Derivation and characterization of blastocyst stem cells
To analyze the role of the developmental stage of the embryo on the developmental potential of embryo-derived stem cells, we explored whether novel cell lines could be derived from blastocysts under similar growth factor conditions as previously described for EpiSC- and human ES cell cultures (bFGF, ActivinA and BIO). Initial experiments using matrigel as substrate were unsuccessful, but when instead, blastocysts were hatched on MEFs in the presence of bFGF, ActivinA and BIO and a blocking antibody against murine LIF we were able to derive stable cell lines with novel properties, which we designated FAB-SCs, for bF
ctivin and B
IO-derived stem cells. If the zona pellucida was left intact, only 10% of the blastocysts hatched and upon trypsinization and passaging of the ICM outgrowths, a third of the hatched blastocysts yielded stable cultures that were homogenous in appearance (n=184). Removal of the zona pellucida prior to plating of the embryos improved the derivation frequency significantly, as 80% of the embryos demonstrated robust ICM outgrowth and upon passaging 30% of the original blastocysts yielded stable FAB-SC lines (n=99). It is interesting to note that under FAB-SC conditions ICM expansion was noticeable within 2 days after plating. In contrast, when blastocysts are plated under mES cell conditions, ICM expansion is delayed and occurs several days later. The difference in ICM outgrowth is not due to differences in cell proliferation rates, since FAB-SCs and mES cell proliferation rates are similar (not shown). Instead, the delay in ICM outgrowth under ES cell conditions may indicate that ES cell lines are derived from a small subpopulation of cells within the ICM while FAB-SC conditions allow the entire ICM to expand. Alternatively, the delayed ICM outgrowth in ES cell conditions may reflect a pause in cell proliferation associated with epigenetic reprogramming events that are essential for the derivation of mES cells (Kaji et al., 2006
). While speculative, this latter option would imply that such reprogramming does not occur under FAB-SC conditions.
FAB-SCs share features with EpiSCs and mES cells, yet are distinct from both
Unlike mES cell colonies, which have a characteristic 3-dimensional appearance of tight shiny colonies, FAB-SCs grew as monolayer colonies reminiscent of EpiSCs (). Q-PCR analysis of Oct4, Sox2 and Nanog demonstrated that all three pluripotency transcription factors were expressed in FAB-SC lines as well as in traditional mES cells (). We further confirmed the homogeneous expression and nuclear localization of these transcription factors using immunohistochemistry (). In addition, we confirmed the expression of the cell surface marker SSEA1 on the FAB-SCs (not shown) further demonstrating that the FAB-SC cultures homogeneously express molecular hallmarks of pluripotent cells. Although FAB-SCs are derived on a feeder layer of MEFs, in the presence of a blocking antibody to LIF, we cannot formally exclude that very low residual levels of LIF signaling are required for FAB-SC derivation. However, established FAB-SC lines can be maintained on gelatin or Matrigel coated dishes in serum-free media in the absence of LIF or BMP4, with sustained expression of pluripotent markers, demonstrating that these growth factors are not required for the maintenance of these cells. Finally, as we will demonstrate below, stimulation of FAB-SCs with LIF and BMP4 induces profound permanent phenotypic changes in these cells, arguing that FAB-SCs do not experience these growth factors during their derivation.
Blastocyst-derived stem cells (FAB-SC)
FAB-SCs are derived under culture conditions similar to those recently reported for the derivation of murine EpiSCs (Brons et al., 2007
; Tesar et al., 2007
), but FAB-SCs and EpiSCs originate from different developmental stages of the embryo. To chart similarities and differences among FAB-SCs, ES cells and EpiSCs, we performed global gene- and microRNA (miRNA) expression analysis on these cells.
Using a Luminex bead platform (Lu et al., 2005
), we analyzed the expression levels of >430 miRNAs in independent ES, EpiSC and FAB-SC clones. Heatmap analysis of these samples revealed clear differences in the global miRNA profiles of these three cell lines (). Interestingly, FAB-SCs express miRNAs recently shown to be ES cell specific, such as the miR-290 cluster (Houbaviy et al., 2003
), or enriched in self-renewing ES cells, such as miR-18a, miR-19a and miR- 20a (Hayashi et al., 2008
). In contrast, EpiSCs express low levels of these miRNAs. These data demonstrate that FAB-SCs express miRNAs typical of ES cells, reflecting the blastocyst origin of these cells. EpiSCs on the other hand, express several miRNAs associated with post-implantation development including miR-1 and miR-206 (associated with muscle development), and miR-150 and miR-142 (hematopoietic differentiation) (Chen et al., 2004
; Xiao et al., 2007
). The microRNA expression profiling of these pluripotent stem cell lines underscores the unique character of FAB-SCs and reflects their early developmental origin. EpiSCs on the other hand demonstrate the expression of miRNAs associated with early lineage commitment, in line with the post-implantation epiblast origin of these cells.
To further interrogate the similarities and differences between FAB-SCs, mES cells and EpiSCs, we performed microarray analysis of the transcriptional profile of these different cell lines. FAB-SCs express several known pluripotency factors, including Oct4, Sox2 and Nanog, at similar levels as mES and EpiSCs (). Yet, the expression of epiblast markers is absent or low in FAB-SCs compared to EpiSCs, demonstrating that while these cells are propagated under similar growth factor conditions, they are not the same (). FAB-SCs distinguish themselves from ES cells as well, as they do not express many of the genes associated with germ cell differentiation, such as Stella, Blimp1 or Dazl, which are commonly expressed in mES cells (). The mRNA expression analysis further demonstrates that FAB-SCs represent an alternative stable stem cell state that is distinct from both mES cells and EpiSCs.
FAB-SCs fail to demonstrate pluripotency in assays of development
We next tested the ability of FAB-SCs to generate derivatives of all three germ layers in in vitro
and in vivo
assays of development. Embryoid body (EB) formation is a simple and widely used method in which aggregates of pluripotent cells initiate a differentiation program that is reminiscent of early embryonic development (Doetschman et al., 1985
; Leahy et al., 1999
). In the context of the EB, molecular interactions that drive early embryonic development are recapitulated and cells differentiate to form ectoderm, endoderm and mesoderm derivatives.
Surprisingly, and in stark contrast to ES cells, EBs made from FAB-SCs remained small and failed to expand. We next interrogated the ability of FAB-SCs to form teratomas when injected into immunodeficient mice. FAB-SCs failed to form any teratomas at 3 months after injection (n=20) whereas teratomas formed within 1 month in all mice injected with ES cells (n=5).
Pluripotency is characterized by the ability of a stem cell to self renew indefinitely while maintaining the capacity to differentiate into derivatives of all three germ layers. While FAB-SC cultures display sustained expression of hallmark molecular markers of pluripotency >30 passages (), the cells fail to pass standard in vitro
and in vivo
tests of pluripotency such as EB differentiation or teratoma formation. As such, FAB-SCs are also molecularly and functionally distinct from primitive epiblast-like cells (EPL cells) that are derived when ES cells are cultured in the presence of HEPG2 conditioned medium (Rathjen et al., 1999
). Epiblast marker genes, which are expressed in EPL cells are low or absent in FAB-SCs and in contrast, FAB-SCs express the ICM marker Gbx2 and EPL cells do not. In addition, EPL cells are capable of forming teratomas, while FAB-SCs are not, excluding the possibility that FAB-SCs are EPL cells.
Growth factor stimulation induces FAB-SC pluripotency
To examine the influence of the growth factor milieu on FAB-SC pluripotency, we explored the effect of LIF and BMP4 stimulation on the ability of FAB-SCs to generate teratomas ().
LIF and BMP4 stimulate FAB-SC teratoma formation
To ensure a homogeneous FAB-SC population, we used flow-cytometry to sort single cell clones of FAB-SCs containing a GFP transgene into 96-well plates, and visually confirmed the presence of a single cell in each well. Clonal FAB-SCs were stimulated with LIF (100ng/ml) and BMP4 (40 ng/ml) for 1 week and injected subcutaneously into NOD-SCID mice (1×106 cells, n=7) to assess their teratoma-forming potential. While none of the native FAB-SC clones gave rise to teratomas, injection of LIF/BMP4 stimulated FAB-SC clones resulted in the formation of teratomas in all recipients (3 independent clones, n=7 for each clone). H&E staining and immunofluorescent detection of markers of germlayer differentiation demonstrated that the teratomas generated by the LIF/BMP4 stimulated FAB-SCs displayed derivatives of all three embryonic germlayers (, left panels), demonstrating that brief culture in LIF/BMP4 induces FAB-SC developmental potential. Interestingly, when the LIF/BMP4 stimulated FAB-SCs were returned back to the original FAB-SC growth factor conditions and cultured for another week, these “reverted” FAB-SCs retained the ability to generate teratomas (, right panels). Thus, the FAB-SC pluripotent state, induced by transient LIF/BMP4 stimulation, is retained even when the LIF/BMP4 stimulus is subsequently removed.
Blastocyst contribution by FAB-SCs
To further explore our observation that LIF/BMP4 stimulation induced FAB-SC pluripotency, we examined the effect of growth factor stimulation on the ability of FAB-SCs to form chimeras upon transfer into recipient blastocysts. Control- and LIF/BMP4 stimulated FAB-SCs, expressing the GFP transgene described above, were injected into blastocyst embryos and their integration into the recipient blastocyst was monitored over time. While the LIF/BMP4-exposed FAB-SCs integrated with the cells of the ICM within 6 hours after injection, unstimulated FAB-SCs remained dispersed in the blastocyst cavity, revealing that integration of FAB-SCs into the recipient embryos was impaired ().
Growth factor stimulation induces FAB-SC chimera formation and germline contribution
To further analyze the developmental potential of FAB-SCs before and after growth factor stimulation, we transferred the embryos into pseudopregnant females and analyzed chimerism by the expression of the GFP transgene and/or coat color. No chimerism was observed in any of the >320 pups from blastocysts injected with 10–12 FAB-SCs each. Sectioning and immunohistochemistry staining of the embryos using an anti-GFP antibody revealed no GFP contribution to the recipient embryos at mid-gestation (E9.5–E11.5). In contrast, even brief 48 hour stimulation of FAB-SCs with LIF and BMP4 induced the ability of these cells to form chimeras. Although the chimeric frequency was low (7 out of 254 pups), the chimeras demonstrated high (40–90%) FAB-SC contribution (, top panel). Mating with a wild-type female showed transmission of the FAB-SC-derived GFP transgene to the offspring, demonstrating that LIF/BMP4 stimulated FAB-SCs are capable of germline contribution as well (, middle panel). These embryo chimerism experiments were repeated with single cell-derived clonal FAB-SC lines. Again, no chimerism was observed upon blastocyst transfer of clonal FAB-SCs (n=160). However, LIF/BMP4 stimulation of clonal FAB-SCs for 7 days induced the ability of these cells to contribute to recipient embryos with a frequency similar to the parental cell line (not shown).
Finally, we analyzed whether FAB-SCs exposed to LIF/BMP4 for 1 week would contribute to chimera formation after further culture in bFGF, ActivinA and BIO. Clonal LIF/BMP4 stimulated FAB-SCs were cultured for an additional 7 days in FAB-SC media containing bFGF, ActivinA, BIO and anti-LIF antibody, and subsequently injected into recipient blastocyst embryos. GFP contribution by the injected reverted FAB-SCs was detected in 4 out of 123 embryos analyzed, with chimerism ranging from 20–80% (), demonstrating that pluripotency is retained even after removal of the LIF/BMP4 signal.
The above data demonstrate that LIF/BMP4 stimulation of FAB-SCs induces their ability to contribute to chimera formation. The relatively low number of chimeras obtained suggests that only a fraction of the FAB-SCs undergoes full growth-factor mediated conversion to the pluripotent state. Robust contribution by cells that do successfully undergo pluripotent conversion, including contribution to the germline, indicates however that the induced cells are truly pluripotent. Importantly, the ability of FAB-SCs to display this effect at the clonal level demonstrates that the LIF/BMP4 stimulated chimera formation is the result of an induction of FAB-SC pluripotency rather than clonal selection of “competent” cells from a heterogeneous starter population.
E-Cadherin is induced by transient LIF/BMP4 stimulation of FAB-SCs
The unique properties of FAB-SCs allowed us to further probe the molecular mechanism behind the induction of FAB-SC pluripotency by growth factor stimulation. Microarray comparison of the FAB-SC gene expression profile of three independent clonal FAB-SC lines, with the expression profile of LIF/BMP4 stimulated FAB-SCs demonstrated profound changes in gene expression (, upper panel). Comparison of FAB-SCs to cells that were transiently stimulated with LIF/BMP4 and subsequently reverted to the original growth conditions of bFGF, ActivinA and BIO enabled us to focus on gene expression changes linked to the induction of pluripotency (, lower panel). We compared the gene expression profiles of FAB-SCs, the LIF/BMP4 stimulated FAB-SC and the growth factor reversed FAB-SCs and searched for genes that were up- or downregulated at least 3-fold by LIF/BMP4 stimulation, and remained altered in the reverted FAB-SCs. Only a handful of genes displayed this expression profile consistently in 3 independent FAB-SC clones (). Four “hits” were genes involved in RNA translation and two were genes with unknown function. Cdh1 (E-Cadherin) displayed the most profound induction, as it demonstrated a 4–6 fold upregulation on all seven features of the microarray. Western analysis of E-Cadherin expression in FAB-SCs, LIF/BMP4 stimulated FAB-SCs and reverted FAB-SCs demonstrated that while FAB-SCs express low levels of E-Cadherin, LIF/BMP4 stimulation resulted in upregulation of E-Cadherin expression to comparable levels as those observed in mES cells, which was sustained in the reverted FAB-SCs ().
E-Cadherin (Cdh1) is induced by LIF/BMP4 stimulation of FAB-SC
E-Cadherin is a critical regulator of FAB-SC pluripotency
To further analyze the function of E-Cadherin in pluripotent stem cells, we generated lentiviral shRNA hairpins to examine the effect of downregulation of E-Cadherin expression on the ability of FAB-SCs to generate teratomas. shRNA hairpins were tested to functionally downregulate E-Cadherin expression in mES cells (Supplemental figure 1A
). While E-Cadherin expression was unaffected in the control hairpin, two E-Cadherin hairpins (Cdh1-SH3 and Cdh1-SH4) demonstrated >90% downregulation of E-Cadherin expression and were selected for further experiments. Functional knockdown of E-Cadherin expression was tested in a cell aggregation assay. Cadherins mediate cell-cell adhesion through homotypic interactions. Two cell populations expressing similar levels of the same cadherin will form homogeneous aggregates, while dissimilarities in the nature or level of cadherin expression will result in segregation of the two cell types (Gibralter and Turner, 1985
; Takeichi et al., 1981
). While ES cells transduced with the control shRNA hairpin formed homogeneous aggregates when mixed with wild-type ES cells, ES cells expressing the E-Cadherin hairpins did not mix with the wild-type cells, but instead aggregated in spatially separate domains, demonstrating functional consequences of E-Cadherin knock-down in these cell lines (Supplemental figure 1B
). Next we explored the effect of knockdown of E-Cadherin expression on LIF/BMP4 stimulated FAB-SCs, but we were unable to establish stable clones using the E-Cadherin knockdown hairpins. Transduction of LIF/BMP4 stimulated FAB-SCs with the control vector yielded stable clones expressing the tdTomato reporter gene present in the lentiviral shRNA vector (). Loss of E-Cadherin has been reported to induce anoikis mediated apoptosis in certain cancer cell lines, yet no difference in apoptosis was observed between the control and E-Cadherin knockdown samples (not shown). When we analyzed the transduced cells by fluorescent microscopy however, we observed a striking difference in the morphology of the E-Cadherin knockdown cells compared to control. In the control sample, tdTomato-positive (Lentiviral transduced) cells proliferated as undifferentiated colonies (, lower panels). In contrast, stimulated FAB-SC cells transduced with the E-Cadherin hairpins had a fibroblast-like morphology, demonstrating that downregulation of E-Cadherin resulted in rapid FAB-SC differentiation (, upper panels, arrows).
E-Cadherin regulates FAB-SC pluripotency
Induction of FAB-SC pluripotency by ectopic expression of E-Cadherin
The upregulation of E-Cadherin expression in FAB-SCs following LIF/BMP4 stimulation correlates with the induction of the ability of these cells to form teratomas containing derivatives of all three germ layers. We next examined whether this upregulation of E-Cadherin expression is sufficient to induce the teratoma forming potential of FAB-SCs. FAB-SCs transduced with a lentiviral vector expressing E-Cadherin formed teratomas in immunodeficient mice after one month (5 out of 7), whereas none of 7 mice injected with control FAB-SCs developed teratomas. H&E staining and immunofluorescent detection of markers of germlayer differentiation demonstrated that the teratomas generated by the E-Cadherin FAB-SCs displayed derivatives of all three embryonic germ layers (). These data demonstrate that overexpression of E-Cadherin alone is sufficient to induce robust teratoma-forming potential in FAB-SCs and suggest that a key target of LIF/BMP4 stimulation is upregulation of E-Cadherin expression.
Accelerated ES cell differentiation in the absence of E-Cadherin expression
Above data demonstrate that E-Cadherin plays an important role in regulating the FAB-SC pluripotent state. To test whether abrogation of E-Cadherin expression would compromise the pluripotency of mES cells, we generated stable mES cell lines expressing the control or Cdh1 knockdown hairpins. Downregulation of E-Cadherin expression in mES cells changes the morphology of the cells from the tight, 3-dimensional colony shape to a more flattened colonies of loosely connected cells, very much resembling FAB-SCs (Supplemental Figure 2A
). ES cells stably expressing the E-Cadherin hairpins could be propagated for over 20 passages and the proliferation and apoptotic rates between the control and E-cadherin knockdown ES cell lines were comparable (see below) suggesting knockdown of E-cadherin did not impair ES self-renewal, proliferation or apoptosis. Using immunohistochemistry we analyzed the expression of Oct4 in the control and E-Cadherin knockdown ES cell lines (Supplemental Figure 2B
). No differences in the level, localization or percentage of Oct4 expression were observed between control and E-Cadherin knockdown ES cells. Furthermore, even cells that demonstrated a rounded-up clustered colony morphology were expressing Oct4 (Arrowheads, supplemental Figure 2B
). Thus, while knockdown of E-Cadherin expression in traditional mES cells results in a change in morphology and loss of tight adhesion of the ES cells, loss of E-Cadherin does not impair ES cell self-renewal. Like FAB-SCs, the E-Cadherin knock-down ES cells formed small EBs (). Moreover, whereas E-Cadherin knock-down ES cells form teratomas upon subcutaneous injection into NOD-SCID mice, loss of E-Cadherin expression results in a profound reduction in teratoma size, as measured by the weight of the teratomas (). Analysis of the teratomas revealed multilineage differentiation however, indicating that differentiation of the Cdh1 knockdown ES cells per se was not impaired (not shown). The reduced size of the E-Cadherin knock-down EBs was not due to decreased proliferation or increased apoptosis of these cells (). Finally we analyzed the expression of pluripotency markers Oct4, Sox2 and Nanog before and during differentiation of control ES cells or the E-Cadherin knockdown cell lines. While we observed no difference in the downregulation of Oct4 and Sox2 expression, Western blot analysis revealed an accelerated loss of Nanog protein expression in the absence of E-Cadherin expression as compared to control cells (). The rapid loss of Nanog expression in the E-Cadherin knockdown cells was also observed when we monitored Nanog RNA levels by Q-PCR (). Oct4, Sox2 and Nanog have been shown to bind to promoter elements of genes involved in early cell fate decisions (Boyer et al., 2005
; Boyer et al., 2006
). The concerted binding of these transcription factors mediates the recruitment of the polycomb silencing complex, thereby suppressing gene expression. Thus part of the role Oct4, Sox2 and Nanog play in ES cell self-renewal is to prevent the expression of genes associated with differentiation, such as genes in the Hox-cluster. Since loss of E-Cadherin results in early downregulation of Nanog upon cell differentiation, we analyzed the expression of HoxA1 and HoxB1 in these cells. Upregulation of HoxA1 and HoxB1 is a hallmark sign of cell differentiation toward the somatic lineages and distinguishes pluripotent cells from early somatic cells in the primitive epiblast (Saitou et al., 2002
; Yabuta et al., 2006
). While we observed no upregulation of HoxA1 expression during early differentiation of control, or E-Cadherin knockdown ES cells, HoxB1 expression was upregulated prematurely in both E-Cadherin knockdown cell lines compared to control (). Together our data demonstrate that loss of E-Cadherin expression results in rapid differentiation of FAB-SCs, and while mES cells can maintain their pluripotent state in the absence of E-Cadherin expression, they demonstrate an accelerated loss of Nanog expression and premature upregulation of HoxB1 expression upon induction of differentiation.
Loss of E-Cadherin expression compromises EB- and teratoma formation by accelerating ES cell differentiation