation protocols or to provide an overview of the instructive
signals needed for differentiation. This chapter will first provide an overview focus-
ing on XEN cells as a model for primitive endoderm after which it will discuss the
potential of using these cells as a therapeutic tool in cardiac regeneration.
8.1. I NTRODUCTION
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8.1. I NTRODUCTION : E XTRAEMBRYONIC ENDODERM DEVELOP -
MENT
The mature mouse blastocyst (4.5 days post-coitum; dpc) consists of three distinct cell types: the trophectoderm, which gives rise to the trophoblast and extraem- bryonic ectoderm (ExEc), the pluripotent cells of the epiblast, and the primitive or extraembryonic endoderm (ExEn), an epithelial layer of cells on the surface of the epiblast. The primitive endoderm gives rise to 1) visceral endoderm (VE) that surrounds the epiblast and the ExEc and 2) parietal endoderm (PE) that interacts with the trophoblast giant cell layer. PE cells migrate along the inner surface of the trophectoderm and together with trophoblast giant cells form the parietal yolk sac (Hogan et al., 1980). The PE, as well as the VE, mediates nutrient-waste exchange for the developing embryo. Initially the VE overlays only the epiblast, but as the ExEc increases in size the VE quickly expands to also cover the ExEc. The VE overlay- ing the epiblast becomes molecularly and morphologically distinct from the VE in contact with the ExEc around 5.0 dpc, representing the embryonic VE (emVE) and extraembryonic VE (exVE), respectively. The cells from the exVE are columnar and cuboidal, while the emVE cells are flatter and more epithelial in shape (Takito and Al-Awqati, 2004).
Around 5.5 dpc a group of cells at the distal tip of the epiblast differentiates into a morphologically distinguishable subset of emVE, the distal visceral endo- derm (DVE) (Srinivas et al., 2004; Rivera-Perez et al., 2003). This marks formation of the first axis of the body, the distal-proximal axis. Within 4-5 hours (between ≈5.75 and ≈6.0 dpc) the DVE migrates proximally as a continuous epithelial sheet to the prospective anterior pole of the embryo. The underlying mechanism of this mi- gration is yet to be fully characterized and both active migration and differences in the proliferation rate of the anterior versus posterior epiblast have been sug- gested (Srinivas et al., 2004; Stuckey et al., 2011; Trichas et al., 2011; Migeotte et al., 2010). The unilateral movement of the DVE changes the distal-proximal axis into the anterior-posterior axis of the embryo and the DVE is now called anterior visceral endoderm (AVE).
The VE and its derivatives, play critical roles in organization and differentiation
of the epiblast. The VE is the first site of haematopoiesis (Toles et al., 1989; McGrath
and Palis, 2005) and induces through the expression of Indian hedgehog and Vas-
cular endothelial growth factor the formation of blood islands and endothelial cells
(Dyer et al., 2001; Byrd et al., 2002; Damert et al., 2002). In addition, the proximal
VE was shown to be involved in early primordial germ cell differentiation (de Sousa
Lopes et al., 2007; de Sousa Lopes et al., 2004). Finally, microsurgical removal of AVE
resulted in anterior neural structures truncations (Thomas and Beddington, 1996)
and its derived BMP2 signals have been shown to take part in heart positioning and
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164 8.2. E X E N - DERIVED STEM CELLS
foregut invagination (Madabhushi and Lacy, 2011).
Interestingly, the lineage distinction between embryonic and ExEn tissue was marked by the assumption that the VE that surrounds the epiblast was displaced by the definitive endoderm. Recently it has been shown that cells from the VE persist within the definitive endoderm layer of the embryo and contribute to the early gut tube. This suggests that the distinction between extraembryonic and embryonic tissues is not as strict as believed and the lineage that was previously considered to be exclusively embryonic has extraembryonic contributions (Kwon et al., 2008).
An interesting question is whether, within the definitive endoderm and potentially its derivative tissues, there are molecular and functional differences between the ExEn- and epiblast-derived cells which may be studied by in vitro culturing and manipulating cells representative of the ExEn.
8.2. D ERIVATION , PROPERTIES AND APPLICATIONS OF E X E N -
DERIVED STEM CELLS
Stem cells can be derived from each of the primary lineages of the mammalian em- bryo (Figure 1). ES cells from the inner cell mass (ICM) or early epiblast, trophoblast stem (TS) cells from the trophectoderm layer and XEN stem cells from the primitive endoderm (or ExEn). Most importantly each one of these stem cell systems are ca- pable of indefinite self-renewal in culture and once reintroduced into the mouse embryo will display lineage restricted contributions in the resulting chimeric em- bryos that are consistent with their lineage of origin (Beddington and Robertson, 1989; Tanaka et al., 1998; Kunath et al., 2005). Interestingly, in vivo, XEN cells can only repopulate the PE, rarely the VE (Kunath et al., 2005; Kruithof-de Julio et al., 2011).
Initially, parietal endoderm cell (PEC) lines have been isolated by Fowler et al.
in 1990. In vitro studies have shown that these cells have characteristics of PE, closely resembling the basement membrane matrix of Reichert’s membrane. How- ever, their chimera contribution potential was not assessed (Fowler et al., 1990).
PECs morphologically resemble the more recently isolated XEN cells (Kunath et al.,
2005) (reviewed in Rossant, 2007) being round and refractile or stellate or epithelia-
like cells. Several methods of isolation have been proposed for XEN cell derivation
which lead all to cells with similar morphological characteristics, however their re-
sponse to growth factors seems to be influenced by the derivation process. In this
context, it is essential to appreciate that primitive endoderm already exhibits het-
erogeneous expression of some of the DVE/AVE markers, such as Cer1 and Lefty1
(Torres-Padilla et al., 2007; Takaoka et al., 2006; Takaoka et al., 2011). Furthermore,
Nodal/Activin signaling can be differentially perceived within the primitive endo-
derm (Granier et al., 2011). This would imply that, at the time of signaling pathway
8.2. E X E N - DERIVED STEM CELLS
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Trophectoderm
Inner cell mass
Primitive endoderm
4.5dpc In vitro In vivo contribution 5-7dpc
Placenta
Epiblast
Visceral endoderm Parietal endoderm
Definitive endoderm Ectoderm Mesoderm
EmVE (DVE/AVE) ExVE
TS cells
ES cells
XEN (or PEC) cells 6.5dpc 15.5dpc Trophectoderm
Inner cell mass / epiblast Primitive endoderm / ExEn
Figure 8.1: Primary lineages of the mammalian embryo At 3.5–4.5 days post-coitum (dpc), tro- phoblast stem (TS) cells, embryonic stem (ES) cells and extraembryonic endoderm (XEN)/ parietal endoderm cells (PEC) can be derived from the trophectoderm, inner cell mass (ICM) and primitive endoderm, respectively, and propagate indefinitely in culture. In chimeras, TS, ES and XEN cells con- tribute to their lineage of origin. The TE will give rise to the placenta; the ICM will form the three germ layers of the embryo and the primitive endoderm will develop into the visceral endoderm (VE) and parietal endoderm (PE) lineages.
maturation, primitive endoderm cells are sensitive to even minor changes in sig- naling intensity. Interestingly, primitive endoderm-progenitors expressing Oct4 ex- hibit greater developmental plasticity than Oct4-expressing epiblast progenitors at a similar stage (Grabarek et al., 2012) which could reflect the in vitro heterogeneous nature of XEN cells.
Extensive microarray analysis on XEN cells performed by several groups all agree in the expression of primitive endoderm markers SOX7, GATA4, GATA6 and the VE markers hHEX and DKK1. Furthermore, XEN cells are characterized by the lack of AFP expression as well as the absence of definitive endoderm markers (Brown et al., 2010b; Kunath et al., 2005; Kruithof-de Julio et al., 2011). Given their reproducible derivation from the ExEn and their expression profile similar to this extraembryonic tissue, XEN cells can be a powerful tool to study inductive effects attributed to the AVE. Brown et al. (2010) have undertaken an extensive array anal- ysis of three cell lines that are similar to the heart-inducing AVE: two embryonal carcinoma-derived (END2 and PYS2) and the XEN cells. By comparing the gene ex- pression profiles they have identified a discrete set of genes that could support my- ocardial differentiation. In addition, by using XEN cell-derived conditioned media on embryoid bodies they were able to enhance cardiogenesis (Brown et al., 2010a).
These are interesting observations with potential therapeutic properties. In order
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166 8.3. C ONCLUSION
to obtain the appropriate cell products (cardiomyocytes for cardiac repair), it is of importance to stepwise direct the fate of the stem cell of interest with the proper instructive signals in vitro. During development, the VE interacts with the nascent mesoderm to induce the cardiac fate. Therefore, XEN cells could be used to recapit- ulate this developmental process in vitro and induce and accelerate differentiation of stem cells (e.g. cardiac progenitor cells) into cardiomyocytes which will be dis- cussed in appendix 3.
Although derived from primitive endoderm, XEN cells contribute efficiently in chimeras to PE but not to VE (Kunath et al., 2005; Kruithof-de Julio et al., 2011).
This lack of contribution to the VE lineage could be caused by many factors among them the preferential interaction with the mural trophectoderm (Artus et al., 2012) (Kruithof-de Julio and Shen, unpublished observation), the alteration of a signaling pathway in the establishment of the cell line based on the derivation process (XEN cells derived in the presence of LIF poorly respond to growth factor stimulation) or the derivation of a ‘committed’ cell that has lost potency. In support of the later, extraembryonic endoderm precursor (XEN-P) stem cells have been derived from rat blastocyst (Debeb et al., 2009; Galat et al., 2009). These cells are characterized by a less ‘endoderm’ defined gene expression profile; they express the ES markers OCT4, REX1, AP, and SSEA1 and contribute to the PE as well as the VE lineages in chimeras.
The authors suggest they are precursor endoderm cells as they could represent the first committed step of the ExEn.
Finally, with respect to the plasticity or commitment of stem cells, recently Cho et al. (2012) was able to derive XEN cells from mouse ES cells via the addition of exogenous retinoic acid and Activin. These XEN cells are indistinguishable from embryo-derived XEN cells, including their differentiation capacity (Cho et al., 2012).
These data show a high degree of plasticity within the ICM and provide the possibil- ity of deriving XEN cell lines from the various mutant ES cell lines available, thereby shedding light on the factors required for XEN cell derivation and the development of ExEn.
8.3. C ONCLUSION
Multipotent stem cells have the potential to develop into different cell types in the body during early life and growth. They are distinguished from other cell types by two important characteristics. First, they can self-renew and second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions.
In order to fulfil the desire to generate a cell-based therapy, it is necessary to ma-
nipulate the cells, in vitro, to successfully differentiate them towards the cell-type of
interest. The meticulous application of developmental principals to stem cell cul-
ture systems is the basis of this kind of research. The major limitation is the very
8.3. C ONCLUSION
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low frequency of differentiated cells identified and the cellular heterogeneity. Only when large numbers of highly enriched progenitors are accessible, methods can be defined for their maturation and their functional capacity rigorously tested in ani- mal models. XEN cells might give the missing link in this differentiation process.
Extraembryonic and embryonic tissues interact to specify each other commit- ment. By using XEN cells as inductive feeder layer for ES cells, epiblast stem cells (EpiSC) or their multipotent progeny, a particular developmental or differentiation fate can be induced (Kruithof-de Julio, Moerkamp and Goumans, unpublished ob- servations). In this context, XEN cells may not only be a model to understand ExEn development, but also to elucidate the inductive role of the ExEn in embryonic tis- sue specification, for example the formation of heart and blood lineages (Artus et al., 2012; Brown et al., 2010a). Once established, their roles in these inductive pro- cesses, mutant lines can be derived to pin point out the specific genes involved.
Thereby, XEN cells may be a powerful in vitro tool (Figure 2), by either the use of conditioned medium or co-culture, for providing an overview of the factors that support the differentiation during developmental processes, like cardiogenesis and hematopoiesis. The identification of the exogenous differentiation stimuli may be translated into clinical practice for stem cell-based approaches to efficiently obtain a high number of the differentiated cell type of interest. For cell-based therapies, XEN cells are preferably derived from human embryos (appendix 3). The derivation from human embryos was only partly supplemented by the SOX7 overexpressing hES cells, however, remains subject to future studies. Alternatively, for the iden- tification of developmental stimuli, XEN cells could be derived from the mouse postimplantation epiblast. Mouse EpiSC are highly similar to hES cells in their pluripotent state and growth factor requirements and therefore its derived XEN cells may resemble human ExEn development more closely. Cho et al. (2012) was unable to derive XEN cells from mouse EpiSC by using the same protocol as for the deriva- tion of XEN cells from mouse ES cells (Cho et al., 2012). In the case of EpiSC, due to their epigenetic state, XEN cell derivation may require different exogenous stimuli.
However, there remains the possibility that EpiSC are indeed unable to convert their developmental fate. Finally, although it has not been shown to date, XEN cells may be derived from somatic cells via direct reprogramming, thereby circumventing the requirement of human embryos.
Although one must keep in mind the highly heterogeneous population and the
existence of species-specific differences, which may be due to the stage and deriva-
tion protocols, XEN cells can also be a model to study extraembryonic contribution
to definitive endoderm and its differentiated derivatives. This raises the question as
to whether XEN cells per se can indeed contribute to the primitive gut tube or must
they be prior differentiated towards a definitive endodermal lineage. In later stage
chimeras derived from Nodal, Cripto or untreated XEN cells contribution to defini-
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168 8.3. C ONCLUSION
tive endoderm has never been observed (Kruithof-de Julio and Shen, unpublished observation) only contribution to the visceral yolk sac.
In summary, XEN cells provide a valuable cell culture model to dissect the ex- traembryonic endodermal lineage and its potential contribution to the embryonic one. Thereby, XEN cells may increase our understanding of the molecular mecha- nism behind endoderm behavior and function during development. Furthermore, XEN cells may give an inside in the inductive factors underlying mesoderm spec- ification, like cardiogenesis and hematopoiesis, which may be reflected into stem cell-based therapeutic approaches.
- To identify factors required for an undifferentiated state and stimuli that direct VE and PE differentiation
- To study extraembryonic contribution to definitive endoderm
- To study the interaction between the visceral endoderm and nascent mesoderm, like their ability to promote proliferation of primitive erythroid cells
A tool to study developmental processes A tool for stem cell based therapy
Directed differentiation of the susceptible stem cells in a patient specific manner
XEN cells or CDM
XEN cells
Figure 8.2: Primary lineages of the mammalian embryo At 3.5–4.5 days post-coitum (dpc), tro-
phoblast stem (TS) cells, embryonic stem (ES) cells and extraembryonic endoderm (XEN)/ parietal
endoderm cells (PEC) can be derived from the trophectoderm, inner cell mass (ICM) and primitive
endoderm, respectively, and propagate indefinitely in culture. In chimeras, TS, ES and XEN cells con-
tribute to their lineage of origin. The TE will give rise to the placenta; the ICM will form the three germ
layers of the embryo and the primitive endoderm will develop into the visceral endoderm (VE) and
parietal endoderm (PE) lineages.
8.4. R EFERENCES
8
169
8.4. R EFERENCES
Artus, J., Douvaras, P., Piliszek, A., Isern, J., Baron, M.H., and Hadjantonakis, A.K. (2012) BMP4 sig- naling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm iden- tity. Dev Biol 361:245-262.
Beddington, R.S., and Robertson, E.J. (1989) An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105:733-737.
Brown, K., Doss, M.X., Legros, S., Artus, J., Hadjantonakis, A.K., and Foley, A.C. (2010a) eX- traembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation. PLoS One 5:e13446.
Brown, K., Legros, S., Artus, J., Doss, M.X., Khanin, R., Hadjantonakis, A.K., and Foley, A. (2010b) A comparative analysis of extra-embryonic endoderm cell lines. PLoS One 5:e12016.
Byrd, N., Becker, S., Maye, P., Narasimhaiah, R., St-Jacques, B., Zhang, X., McMahon, J., McMa- hon, A., and Grabel, L. (2002) Hedgehog is required for murine yolk sac angiogenesis. Development (Cambridge, England) 129:361-372.
Chapman, V., Forrester, L., Sanford, J., Hastie, N., and Rossant, J. (1984) Cell lineage-specific un- dermethylation of mouse repetitive DNA. Nature 307:284-286.
Chazaud, C., Yamanaka, Y., Pawson, T., and Rossant, J. (2006) Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 10:615-624.
Cho, L.T., Wamaitha, S.E., Tsai, I.J., Artus, J., Sherwood, R.I., Pedersen, R.A., Hadjantonakis, A.K., and Niakan, K.K. (2012) Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states. Development 139:2866-2877.
Clements, M., Pernaute, B., Vella, F., and Rodriguez, T.A. (2011) Crosstalk between Nodal/activin and MAPK p38 signaling is essential for anterior-posterior axis specification. Curr Biol 21:1289-1295.
Damert, A., Miquerol, L., Gertsenstein, M., Risau, W., and Nagy, A. (2002) Insufficient VEGFA ac- tivity in yolk sac endoderm compromises haematopoietic and endothelial differentiation. Develop- ment (Cambridge, England) 129:1881-1892.
de Sousa Lopes, S.M., Hayashi, K., and Surani, M.A. (2007) Proximal visceral endoderm and ex- traembryonic ectoderm regulate the formation of primordial germ cell precursors. BMC developmen- tal biology 7:140.
de Sousa Lopes, S.M., Roelen, B.A., Monteiro, R.M., Emmens, R., Lin, H.Y., Li, E., Lawson, K.A., and Mummery, C.L. (2004) BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes & development 18:1838-1849.
Debeb, B.G., Galat, V., Epple-Farmer, J., Iannaccone, S., Woodward, W.A., Bader, M., Iannaccone, P., and Binas, B. (2009) Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.
PLoS One 4:e7216.
Dyer, M.A., Farrington, S.M., Mohn, D., Munday, J.R., and Baron, M.H. (2001) Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the mouse embryo. Development (Cambridge, England) 128:1717-1730.
Fowler, K.J., Mitrangas, K., and Dziadek, M. (1990) In vitro production of Reichert’s membrane by mouse embryo-derived parietal endoderm cell lines. Exp Cell Res 191:194-203.
Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S., Nakao, K., Miyazaki Ji, J., and Niwa, H. (2002) Differentiation of embryonic stem cells is induced by GATA factors. Genes Dev 16:784-789.
Galat, V., Binas, B., Iannaccone, S., Postovit, L.M., Debeb, B.G., and Iannaccone, P. (2009) Devel- opmental potential of rat extraembryonic stem cells. Stem Cells Dev 18:1309-1318.
Gardner, R.L., and Davies, T.J. (1992) Environmental factors and the stability of differentiation in mammalian development. C R Acad Sci III 314:67-69.
Grabarek, J.B., Zyzynska, K., Saiz, N., Piliszek, A., Frankenberg, S., Nichols, J., Hadjantonakis, A.K.,
and Plusa, B. (2012) Differential plasticity of epiblast and primitive endoderm precursors within the
8
170 8.4. R EFERENCES
ICM of the early mouse embryo. Development (Cambridge, England) 139:129-139.
Granier, C., Gurchenkov, V., Perea-Gomez, A., Camus, A., Ott, S., Papanayotou, C., Iranzo, J., Moreau, A., Reid, J., Koentges, G., Saberan-Djoneidi, D., and Collignon, J. (2011) Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse em- bryo. Developmental biology 349:350-362.
Hogan, B.L., Cooper, A.R., and Kurkinen, M. (1980) Incorporation into Reichert’s membrane of laminin-like extracellular proteins synthesized by parietal endoderm cells of the mouse embryo. Dev Biol 80:289-300.
Kruithof-de Julio, M., Alvarez, M.J., Galli, A., Chu, J., Price, S.M., Califano, A., and Shen, M.M.
(2011) Regulation of extra-embryonic endoderm stem cell differentiation by Nodal and Cripto signal- ing. Development 138:3885-3895.
Kunath, T., Arnaud, D., Uy, G.D., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R.L., Avner, P., and Rossant, J. (2005) Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132:1649-1661.
Kwon, G.S., Viotti, M., and Hadjantonakis, A.K. (2008) The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages. Dev Cell 15:509- 520.
Lawson, K.A., Dunn, N.R., Roelen, B.A., Zeinstra, L.M., Davis, A.M., Wright, C.V., Korving, J.P., and Hogan, B.L. (1999) Bmp4 is required for the generation of primordial germ cells in the mouse embryo.
Genes Dev 13:424-436.
Lim, C.Y., Tam, W.L., Zhang, J., Ang, H.S., Jia, H., Lipovich, L., Ng, H.H., Wei, C.L., Sung, W.K., Robson, P., Yang, H., and Lim, B. (2008) Sall4 regulates distinct transcription circuitries in different blastocyst-derived stem cell lineages. Cell Stem Cell 3:543-554.
Madabhushi, M., and Lacy, E. (2011) Anterior Visceral Endoderm Directs Ventral Morphogenesis and Placement of Head and Heart via BMP2 Expression. Developmental cell 21:907-919.
Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T., Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J., Calabrese, J.M., Dennis, L.M., Volkert, T.L., Gupta, S., Love, J., Hannett, N., Sharp, P.A., Bartel, D.P., Jaenisch, R., and Young, R.A. (2008) Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134:521-533.
McGrath, K.E., and Palis, J. (2005) Hematopoiesis in the yolk sac: more than meets the eye. Exp Hematol 33:1021-1028.
Migeotte, I., Omelchenko, T., Hall, A., and Anderson, K.V. (2010) Rac1-dependent collective cell migration is required for specification of the anterior-posterior body axis of the mouse. PLoS biology 8:e1000442.
Monk, M., Boubelik, M., and Lehnert, S. (1987) Temporal and regional changes in DNA methy- lation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development.
Development (Cambridge, England) 99:371-382.
Morrisey, E.E., Tang, Z., Sigrist, K., Lu, M.M., Jiang, F., Ip, H.S., and Parmacek, M.S. (1998) GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev 12:3579-3590.
Niakan, K.K., Ji, H., Maehr, R., Vokes, S.A., Rodolfa, K.T., Sherwood, R.I., Yamaki, M., Dimos, J.T., Chen, A.E., Melton, D.A., McMahon, A.P., and Eggan, K. (2010) Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev 24:312-326.
Paca, A., Seguin, C.A., Clements, M., Ryczko, M., Rossant, J., Rodriguez, T.A., and Kunath, T. (2012) BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm. Dev Biol 361:90-102.
Rivera-Perez, J.A., Mager, J., and Magnuson, T. (2003) Dynamic morphogenetic events character-
ize the mouse visceral endoderm. Developmental biology 261:470-487.
8.4. R EFERENCES
8
171
Rossant, J. (2007) Stem cells and lineage development in the mammalian blastocyst. Reproduc- tion Fertility Development 19(1):111-8.
Rugg-Gunn, P.J., Cox, B.J., Ralston, A., and Rossant, J. (2010) Distinct histone modifications in stem cell lines and tissue lineages from the early mouse embryo. Proceedings of the National Academy of Sciences of the United States of America 107:10783-10790.
Seguin, C.A., Draper, J.S., Nagy, A., and Rossant, J. (2008) Establishment of endoderm progenitors by SOX transcription factor expression in human embryonic stem cells. Cell Stem Cell 3:182-195.
Shimosato, D., Shiki, M., and Niwa, H. (2007) Extra-embryonic endoderm cells derived from ES cells induced by GATA factors acquire the character of XEN cells. BMC Dev Biol 7:80.
Spruce, T., Pernaute, B., Di-Gregorio, A., Cobb, B.S., Merkenschlager, M., Manzanares, M., and Rodriguez, T.A. (2010) An early developmental role for miRNAs in the maintenance of extraembryonic stem cells in the mouse embryo. Dev Cell 19:207-219.
Srinivas, S., Rodriguez, T., Clements, M., Smith, J.C., and Beddington, R.S. (2004) Active cell mi- gration drives the unilateral movements of the anterior visceral endoderm. Development (Cambridge, England) 131:1157-1164.
Stuckey, D.W., Clements, M., Di-Gregorio, A., Senner, C.E., Le Tissier, P., Srinivas, S., and Ro- driguez, T.A. (2011) Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo. Development (Cambridge, England) 138:1521-1530.
Takaoka, K., Yamamoto, M., and Hamada, H. (2011) Origin and role of distal visceral endoderm, a group of cells that determines anterior-posterior polarity of the mouse embryo. Nature cell biology 13:743-752.
Takaoka, K., Yamamoto, M., Shiratori, H., Meno, C., Rossant, J., Saijoh, Y., and Hamada, H. (2006) The mouse embryo autonomously acquires anterior-posterior polarity at implantation. Developmen- tal cell 10:451-459.
Takito, J., and Al-Awqati, Q. (2004) Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin. J Cell Biol 166:1093-1102.
Tanaka, S., Kunath, T., Hadjantonakis, A.K., Nagy, A., and Rossant, J. (1998) Promotion of tro- phoblast stem cell proliferation by FGF4. Science 282:2072-2075.
Thomas, P., and Beddington, R. (1996) Anterior primitive endoderm may be responsible for pat- terning the anterior neural plate in the mouse embryo. Curr Biol 6:1487-1496.
Toles, J.F., Chui, D.H., Belbeck, L.W., Starr, E., and Barker, J.E. (1989) Hemopoietic stem cells in murine embryonic yolk sac and peripheral blood. Proceedings of the National Academy of Sciences of the United States of America 86:7456-7459.
Torres-Padilla, M.E., Richardson, L., Kolasinska, P., Meilhac, S.M., Luetke-Eversloh, M.V., and Zernicka-Goetz, M. (2007) The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation. Develop- mental biology 309:97-112.
Trichas, G., Joyce, B., Crompton, L.A., Wilkins, V., Clements, M., Tada, M., Rodriguez, T.A., and Srinivas, S. (2011) Nodal dependent differential localisation of dishevelled-2 demarcates regions of differing cell behaviour in the visceral endoderm. PLoS biology 9:e1001019.
Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J.A., and Kosik, K.S. (2009) MicroRNA-145
regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell
137, 647–658.
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A. A PPENDIX : E XTRAEMBRYONIC E NDODERM CELLS AS AN in vitro TOOL FOR CARDIOGENESIS
Moerkamp A.T., Lodder K., Kruithof-de Julio M., Goumans M.J.
Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands.
I NTRODUCTION
Due to the therapeutic value of cardiac progenitor cells (CPCs), it is critical to un- derstand the signaling networks that direct their maintenance and differentiation.
In order to obtain a homogenous population of cardiomyocytes for cardiac repair, it is of importance to stepwise direct the fate of CPCs with the proper instructive signals in vitro.
During development, the visceral endoderm (VE), and specifically the anterior visceral endoderm (AVE), interacts with the nascent mesoderm to induce the car- diac fate. Extraembryonic endoderm (XEN) cells, as derivatives of the primitive en- doderm, could be used to recapitulate this developmental process in vitro as an alternative differentiation strategy and/or accelerate the current available cardiac differentiation protocols. Furthermore, communication between XEN cells and hu- man CPCs could give us an insight into the instructive signals needed for human cardiogenesis.
The VE as heart-inducing signaling center was the basis for the study by Mum- mery et al., (2003) showing that co-culture of human embryonic stem (ES) cells with VE-like cells, the END-2 cell line, results in differentiation into spontaneously beat- ing cardiomyocytes [6]. In addition, Brown et al., (2010) used XEN derived condi- tioned medium (CDM) to enhance the cardiogenesis potential of mouse ES cells [1]. Altogether this raised the question whether XEN-like cells could also be used in a more clinically relevant model: human CPC to cardiomyocyte differentiation.
Therefore, this chapter will provide an overview of some preliminary data focus- ing on the possibilities of using XEN cells as a tool in human CPC to cardiomyocyte differentiation.
R ESULTS AND DISCUSSION
XEN DERIVED CDM INEFFICIENTLY DIRECT HUMAN CPC S TOWARDS A CARDIOMY -
OCY TE FATE .
We have explored two approaches to investigate if XEN cells influence Sca-1+ CPC
to cardiomyocyte differentiation: 1) co-culture (contact-dependent differentiation)
and 2) XEN derived CDM (XEN-CDM; contact-independent differentiation).
A. A PPENDIX 3
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173 A
B
D
PKH GFP
PKH+ vesicles H um an C PC s
C
E cTnI
TG Fβ /A A XE N -C D M /A A XE N -C D M /A A
CTRL TGFβ AA XEN
TGFβAA XENAA AA 0.0
0.5 1.0 1.5
cTnT expression
Fold increase
TGFβAA XEN TGFβAA
XENAA AA 0.000
0.001 0.002 0.003 0.0040.81.0 1.2 1.4 1.6
cTnT expression
Fold increase
CTRL TGFβ AA XEN
TGFβAA XENAA AA 0.0
0.5 1.0 1.5 2.0
MEF2C expression
Fold increase
CTRL TGFβ AA XEN
TGFβAA XENAA AA 0
2 4 6
GATA4 expression
Fold increase
TGFβAA XEN TGFβAA
XENAA AA 0.0
0.2 0.4 0.6 0.8 1.0 1.2
GATA4 expression
Fold increase
TGFβAA XEN TGFβAA
XENAA AA 0.0
0.2 0.4 0.6 0.8 1.0 1.2
MEF2C expression
Fold increase
Figure 1: XEN derived CDM inefficiently direct human CPCs towards a cardiomyocyte fate. (A) Hu- man Sca-1+ CPCs were treated for 24 hours with PKH26 labeled XEN derived GFP+ extracellular vesi- cles, showing that the mouse derived vesicles are taken up by the human cells. (B-C) qRT-PCR analysis for the early cardiac transcription factors GATA4 and MEF2C and the mature cardiac marker cTnT. (B) and (C) are two independent differentiation experiments and the error bars represent technical repli- cates. (D) Bright field pictures of cells either treated with TGFβ or XEN-CDM. (E) Immunofluorescent staining for cTnI of a cardiomyocyte-like cell derived by treatment with XEN-CDM (Scale bar: 50 µM).
Co-culture of XEN cells and Sca-1+ CPCs was unsuccessful due to the high
proliferation rate of XEN cells compared to non-proliferating/differentiating CPCs
(data not shown). Therefore, we focused on stimulating CPCs with XEN-CDM. Ex-
tracellular vesicles present in the CDM were labelled with PKH26 as desribed in
[9]. Upon adding XEN-CDM to human Sca-1+ CPCs, a clear uptake of vesciles was
seen, demonstrated by the intracellular presence of GPF and PHK26 (Figure 1A).
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174 A. A PPENDIX 3
This shows that mouse derived vesicles are taken up by human CPCs.
Subsequently, we investigated if XEN-CDM could influence the differentiation of human CPCs. Sca-1+ CPCs were treated with 5-azacytidine for 72 hours, as previ- ously described, followed by 3 weeks of culture in either differentiation medium [8]
or XEN-CDM. Medium was either left empty (CTRL) or supplemented with TGFβ3 and Asorbic Acid (TGF β/AA) or only Asorbic Acid (AA). Our data suggest that XEN- CDM does not induce efficient cardiomyocyte differentiation evident by the ab- sence or low expression of cardiac troponin T2 (cTnT; Figure 1B-C). The cells in all conditions remained positive for the cardiac transcription factors GATA4 and MEF2C (Figure 1B-C). In addition, cells cultured in the presence of XEN-CDM looked very different from cells subjected to the differentiation protocol as de- scribed in [8] (Figure 1D). However, cardiac troponin I3 (cTnI) immunofluorescent staining show the faint appearance of sarcomeric structures upon XEN-CDM treat- ment (Figure 1E).
In conclusion, our data suggest that XEN-CDM, within this setting, is not as po- tent as TGF β in the induction of cardiomyocyte differentiation. This may be due to the concentration of XEN-CDM used causing a misbalance in inductive signals.
Furthermore, although XEN cells express markers attributed to the AVE [2], accord- ing to the embryonic timeline, they are derived from the extraembryonic endoderm which is before the initiation of AVE. Therefore, XEN cells may not be fully mature in their ability to induce cardiomyocyte differentiation. Alternatively, CPCs may not be in the proper developmental stage in order to respond to XEN derived signals. Fi- nally, cardiomyocyte differentiation is sensitive to timing and CPC may be only for a short period of time responsive to XEN-derived signals.
D ERIVATION OF HUMAN XEN- LIKE CELLS .
Preferably the XEN cells used for induction of human cells towards the cardiomy- ocyte fate are from human origin. Therefore, we derived human XEN-like cells from human fetal extraembryonic tissue. Two clones were derived from a human week 17 extraembryonic membrane (Figure 2A) which was processed into a single cell suspension and cultured on culture plastic or 0.1% gelatin in XEN-medium con- taining 20 ng/ml Activin A. The clones had a cobblestone morphology (Figure 2B) and expressed the VE markers GATA6, HNF4 α and SOX7 (Figure 2C). However, fu- ture should repeat and confirm these findings and microarray analysis should be done to trace their identity.
M ETHODS
CPC ISOL ATION , CULTURE AND CARDIOMYOCY TE DIFFERENTIATION
Sca-1+ CPCs were isolated and cultured as previously described [8]. During car-
diomyocyte differentiation [3,7], cells were cultured in differentiation medium
A. A PPENDIX 3
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175
A B Culture plastic Gelatin
C
W17 (1) W17 (2) 0.0
0.2 0.4 0.6 0.8 1.0
GATA6
Relative expression (AU)
W17 (1) W17 (2) 0.00
0.02 0.04 0.06 0.08
HNF4α
Relative expression (AU)
W17 (1) W17 (2) 0.00
0.01 0.02 0.03 0.04
SOX7
Relative expression (AU)
