Klf4在维持胚胎干细胞干性方面的研究

1、Klf4在维持胚胎干细胞干性方面的研究Epiblast Mammalian embryos produce extraembryonic cells prior to defining the founder population for the embryo proper (Gardner,1983; Selwood and Johnson, 2006). The primary role of the extraembryonic lineages is to mediate uterine implantation and subsequent maternal sustenance o

2、f the growing embryo and fetus. In recent years, it has been discovered that extraembry-onic tissues also supply powerful inductive signals that specify and pattern early development (Beddington and Robertson,1999). To form the embryo, a pool of uncommitted cells must be established and poised to re

3、spond to those signals. This population is the epiblast. The newly formed epiblast is a cluster of 1020 unspecialized cells sandwiched between the trophoblast and the hypoblast.The Limitation of Totipotency Because it can give rise to an entire embryo, the mammalian egg is often described as totipot

4、ent. However, this description does not mean that the egg itself has the ability to differentiate into all cell types. Thus, the egg and blastomeres produce directly only two cell types, the trophoblast and the inner cell mass (ICM).For subsequent development, cells within the ICM must acquire the c

5、apacity to generate other cell types and to do so in a flexible manner (Gardner and Beddington, 1988). The ICM produces a second extraembryonic lineage, the hypoblast, and around the same time, the remaining cells develop into pluripotent epiblast.The epiblast is functionally and molecularly distinc

6、t from blastomeres and early inner cell mass (Gardner, 1998;Kaji et al., 2007; Kurimoto et al., 2006). Thus, rather than representing a diminution in potency from the egg,we suggest that the epiblast constitutes the ground state, meaning a fully unrestricted population that harbors the requisite dev

7、elopmental potency and flexibility to produce all embryonic lineages. l Nichols and Smith have suggested that the two types of ESC differ fundamentally in the gene networks that maintain their pluripotency and named the LIF-dependent ESC “naive”and the FGF2-dependent type “primed.”The Ground State a

8、nd True Embryonic Stem Cells The newly formed epiblast is a cluster of 1020 unspecialized cells sandwiched between the trophoblast and the hypoblast. The epiblast generates the entire fetus and single mouse epiblast cells, isolated at this stage and microinjected into another blastocyst, can contrib

9、ute to all lineages(Gardner, 1998). Functionally, therefore, preimplantation epiblast is the developmental ground state. Embryonic stem cells (ESCs) can be derived at this point(Evans and Kaufman, 1981; Martin, 1981). ESCs represent immortalization of the naive epiblast. Under appropriate conditions

10、, they exhibit unlimited selfrenewal capacity while retaining the attributes of preimplantation epiblast identity and potency. Specifically, when returned to the blastocyst, ESCs are readily incorporated into the epiblast and re-enter embryonic development to produce functional soma and germ cells (

11、Bradleyet al., 1984). ESCs also share an epigenetic feature with preimplantation epiblast. This trait is the presence of two active X chromosomes in female cells. In female embryos, the paternally inherited X chromosome is silenced during cleavage and remains silent in extraembryonic lineages. React

12、ivation occurs transiently in the pluripotent lineage prior to implantation (Heard,2004). The Primed Pluripotent State After uterine implantation, the rodent epiblast converts into a single-cell layer of columnar epithelium (Kaufman, 1992). This conversion is associated with a morphological transfor

13、mation into a cup-shaped structure known as the egg cylinder . The epiblast is displaced downward after implantation due to proliferation of the trophecto-derm-derived extraexembryonic ectoderm and the constraint of the uterinewall. In XX embryos, one of the X chromosomes undergoes random inactivati

14、on in early egg cylinder epiblast cells (Heard, 2004). The epiblast is then subject to a systematic topological bombardment with inductive factors emanating from the adjacent yolk sac and trophoblast tissue (Beddington and Robertson, 1999). Egg cylinder epiblast cells, therefore, become instructivel

15、y specified according to their location. However, postimplantation epiblast cells cannot contribute to blastocyst chimeras (Rossant, 2008), nor can they give rise to ESCs. Cell lines have now been derived from postimplantation mouse epiblasts using culture conditions without Lif but including Fgf an

16、d activin (Brons et al., 2007; Tesar et al., 2007). These cells, termed EpiSCs, express core pluripotency factors,Oct4, Sox2, and Nanog, but differ from ESCs in expression of several other transcripts. However, EpiSCs are not competent to contribute to blastocyst chimeras (Guo et al., 2009; Tesar et

17、 al., 2007) and are, therefore, developmentally and functionally distinct from naive epiblast and ESCs . EpiSCs can also be produced from ESCs in culture (Guo et al.,2009).Consistent with a true differentiation event, one copy of the X chromosome in XX cells is epigenetically silenced as ESCs become

18、 EpiSCs. However,EpiSCs still express the canonical pluripotency factors and canbe reprogrammed to naive pluripotency by transfection with just a single factor, Klf4 (Guo et al., 2009). The resulting iPSCs show reactivation of the X chromosome, exhibit the ESC-specific transcriptional profile, produ

19、ce high contribution somatic chimeras, and give germline transmission. Ground state naive pluripotency is established in the epiblast of the mature blastocyst and may be captured in vitro in the form of ESCs. Shortly after implantation, the epiblast transforms into a cup-shaped epithelium and become

20、s primed for lineage specification and commitment in response to stimuli from the extraembryonic tissues. EpiSCs are the in vitro counterpartof primed epiblast. ESCs can be induced to differentiate into EpiSCs by exposure to activin and Fgf, but the reverse transition requires transfection with Klf4

21、 or other reprogramming factors. INTRODUCTIONES and EpiSCsSimilarities:l Both ES cells and EpiSCs are capable of multilineage differentiation in vitro and can form teratomas when grafted into adult mice (Brons et al., 2007; Tesar et al., 2007). l Both cell types express the three transcriptionalregu

22、lators, Oct4 (Pou5f1 MouseGenome Informatics), Sox2 and Nanog, that are generally considered to constitute the core pluripotency network (Boyer et al.,2005; Loh et al., 2006; Wang et al., 2006).Differences:l However, there are significant differences in gene expression between ES cells andEpiSCs (Te

23、sar et al., 2007). l Furthermore, the culture conditions for maintaining the two cell types are quite distinct. l ES cells self-renew in response to the cytokine leukaemia inhibitory factor (Lif) (Smithet al., 1988; Williams et al., 1988) and either serum, bone morphogenetic protein, or the inhibiti

24、on of Mek/Erk signalling(Burdon et al., 1999; Ying et al., 2003; Ying et al., 2008). They aredriven into differentiation by FGF/Erk signalling (Kunath et al.,2007; Stavridis et al., 2007). l EpiSCs, by contrast, are maintained byFGF and activin (Brons et al., 2007).MATERIALS AND METHODSEpiSC derivat

25、ion and cultureEmbryonic stem cell and induced pluripotent stem (iPS) cell culturePiggyBac vector transfectioniPS cell induction and propagationRT-PCRTaqman probesChimaera productionRESULTS AND DISCUSSION(A) Phase contrast and fluorescence images of established EpiSC line We derived EpiSCs from E5.7

26、5 mouse embryos carrying the Oct4GiP transgene (Ying et al., 2002). Cell lines were established and maintained without feeders in serum-free N2B27 medium (Ying and Smith, 2003) supplemented with activin A and Fgf2 (bFGF)(Brons et al., 2007). Fig. 1. EpiSCs are distinct from, and do not spontaneously

27、 convert to, ES cells. (B) qRT-PCR analysis of marker gene expression in ES cells and EpiSCs. ES, ES cells in 2i/Lif. Epi6 and Epi7 are two independent EpiSC lines. y-axis, relative expression normalised to Gapdh.(C) Immunostaining of male and female EpiSCs for me3H3K27 and Oct4. White arrow indicat

28、es focus of staining diagnostic of an inactive X chromosome.(D) EpiSCs lose Oct4 expression and differentiate or die in 2i/Lif. AF, EpiSCcultured in activin A plus Fgf2We conclude that the EpiSC represents a stable cell state that does not naturally revert to nave pluripotent status.(E) qRT-PCR anal

29、ysis of ES cell differentiation into EpiSCs upon culture in Fgf2 and activin. Epi3 and Epi10 indicate cells cultured in Fgf2 and activin for three and ten passages, respectively. y-axis, relative expression normalised to Gapdh.(F)Oct4 and me3H3K27 immunostaining of female ES cell-derived EpiSCs. Epi

30、SCs both express Oct4 and exhibit a nuclear body indicative of the inactive X (white arrow).Blue arrow indicates a dividing cell. (A) qRT-PCR analysis of Lif induction of Klf4 in ES cells but not in EpiSCs. Cells were stimulated with Lif (+LIF) for 1 hour.(B) ES cells constitutively expressing Klf4

31、acquire an EpiSC marker profile in Fgf2 plus activin A. MT, empty vector transfectants. P0, P2 and P10 indicate passage numbers in Fgf2/activin.Fig. 2. Klf4 neither prevents ES cell differentiation into EpiSCs nor converts an EpiSC population into ES cells in the presence of activin and FGF. This in

32、dicates that forced expression of Klf4 does not preventconversion into EpiSCs. (C) Constitutive Klf4 expression permits continued recovery of ES cell colonies after culture in activin and Fgf2. One thousand cells were plated for each sample in triplicate at the indicated passage (P) number. MT, empt

33、y vector transfectants; K4, Klf4 transfectants.Therefore, constitutive Klf4 either allows long-term persistence of a small fraction of undifferentiated ES cells,or enables a fraction of EpiSCs to dedifferentiate and regain the ground state.(D) PiggyBac vector for expression of Klf4 (pGG137Klf4), and

34、 control PiggyBac vector (pGG131). Arrows (P) indicate PCR primers used to amplify the PB LTR fragment after Cre-mediated recombination.(E) Hygromycin-selected Klf4 and control vector-transfected EpiSCs.(F) qRT-PCR analysis showing that forced Klf4 expression does not induce ES cell marker gene expr

35、ession in EpiSC culture. ES, ES cells; Epi, EpiSCs; Vec, EpiSC transfected with control vector pGG131; Klf4, EpiSCs transfected with pGG137Klf4. y-axis, relative expression normalised to Gapdh. We conclude that the expression of Klf4 at a similar RNA level to that present in ES cells is not alone su

36、fficient to reset EpiSCs and instate full pluripotency in cells maintained in activin and Fgf2.Fig. 3. EpiSCs transfected with Klf4 can convert to ground state pluripotency. (A)Oct4-positive colonies obtained by transfection with Klf4 and transfer to 2i/Lif after 72 hours. Images were taken after 9

37、days in 2i/Lif.(B) qRT-PCR analysis of marker gene expression in ES cells, EpiSCs and derivative Epi-iPS cells isolated in 2i/Lif. y-axis, relative expression normalised to Gapdh. qRT-PCR analysis showed the marker profile of ES cells, with upregulation of Stella and Klf2.Conversely, Fgf5 and brachy

38、ury mRNAs were lost.(C)me3H3K27 staining of female EpiSCs and derivative Epi-iPS cells. We examined me3H3K27 immunostaining and found that the nuclear body corresponding to the inactive X chromosome was lost in Oct4-GFP-positive cells after transfer to 2i/Lif (Fig. 3C). However, in each of the GFP-p

39、ositive clones we observed partial or complete loss of visible DsRed expression (Fig. 3D,E), although qRT-PCR analysis revealed that the transgenes were not completelysilenced (Fig. 3F).(G) Chimeric mouse produced from the K4C12 Epi-iPS clone and agouti germline offspring.This confirms that the deve

40、lopmental capacity has been fully derestricted and the authentic pluripotent state established. These cells should therefore be considered as EpiSC-derived iPS cells, or Epi-iPS cells.Fig. 4. Retention of ground state pluripotency after transgene excision. (A) Splinkerette-PCR reveals 1-3 PB inserti

41、ons in each iPS clone.lWe chose two DsRed-positive clones and transfected each with a Cre expression plasmid. After 5 days, cells that no longer expressed DsRed were isolated using flow cytometry with single-cell deposition into 96-well plates (Fig. 4B). (B) Flow cytometry showing the DsRed-negative

42、 population in the K4C3 line before and after Cre transfection. (C)Genomic PCR showing loss of the Klf4 transgene and gain of the PB-LTR fragment in two revertant clones.(D) RT-PCR analysis showing the lack of Klf4 transgene and DsRed expression in expanded Cre-reverted cells.lTwo thirds of the expa

43、nded clones retained only the PB terminal repeats. RT-PCR analysis failed to detect expression of the Klf4 transgene or DsRed from these revertants (Fig. 4D). lThey retained ES cell morphology, Oct4-GFP expression and ES cell marker profile(Fig. 4E,F). (F)Maintained morphology and Oct4-GFP expressio

44、n in a Cre-reverted Epi-iPS cell line.(G)me3H3K27 staining of Klf4 transgene-deleted iPScells as compared with parental EpiSCs. (H) Chimeric mouse made with revertant K4C3-A3 cells, and agouti offspring denoting germline transmission.l Female chimaeras from two out of three clones produced agouti of

45、fspring in their first litter (Fig. 4H),indicative of transmission of iPS cell-derived oocytes. lTherefore,complete removal of the Klf4 transgene does not destabilise the induced ground state. This establishes that reprogramming has been finalised and does not depend upon ongoing transgene expressio

46、n or insertional mutagenesis. Human and mouse embryonic stem cells (ESCs) are derived fromblastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). mEpiSCs and hESCs share a flattened morphology, intolerance to passaging as single cells, dependence on TGF/Activin signaling (15), inactivation of the X chromosome in most female cell lines isolated (16), and a high propensity todifferentiate into PGCs in