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WO2018138281A1 - Cellules souches pluripotentes - Google Patents

Cellules souches pluripotentes Download PDF

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WO2018138281A1
WO2018138281A1 PCT/EP2018/051990 EP2018051990W WO2018138281A1 WO 2018138281 A1 WO2018138281 A1 WO 2018138281A1 EP 2018051990 W EP2018051990 W EP 2018051990W WO 2018138281 A1 WO2018138281 A1 WO 2018138281A1
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cells
formative
stem cell
stem cells
cell
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WO2018138281A8 (fr
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Austin Smith
Masaki Kinoshita
Ge Guo
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Cambridge Enterprise Ltd
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Cambridge Enterprise Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates generally to methods and materials for use in deriving and propagating pluripotent stem cells in the "formative" state.
  • the invention further provides such cells, and uses thereof.
  • Mammalian embryos develop from pluripotent cells that are first set aside as "naive" founders in the blastocyst, subsequently gain competence for multi-lineage induction, and finally acquire lineage-specific "priming" prior to commitment.
  • ES embryonic stem
  • EpiS epiblast-derived stem
  • ES embryonic stem
  • EpiS post- implantation epiblast-derived stem
  • Naive pluripotent stem cells such as mouse ES cells
  • Primed pluripotent stem cells show features of gastrulating epiblast cells. They are considered to represent a late form of pluripotency poised for
  • ES and EpiS cells are therefore considered to represent distinct naive and primed stem cell states respectively (Nichols and Smith, 2009).
  • Naive and primed pluripotent stem cells show global differences in gene expression, epigenetic features, metabolism, embryo colonisation potential, and various other properties. They have distinct culture requirements, for example, dual inhibition of MEK and GSK3 plus the cytokine leukaemia inhibitory factor ("2i+LIF") for mouse ES cells (Wray et al., 201 1 ; Ying et al., 2008) and high dose activin with fibroblast growth factor and optionally Wnt inhibition (“AhiF+XAV”) for primed EpiS cells (Kurek et al., 2015; Sumi et al., 2013; Tsakiridis et al., 2014).
  • 2i+LIF cytokine leukaemia inhibitory factor
  • ES cells are self-renewing pluripotent cell lines derived from pre-implantation embryos (Evans and Kaufman, 1981 ; Martin, 1981 ). They undergo continuous division in culture while retaining the capacity to enter into multi-lineage differentiation both in vitro and upon return to the embryo.
  • ES cells can be propagated at scale in a substantially homogeneous condition termed the naive ground state in 2i+LIF medium. Naive ES cells are thereby suspended in a specific time window of early development (Boroviak et al., 2014; hackett and Surani, 2014). Naive cells such as ES cells can be differentiated into primed cells in culture while primed cells can be reverted to naive status by genetic manipulation and/or culture perturbations.
  • pluripotent cells become responsive to germline induction either by cytokines or by forced transcription factor expression (Hayashi et al., 201 1 ; Nakaki et al., 2013).
  • naive ES cells cannot respond to such stimuli but must be differentiated for 24-48hrs into a post-implantation epiblast-like cell (EpiLC) state to acquire responsiveness.
  • the period of formative pluripotency in the mouse embryo is postulated to extend from the late pre-implantation epiblast at around E4.75 until regionalised patterning becomes evident in the egg cylinder from around E5.75.
  • This window epiblast cells remain unpatterned and without expression of exclusive lineage-specification factors. They have completely down-regulated the naive pluripotency transcription factor circuitry and can no longer give rise directly to ES cells (Boroviak et al., 2014; Boroviak et al., 2015; Brook and Gardner, 1997).
  • Primed pluripotent EpiS cells have been derived by culture in medium supplemented with Knockout serum replacement (KSR) or high levels of activin (10-20 ng/ml) together with fibroblast growth factor (FGF, 10-12.5ng/ml) on a feeder cell layer (Brons et al., 2007; Tesar et al., 2007) or without feeders (Guo et al., 2009).
  • KSR Knockout serum replacement
  • FGF fibroblast growth factor
  • established EpiS cell lines are distinct from E5.5 epiblast. Notably they display global transcriptome similarity to gastrula-stage primed epiblast at E7.0, from which they can also be derived efficiently (Kojima et al., 2014; Osorno et al., 2012).
  • EpiS cells do not respond to primordial germ cell induction unlike E5.5 epiblast or EpiLC (Hayashi et al., 201 1 ; Murakami et al., 2016; Ohinata et al
  • the present inventors have investigated whether pluripotency progression may be paused at the intermediate "formative" phase described above. They hypothesised that global regulatory remodelling during the formative transition instates the molecular machinery for multi-lineage commitment and subsequent differentiation. Notably, the abrupt elimination of naive transcription factors which control the ES cell state means they no longer play a role.
  • the formative remodelling of pluripotency is proposed to generate a group of cells that are uniformly equipped to respond to patterning and lineage specification cues.
  • formative pluripotency may be manifest in profound transcriptional and epigenetic resetting relative to naive pluripotency, lack of lineage-affiliated transcriptional activity characteristic of primed pluripotency, and different requirements for propagation compared to earlier naive or later primed pluripotency.
  • the disclosure herein provides methods for generating a discrete pluripotent cell population that is molecularly and functionally distinguishable from previous
  • heterogeneous pluripotent cultures and from defined naive ES cells or primed EpiS cells. It is believed that these culture conditions described herein sustain a stable pluripotent stem cell state corresponding to the formative phase of pluripotency in the embryo.
  • the invention thus provides cells which possess the expected core features of formative pluripotency, but which can be maintained as continuous stem cell cultures. These cells and lines are termed formative stem (FS) cell lines herein.
  • FS formative stem
  • Provided herein are processes for producing FS cell lines from precursor pluripotent stem cells, and processes for propagating FS cell lines. This is achieved by suppressing signals for lineage specification whilst sustaining proliferation via autocrine FGF stimulation.
  • the inventors have utilised developmental ⁇ appropriate minimal activation of the nodal/activin pathway combined with autocrine stimulation of the FGF/Erk pathway and inhibition of the Wnt pathway.
  • Preferred media further include a pan-retinoic acid receptor inverse agonist, (RARi) and a y-secretase inhibitor.
  • the ability to capture or derive and propagate stable and homogenous cell lines in the formative pluripotent state is unexpected because it was previously considered that the natural formative phase is transient and may not represent a stable attractor state, since formative cells quickly become primed in mouse embryos and there is no precedent for pausing development at this stage in rodents or primates.
  • the formative pluripotent state is sustained by autocrine low level activation of nodal/activin/TGF- ⁇ signalling and of the FGF/Erk MAP kinase pathway and that progression to primed pluripotency is blocked by Wnt inhibition and RAR inhibition.
  • Low level exogenous activin or other TGF( superfamily member) is normally required, potentially together with autocrine nodal.
  • receptor stimulation may be dispensable in the presence of a y-secretase inhibitor.
  • FS cell lines can be provided from different species, including from human cells, and different cell types, including embryonic naive pluripotent stem cells, and other pluripotent cell types, as well as pluripotent cell lines and reprogrammed somatic cells.
  • Formative pluripotent cells as defined herein lack expression of naive pluripotency factors or gastrulation markers. At the whole transcriptome level they cluster apart from naive pluripotent cells and primed pluripotent cells. Functionally they display robust multi- lineage differentiation in vitro and unlike either ES or EpiS cells they respond directly to germ cell induction. Furthermore, mouse formative stem cells show widespread contribution to chimaeras after blastocyst injection.
  • FS cells are believed to retain imprinted gene status.
  • Current human naive pluripotent stem cells do not stably retain imprints.
  • one advantageous embodiment of the invention for obtaining a fully competent human pluripotent stem cell involves transient derivation or resetting to naive status then conversion to formative status before erasure of imprints occurs in the naive state.
  • FS cells may therefore be a superior or alternative source material for chimaera formation or directed differentiation compared with other forms of pluripotent stem cell.
  • Formative pluripotent stem cells may be a desirable intermediate stage for population expansion prior to directed differentiation of human naive pluripotent stem cells.
  • FS cells Further utilities of the FS cells are described hereinafter. While multiple conditions have previously been described for propagating rodent and primate pluri potent stem cells, these primarily result in heterogeneous populations. The notable exception is the 21+LIF culture system for mouse naive ES cells (Ying et al., 2008)) and the recently reported conditions for human naive pluripotent stem cells (Guo et al., submitted; Guo et al., 2016; Takashima et al., 2014; Theunissen et al., 2014)
  • FGF with KSR or high concentrations of activin (or TGF(3) yield cell populations that are exclusively or predominantly primed (Brons et al., 2007; Tesar et al., 2007; Wu et al., 2015a), or are mixed and metastable, such as mouse ES cells in serum (Enver et al., 2009; Filipczyk et al., 2015; Fishell, 1995; Marks et al., 2012).
  • Such cultures may in principle contain minority sub-populations of uncharacterised cells that have features of formative pluripotency, but those cells have not been identified in any previous study, and the mixtures are sub-optimal for initiating multi-lineage differentiation or chimaera formation, and cannot reliably be standardised.
  • the present specification provides conditions for deriving and propagating cells which sustain substantially homogeneous cell populations that: (i) are genetically and phenotypically stable; (ii) exhibit consistent characteristics and behaviour between cell lines; (iii) display robust multi-lineage differentiation; (iv) are distinct from previously well- characterised naive and primed pluripotent stem cell cultures.
  • the invention provides a process for producing a formative stem (FS) cell line, from one or more precursor pluripotent cells, which process comprises:
  • Formative stem cell culture media allows proliferation via autocrine or limited exogenous FGF stimulation while suppressing signals for lineage specification.
  • FS cell culture medium provides developmental ⁇ appropriate moderate stimulation of the FGF/Erk pathway by autocrine ligands, FGF4 and FGF5, optionally supplemented with exogenous FGF (typically less than 10ng/ml e.g. 0.5-5ng/ml, or 0.5 to 3 ng/ml), together with low level activation of the nodal/activing/TGF- ⁇ pathway using less than 5ng/ml exogenous activin A (e.g. 1-4ng/ml, e.g. 1.5-3 ng/ml) or nodal or TGF- ⁇ family member.
  • exogenous FGF typically less than 10ng/ml e.g. 0.5-5ng/ml, or 0.5 to 3 ng/ml
  • exogenous activin A e.g. 1-4ng/ml, e.g. 1.5-3 ng/ml
  • nodal or TGF- ⁇ family member e.g. 1-4ng/ml, e.g. 1.5-3 ng
  • the FS cell culture medium preferably includes an inhibitor of the retinoic acid receptor (RAR) e.g. a selective inverse agonist such as BMS 493 (0.1- 1.0 ⁇ ).
  • RAR retinoic acid receptor
  • a preferred FS cell culture medium comprises:
  • Primed pluripotent stem cells are highly dependent on exogenous stimulation of the nodal/activin/TBF
  • a medium typically used for mouse and human primed cells is supplemented with an activator of the Erk/MAP kinase pathway (typically FGF at 4-12.5ng/ml)) and also of the activin/nodal/TGF- ⁇ pathway (typically activin at 10-20ng/ml, or TGF- ⁇ at 2ng/ml) which may also be provided by MEF feeders (the concentration of activin A secreted by feeders is 6.2-15.0 ng/ml (Kojima et al., 2014) and feeders also secrete TGF-[5s) and/or KSR or serum.
  • an activator of the Erk/MAP kinase pathway typically FGF at 4-12.5ng/ml
  • the activin/nodal/TGF- ⁇ pathway typically activin at 10-20ng/ml, or TGF- ⁇ at 2ng/ml
  • MEF feeders the concentration of activin A secreted by feeders is 6.2-15.0 ng/ml (Kojima
  • pluripotent cells such as early post-implantation mouse epiblast (E5.5) cells can be converted into stem cell lines that retain formative properties by culture on a fibronectin substrate in defined medium and can be expanded long term (>2 passages) in this medium while retaining diploid karyotype.
  • E5.5 early post-implantation mouse epiblast
  • this medium based on 'low activin plus XAV may be termed "A 0 X" herein.
  • a process of the invention may comprise use of a formative stem cell culture medium which comprises:
  • activin at a concentrations of 1 - 4ng/ml, more preferably 1 .5 - 3 ng/ml;
  • a wnt inhibitor to suppress Wnt signalling e.g. XAV939, e.g. at 2 ⁇ , or IWP2 at 2 ⁇ .
  • the formative stem cell culture media comprises no exogenous stimulator of the FGF pathways.
  • the formative stem cell culture media comprises a Wnt inhibitor which is a tankyrase inhibitor e.g. XAV939, optionally at 0.5-10 iiM e.g. 2 ⁇ .
  • retinoic acid (10 "7 M) induced differentiation of FS cells. It is desirable to suppress this endogenous retinoid signalling which can be achieved by addition of a retinoic acid receptor (RAR) inhibitor.
  • RAR retinoic acid receptor
  • addition of RAR inhibitor stabilises formative pluripotent stem cells in the presence of very low concentrations of activin.
  • One preferred inhibitor is a pan-retinoic acid receptor inverse agonist such as BMS 493 applied at a concentration range of 0.1-1 . ⁇ e.g. 0.5 ⁇ .
  • Other RAR inhibitors may include LG100815 (Stavridis et al., 2010). AGN 193109, AGN194310 .
  • inhibitors of the related pan-RXR receptors such as UVI 3003 (0.1 -1. ⁇ ) were not effective in conditions where BMS493 was effective.
  • BMS493 could block neural differentiation of FS cells cultured in A to X with addition of 1 X10 ' M Retinoic acid, whereas UVI3003 could not.
  • a preferred culture condition for FS cells comprises low activin, Wnt inhibition, retinoic acid receptor inhibition and gamma-secretase inhibition.
  • this medium may be termed "A 0 X+RARi+ySi" herein, and may optionally inclusion of a low to moderate concentration of FGF (e.g. ⁇ 5, 4, 3, 2, or 1 ng/ml).
  • FGF e.g. ⁇ 5, 4, 3, 2, or 1 ng/ml
  • the formative stem cell culture media further comprises RARi e.g. BMS 493, optionally at 0.1-1.0 ⁇ .
  • the formative stem cell culture media further comprises a ⁇ - secretase inhibitor e.g. CAS 209984-56-5 at 1 .7 ⁇ -1.0 ⁇ e.g. 1.7nM to 0.1. uM. e.g. 0.1 LiM.
  • a ⁇ - secretase inhibitor e.g. CAS 209984-56-5 at 1 .7 ⁇ -1.0 ⁇ e.g. 1.7nM to 0.1. uM. e.g. 0.1 LiM.
  • the y-secretase inhibitor may be CAS 209986-17-4 or other commercially available agent.
  • the formative stem cell culture media may be based on N2B27 medium (Ying and Smith, 2003) or other media known in the art designed for serum free culture of pluripotent stem cells, such as E6 or SF-03.
  • the formative stem cell culture media may comprise insulin, selenium, transferrin, anti-oxidants and lipid supplements.
  • the FS media may also be characterised by an absence of serum, KSR, BMP or Wnt. These components have all been previously used in various combinations with other components in medium cocktails for culturing ill-defined or heterogeneous pluripotent stem cells. At concentrations normally used in cell culture, such as 10% serum or 20% KSR, they each induce upregulation of somatic genes and/or overt differentiation of FS cells.
  • the inventors observed that cultures differentiated or died upon inhibition of PI3 kinase (Ly294002, 25 ⁇ ), Src (WH-4-023, 1 ⁇ ), JNK (SP-600125, 10 ⁇ ), MEK (PD0325901 , 1 ⁇ ) or FGF receptor (PD173074 , 0.1. uM). Furthermore inhibition of GSK3 with CHIR99021 at 1 ⁇ induced expression of Brachyury, although this effect was not evident at 0.5 ⁇ .
  • the FS media may be characterised by an absence of any one or more of these, although may optionally contain a low level of GSK3 inhibitor (e.g. up to around 0.5 ⁇ ).
  • the formative stem cell culture media may optionally not comprise any one or more of: serum; KSR; BMP; Wnt, retinoic acid, MEF feeders.
  • the formative stem cell medium may optionally not comprise a PI3 kinase inhibitor; a Src inhibitor; a JNK inhibitor; a GSK3 inhibitor, a MEK inhibitor or an FGF receptor inhibitor at concentrations such as those indicated that are standardly used for achieving inhibition in cell culture.
  • Suitable culture conditions in which the novel FS media of the invention may be utilised may be as described herein.
  • E5.5 epiblasts were obtained by dissection and plated individually on fibronectin-coated 4-well plates in N2B27 medium. Cultures were propagated in 7% CO2 and 5% O2. After several days, explants were treated with Accutase for 5-10 seconds at room temp and wash buffer was gently added to detach and fragment the outgrowths into small clumps. Cells were collected by centrifugation and seeded into fresh 4-well plates. Subsequently, expanding cultures were treated with Accutase briefly (30-60 seconds, at room temp) for dissociation into cell clusters by gentle trituration. Established cultures were routinely passaged every 2-3 days and plated onto new fibronectin coated dishes at a split ratio of 1/10-1/15. Medium was changed every other day.
  • a culturing process of the invention comprise one or more of:
  • ROCK inhibitor is optional.
  • FS stem cells as described herein may be derived from a number of different sources.
  • FS stem cells can be derived most directly from embryonic epiblast tissue at the formative phase of development (early post-implantation up to mid-gastrulation in most mammals, including primates), by explant or dissociation into formative culture conditions.
  • FS cells can be derived from naive pluripotent cells either taken directly from embryos, or established as naive embryonic stem cells, or generated as induced pluripotent stem cells by somatic cell reprogramming (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007) coupled with naive resetting.
  • Naive cells are transitioned to the formative phase of pluripotency and then 'captured' by culture in formative stem cell conditions. Transitioning from naive to formative phases will typically entail withdrawal of naive pluripotency maintenance conditions (e.g.
  • FS cells can be derived from primed pluripotent stem cells by resetting to a naive state using methods known in the art (see e.g. published patent application WO2016/027099), then transitioning to the formative phase as described above.
  • the period in the naive state culture conditions may be transient but should be sufficient to achieve epigenetic erasure.
  • 'Formative-like' stem cells can be isolated from primed pluripotent stem cells by resetting or selection/adaptation directly in formative culture conditions. However, since this is expected to occur without comprehensive erasure of epigenetic restrictions or abnormalities, the resulting cells may retain impediments to multi-lineage differentiation.
  • FS cells can also be derived from somatic cells via known factor-based reprogramming methods, reviewed by (Takahashi and Yamanaka, 2015), coupled directly with resetting to a naive state, then treating as described above.
  • FS cells can be provided from desired mammalian or avian species.
  • Preferred mammals include rodent (e.g. mouse), porcine mammals, lagomorph, artiodactyla, or primate (e.g. human).
  • the invention finds utility for, without limitation, mammals which are companion animals, model animals or livestock.
  • Preferred avian species include poultry such as chickens.
  • the processes of the invention utilise precursor pluripotent cells which are already in a formative phase of development e.g. obtained by explant or dissociation from early post-implantation embryonic epiblast tissue.
  • sources of cells at the formative phase of development suitable for culture by the methods of the invention may be identified according to the known embryonic development progression of the species of interest, and the disclosure herein.
  • the FS cells are rodent (e.g. mouse) these may be epiblast cells between E4.75 and E6, preferably E5.0- 5.5 - see e.g. Kalkan, T. and Smith, A. (2014).
  • the formative phase may last for several days.
  • cells with expected features of formative pluripotency are evident at the first stage examined after implantation (E13) and persist during gastrulation up to at least E17 (Nakamura et al., 2016).
  • axis determination and gastrulation commence before implantation. Accordingly formative epiblast in these species is sou reed from the late blastocyst stage prior to or co-incident with formation of the primitive streak.
  • the starting material may for example be a pre-implantation inner cell mass (ICM) explant culture that is matured in vitro to the formative embryonic disc stage (Deglincerti et al., 2016; O'Leary et al., 2012).
  • ICM inner cell mass
  • the precursor pluripotent cells are naive cells. These may be derived, for example, from pre-implantation embryonic epiblast tissue e.g. ICM explant ⁇ dissociation. Alternatively the naive cells maybe from a naive stem cell line. In this case, the cell line may first be cultured in known naive pluripotency maintenance conditions, but these conditions are withdrawn and replaced, directly or indirectly with formative stem cell culture conditions.
  • the inventors have shown that naive mouse ES cells could be used to consistently establish FS cell lines by withdrawal of naive pluripotency maintenance conditions and transfer to formative pluripotency culture conditions, either immediately or after a short period with no factor addition (24-48 hours). For example ES cells could be transferred directly to FS cell culture conditions e.g. by passaging in A
  • human ICM explants may be transferred directly into FS cell conditions.
  • naive epiblast cells could be initially expanded in t2il_Go followed by transfer to FS cell conditions.
  • the naive stem cell line is first cultured in naive pluripotency maintenance conditions t2il_IFGo (Takashima et al., 2014) or an alternative naive culture formulation such as 5il_/A/F or 4iL/A (Theunissen et al., 2016; Theunissen et al., 2014). They may then be transferred either directly to formative stem cell culture media such as A
  • the source pluripotent stem cells may be primed pluri potent stem cells, such as rodent EpiS cells, which are transferred directly or indirectly to formative stem cell culture media, for example by passaging in media having reduced FGF and activin and gradual transition to Ai 0 X+ ARi .
  • This process of adaptation or selection is accompanied by high cell death and differentiation but can nevertheless still be used to obtain FS cells if desired.
  • primed cells examples include mouse EpiS cells and conventional human ES cells (Thomson et al., 1998) or iPSC (Takahashi et al., 2007; Yu et al., 2007).
  • Human primed cells for example cultured on matrigel in E8 medium, may also be converted to FS cells by transfer into A
  • naive cells are provided as the source for FS cells by resetting primed pluripotent cells.
  • the primed pluripotent cells may be, for example, from embryonic tissue or from a cultured cell line.
  • the invention may utilise rodent (e.g. mouse) post-implantation embryonic epiblast tissue during gastrulation, E6 or E7, or mouse EpiS cells.
  • rodent e.g. mouse
  • embryonic epiblast tissue during gastrulation
  • E6 or E7 or mouse EpiS cells.
  • primed human pluripotent stem cells may be used after first resetting to naive status by methods known in the art (Takashima et al., 2014;
  • a resetting medium which comprises a HDAC inhibitor, a MEK inhibitor, and optionally a STAT3 activator, and optionally one or more further inhibitors e.g. a PKC inhibitor, a GSK3 inhibitor, or a Wnt inhibitor.
  • Conventional primed human pluripotent stem cells can be reset to naive state by transgenes or mRNAs (Nanog, KLF2, Klf4, Nr5a1/2) or following a chemical resetting protocol using HDAC inhibitors such as valproic acid or sodium butyrate followed by culture in t2iLGo. After 7 to 9 days resetting, naive-like cells expressing naive makers, KLF17, KLF4, TFCP2L1 , DPPA3 emerge, which maintain pluripotent surface markers,
  • Reset naive cells can be isolated based on expression of EOS-GFP reporter or simply enriched by passaging.
  • a stable naive culture can be established by 3-5 passages in t2ilGo with ROCK inhibitor. The stable reset naive culture can be used to establish human FS cultures as described herein.
  • human naive cells are plated in t2ilGo on geltrex or laminin. After 24 hours FS culture medium is applied. The dome shaped naive colonies acquire a flattened morphology after 4-5 days culture in FS medium. If present, EOS-GFP reporter or other naive reporter expression is down regulated. The culture is passaged by dissociation in the presence of Rock inhibitor and replated. After 2-3 more passages, cells uniformly exhibit an epithelial morphology with lack of naive marker expression, and low or undetectable lineage marker expression, and can then be propagated continuously as FS cell lines.
  • naive cells can be isolated at early stages during resetting. For example, following 7-21 days resetting naive cells can be distinguished by expression of naive markers using a reporter line such as KLF4:GFP, or isolated by expression of surface antigens, for example via a combination of SSEA4 negative and Tra1 -60/Tra1 -81 positive, or expression of novel naive surface markers, such as HAVCR1 (also called
  • the purified resetting cells can be replated to naive medium briefly before transfer to FS medium, or may be plated directly in FS cell medium. This protocol can avoid loss of imprints associated with extended propagation of naive stem cells. Following these protocols, impaired lineage-specific differentiation of primed human pluripotent stem cells may be restored. For example, Shef6 human ES cells show poor neural lineage potential, measured for example by up-regulation of SOX1 and PAX6 in neural induction protocols. Induction of these neural markers is substantially increased following naive resetting and conversion to FS cells.
  • naive cells are provided by reprogramming somatic cells to naive induced pluripotent stem cells (iPSCs).
  • iPSCs naive induced pluripotent stem cells
  • Reprogramming can be done by methods known in the art.
  • Human iPSCs may be derived from different cell types.
  • the cells can be produced from Fibroblasts, keratinocytes, adipose cells, bone marrow stromal cells or urinary epithelial cells.
  • Human iPSCs may be derived from diploid cells which may be a 'wild-type' or non-transformed cell.
  • an iPSC is derived from a transformed (tumour) cell.
  • Cells may be obtained from an individual by standard techniques, for example by biopsy for skin cells.
  • Cells may preferably be obtained from an adult.
  • Methods for generating iPSCs are known in the art, for example as described in: Takahashi et al Nature 2007; Yu et al, Science 2007, reviewed in (Takahashi and Yamanaka, 2015).
  • Stable iPSCs may be reset to naive status then converted to FS cells as above..
  • cultures may be switched to naive resetting medium with HDAC inhibitor, Erk inhibitor, and optionally PKC inhibitor and activation of Stat3 signalling for 5- 10 days.
  • the dome-shaped naive colonies expressing naive markers including KLF17, KLF4 or DNMT3L can be further expanded in t2ilGo medium or may be switched immediately to FS medium for
  • the precursor cell is transiently reset to naive status, then converted to formative status before erasure of imprints occurs in the naive state. It is believed a relatively brief period in naive pluri potency conditions should be sufficient for major epigenome remodelling whilst preserving imprints. Reprogramming somatic cells to naive status then rapidly
  • the FS cells described herein have phenotypic properties quite different from previously characterised ES or EpiS cell lines or iPSC.
  • mouse FS cells can colonise multiple lineages of the developing mouse embryo following blastocyst injection, albeit at lower frequency than ES cells. It is notable that EpiS cells rarely make any significant contribution to blastocyst chimaeras unless they have been genetically manipulated.
  • mouse FS cells can be propagated in the presence of the activin receptor (ALK5) inhibitor A-8301 (0.5-1.0 ⁇ ) ⁇ contrast to EpiSC or primed human piuripotent stem cells.
  • ANK5 activin receptor
  • A-8301 0.5-1.0 ⁇
  • mouse FS cells Unlike naive piuripotent stem cells, mouse FS cells cannot be propagated if FGF/Erk signalling is inhibited, for example in the presence of the Fgfr inhibitor PD-173074 or the Mek inhibitor PD-0325901 , confirming reliance on autocrine FGF.
  • FS cell cultures can expand and maintain undifferentiated morphology in the presence of exogenous FGF (0.1 , 0.5, 1 .0, 2.5, 5.0 and 12.5 ng/ml) although higher concentrations may induce some specification markers.
  • Mouse FS cells maintain expression of Oct4 but differ from ES cells by an absence of naive transcription factors such as Esrrb, Tfcp2l1 , Klf4 and Klf2.
  • FS cells differ from EpiS cells by an absence of early lineage markers such as Tbra, FoxA2, Mixl and Sox1.
  • Markers enriched in the FS cell state appear to include Dppa2, Dppa4 and Wdr86.
  • FS express substantially lower levels of neural, mesoderm and endoderm lineage-affiliated transcripts than EpiS cells, even when EpiS cells are cultured in the presence of XAV939.
  • Human FS cells can be prepared as described above.
  • human FS cells grew robustly with homogeneous morphology and a doubling time of 24-30 hours on plates coated with laminin and fibronectin. They displayed a down-regulation of naive markers (KLF4, KLF17,
  • TFCP2L1 TFCP2L1
  • OCT4 core pluripotency factor
  • OCT6 is not normally expressed in conventional human pluripotent stem cells and thus provides a potential discriminating marker of the formative state.
  • Human FS cells can be differentiated efficiently into neural, mesodermal and endodermal lineages using protocols effective on primed hPSC.
  • Methods of directing differentiation of pluripotent cells are known in the art, and described in the Examples hereinafter, which describe the use of inductive culture systems.
  • neural differentiation can be induced by dual SMAD inhibition using 500 nM LDN 193189 and 1 ⁇ A 83-01
  • Lateral plate mesoderm may be generated by treatment with CHI R99021 (6uM), BMP-4 (40ng/ml), activin A (30ng/ml) for 1 day, and then with BMP-4 (30ng/ml), XAV939 (10uM) and A8301 (1 uM) for 2 days (Loh et al., 2016).
  • Endoderm differentiation is induced by exposure to 100 ng/ml Activin A, 100 nM PI-103, 3 ⁇ CHI R99021 , 10 ng/ml FGF2, 3 ng/ml BMP4 for one day, followed by 2 days in 100ng/ml Activin A, 100nM PI-103, 20ng/ml FGF2, 250nM LDN 193189 (Loh et al., 2014).
  • Human FS cells can also respond to germ cell inductive cytokines (Irie et al., 2015). Following germ cell induction with BMP2, SCF, LI F, EGF and KSR, human FS cells showed expression of surface markers such as TNAP and CD38 between days 4 and 7. Immuno-staining for SOX17, BLIMP1 and OCT4 confirmed PGCLC formation
  • mouse formative stem cells are characterised by one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the following phenotypes:
  • naive transcription factors such as Esrrb, Tfcp2M , Klf4 and Klf2, or early lineage markers such as Tbra, FoxA2 and Sox1 ;
  • (v) can be propagated in the presence of the activin receptor (ALK5) inhibitor A-8301 in the presence of RARi and gamma secretase inhibitor;
  • PD0325901 (1 pM) or the FGF receptor inhibitor PD173074 (0.1 ⁇ );
  • (ix) can expand and maintain undifferentiated morphology in the presence of exogenous FGF (0.1 , 0.5, 1 .0, 2.5, 5.0 ng/ml);
  • human formative stem cells are characterised by one or more (e.g. 1 , 2, 3, 4, 5, or more) of the following phenotypes:
  • a process for propagating an FS cell line which process comprises:
  • the FS cell may optionally be one obtained or derived as described herein.
  • the FS cells are obtained by explant or dissociation from early post- implantation embryonic epiblast tissue or ICM culture, for example as described herein.
  • the FS cell is derived from a different pluripotent stem cell type or somatic cells, for example as described herein.
  • the FS cell culture media and ⁇ or conditions may optionally be as described hereinbefore.
  • the processes of the invention maintains a substantially homogeneous population of FS cells in continuous proliferative culture for at least 30 days. More than 90% of cells, typically 99% of cells, express OCT4 protein as detected by immunostaining. Less than 5% of cells, typically less than 1 % of cells, are
  • substantially homogeneous cell populations of FS cells are genetically and phenotypically stable; (ii) exhibit consistent characteristics and behaviour between cell lines; (iii) display robust multi-lineage differentiation; (iv) are distinct from previously well-characterised naive and primed pluripotent stem cell cultures.
  • an formative stem cell line obtained or obtainable by the processes described herein.
  • Stem cell lines are products of human endeavour, and do not occur in nature. Indeed the combination of FS cell characteristics together with continuous long-term propagation, self-renewal, described herein do not correspond to cell behaviour observed in the organism.
  • the cell line may be "isolated" in the sense of homogeneous and relatively free of other types of cell.
  • the cell line may be present in a suitable artificial culture container e.g. dish or well of glass or plastics material, and in suitable artificial medium as described herein, such as one containing additives such as XAV and RARi that do not exist in the organism.
  • human naive stem cells are unstable in all current culture conditions and even when semi-stable human naive stem cells are established, they show loss of imprinting (Pastor et al., 2016)) which compromises their differentiation competence and utility.
  • naive stem cells reported in the literature are for mice and rats. It appears that rodents may exhibit unusual stability of the naive stem cell state, which may be intrinsically less stable in other species (Martello and Smith, 2014; Takashima et al., 2014)) and consequently more prone to culture stress, genetic instability and loss of imprinting.
  • the FS cell state provides an important alternative stable pluripotent state for species not limited to humans, biomedical model species such as rodents, and livestock.
  • FS cells can be prepared by passing cells rapidly through the erasure process in the naive state and then almost immediately converting them to a population of "clean" but also stable FS cells. Since FS cells have chimaera-forming potential with germline colonisation they provide a route to transgenic modification and genome engineering in non-human species.
  • mammalian or any of the following: primate; non-human mammalian non-human primate; pig; sheep; cat; dog; goat; cow; camel; horse; llama; alpaca etc.
  • the non-human mammalian cell is not a rodent cell.
  • the cell may be avian, for example chicken.
  • Human and non-human pluripotent stems cells have a number of general utilities including: differentiation to create cell culture models of human development and disease that can be applied in drug discovery and development, and in teratogenicity and toxicology testing; source of tissue stem cells and more mature cells for applications in clinical cell therapy; analysis of the relative contributions of genetics and epigenetics to developmental disorders, genetic disease and quantitative traits to facilitate advances in diagnostics, prognostics and patient treatment; generation of tissues and organs for transplantation either by bioengineering in vitro or by lineage/organ specific contribution to human-animal chimaeras.
  • Reset, reprogrammed, or embryo-derived formative state pluripotent stem cells from non- human primates and other mammals can be used for precision genome engineering to enhance or modify germline genetic constitution of animals.
  • Germline modification is achieved by genome engineering or genome editing and clonal selection of ground state cells in culture, followed by production of chimaeras, breeding and screening for transmission of the modified genotype.
  • Desired genetic alterations include single or multiple gene deletion, point mutation, or substitution. Chromosome-scale genome modifications/substitutions are also possible.
  • Applications include: disease models; behavioural models; host compatibility for xenotransplantation and organ substitution; pharmaceutical, antibody and vaccine production; livestock improvement; breeding stock preservation and improvement.
  • Non-human primate formative state cells may also be used in pre-clinical testing and evaluation of cell therapies.
  • the invention provides a process for producing a primed pluripotent cell from an FS cell by withdrawal from FS cell culture medium and transfer to primed cell medium such as E8 for human primed cells or A ,F with or without XAV for mouse EpiS cells.
  • primed cell medium such as E8 for human primed cells or A ,F with or without XAV for mouse EpiS cells.
  • the primed pluripotent cell will be distinguished from the FS cell by virtue of characteristic phenotypes discussed herein.
  • the invention provides a primed pluripotent stem cell obtained or obtainable by this process.
  • the primed pluripotent stem cell may be isolated, or present in artificial context, as described above.
  • FS cells can be induced to form primordial germ cell-like cells (PGCLC) by exposure to inductive cytokines.
  • PPCLC primordial germ cell-like cells
  • An example regime is described in the Examples below. In mouse neither naive ES cells nor primed EpiSC respond to this induction regime. In the
  • FS cells were cultured with hBMP2, mSCF, hLIF, and Egf for 4 days in the presence of Rock inhibitor and 15% KSR in GMEM.
  • PGCLC were detected by expression of the surface marker genes (CD61 and SSEA1 for mouse and Tissue Nonspecific Alkarine Phosphatase (TNAP) and CD38 for human) by FACS, or by fixation and triple immuno-staining for Oct3/4, Stella and Blimpl for mouse and OCT4, SOX17 and BLIMP1 for human.
  • CD61 and SSEA1 for mouse and Tissue Nonspecific Alkarine Phosphatase (TNAP) and CD38 for human
  • FACS fixation and triple immuno-staining for Oct3/4, Stella and Blimpl for mouse and OCT4, SOX17 and BLIMP1 for human.
  • the invention provides a process for producing a PGCLC from an FS cell by cytokine induction.
  • the PGCLC will be distinguished from the FS cell by virtue of characteristic phenotypes discussed herein.
  • the invention provides a PGCLC obtained or obtainable by this process.
  • the PGCLC may be isolated, or present in artificial context, as described above.
  • Multi-lineage differentiation of FS cells may be carried out by use of differentiating protocols or conditions known in the art for differentiating pluripotent cells.
  • An example method comprises aggregated of cells in suspension culture and withdrawal of FS cell culture medium. Aggregates may be maintained in serum-free medium or in the presence of KSR or serum. After several days aggregates may be transferred to adherent dishes for outgrowth of differentiating cells.
  • mouse FS cells could be differentiated and efficiently into the three germ layers in vitro.
  • FS cells could be differentiated using serum free medium in the presence of activin and 3 ⁇ Chiron (CHIR99021 ).
  • For neural differentiation FS cells could be treated in N2B27 optionally with Tgfb receptor inhibitor A83-01 , similarly to primed pluripotent stem cells.
  • the invention provides a process for producing somatic lineage
  • differentiated cells from an FS cell line which process comprises exposing said formative FS cell line to one or more differentiating agents or conditions appropriate to that lineage.
  • the FS stem cell line may be exposed to serum free medium (SF-03) or N2B27 in the presence of 10 ng/ml activin a and 3 ⁇ Chiron.
  • the FS cell line may be exposed to N2B27 optionally with Tgfb receptor inhibitor A83-01 .
  • the differentiated cell will be distinguished from the FS cell by virtue of characteristic phenotypes discussed herein.
  • the invention provides a differentiated cell obtained or obtainable by this process.
  • the differentiated cell may be isolated, or present in artificial context, as described above. Uses of FS cells in chimaera formation
  • the invention provides a process for producing a chimaeric organism, which process comprises introducing a FS cell of the invention into a host embryo at p reimplantation or early post-implantation stages.
  • the invention provides a formative stem cell culture media as described herein.
  • a preferred medium comprises:
  • activin at a concentrations of 1 - 4ng/ml, more preferably 1 .5 - 3 ng/ml;
  • Preferred media are A i0 X, A i0 X+RARi and Ai 0 X+RARi+ySi.
  • the invention provides use of a formative stem cell culture medium of the invention to derive, produce or propagate an FS stem cell line.
  • Human embryonic stem cells for use in the invention may be obtained using methods which do not require destruction of the embryo.
  • embryonic stem cells may be obtained from the human embryo by biopsy. Methods for obtaining embryonic stem cells from the embryo without destruction of the embryo were disclosed for example in Klimanskaya, I ., Chung, Y., Becker, S., Lu, S.J., and Lanza, R., Human embryonic stem cell lines derived from single blastomeres, (2006). Nature 444, 481 -485.
  • the methods and uses do not involve destruction of human embryos. In some embodiments, the methods do not involve or use cells obtained by methods requiring destruction of human embryos.
  • the methods and uses of the invention do not involve use of a human embryo for industrial/commercial purposes. In some embodiments, the methods do not involve cells obtained by methods requiring use of a human embryo for industrial/commercial purposes.
  • the cell is not a human embryonic stem cell. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.
  • FS cells (AioX+RARi, top panel) rapidly differentiate in the presence of either KSR (20%, middle panel) or FCS (10%, bottom panel). Images were taken after 2 days treatment.
  • FS cells and EpiS cells were plated in 10-20 ng of activin a and 3 ⁇ Chiron.
  • Mesoderm genes such as T/Bra, Flk1 and Mesp l are more highly upregulated in FS than EpiS cells.
  • Cells were also analysed by FACS at day 1 and 2.
  • Flk1 positive, Ecadherin negative mesoderm population were induced more efficiently from FS cells than EpiS cells.
  • qRT-PCR and FACS analysis during mesendoderm differentiation FS cells and EpiS cells are plated in 10-20 ng of activin a and 3 uM chiron.
  • Endoderm genes such as Foxa2, Sox/ 7 and Mix 11 were upregulated more in FS than EpiS cells. Endoderm cells were quantified at day 3 by FACS. Cxcr4 and Ecadherin double positive endoderm cells are efficiently induced in FS cells.
  • Endoderm differentiation potency was examined using a directed endoderm differentiation protocol.
  • Cells are treated with 20 ng of activin a and 3 uM chiron for 24 hours, then changed to activin only for 48 hours.
  • Cells were analysed at 72 hours by FACS. Endoderm population was quantified by Cxcr4 and Ecadherin.
  • FS cells
  • naive markers b, in blue
  • primed lineage markers c, in red
  • candidate formative marker OCT6
  • general pluri potency associated marker OCT4 d, in green
  • Figure 3 Porcine formative piuripotent stem cells (a) Outgrowth of pig ICM after 9 days culture in formative stem cell culture medium containing Activin A (5 ng/ml) , XAV939 (2 ⁇ ) and RAR inhibitor BMS 493 (1 ⁇ ).
  • Nodal and the fibroblast growth factors FGF4 and FGF5 are uniformly expressed in the epiblast (Mesnard et al., 2006). These factors are candidates for supporting formative pluripotency but may subsequently contribute to lineage specification.
  • Nodal is known to be down-regulated in vitro so we added activin as a nodal substitute while relying on endogenous FGF production.
  • FS cell cultures can withstand chemical inhibitors of Braf (SB-590885, 0.5 ⁇ ), p38 (SB-202190. 5-30 ⁇ ), Cdc42 (ML141 , 1.0-5.0 ⁇ ), and PTEN (bpV(HOpic), 1.0 ⁇ ).
  • H DAC Histone deacetylase
  • Lsd1 Lysine specific demethylase 1
  • aPKC atypical protein kinase C
  • retinoic acid 10 " 7 M.
  • RARi pan-retinoic acid receptor inverse agonist
  • Fig. 1 c a pan-retinoic acid receptor inverse agonist
  • Fig. 1 c a pan-retinoic acid receptor inverse agonist
  • Fig. 1 c a pan-retinoic acid receptor inverse agonist
  • Fig. 1 c pan-retinoic acid receptor inverse agonist
  • a preferred culture condition for FS cells comprises low activin, Wnt inhibition, retinoic acid receptor inhibition and gamma-secretase inhibition (AloX+RARi+ySi).
  • a low to moderate concentration (0.1-4ng/ml) of FGF may improve consistency of expansion.
  • FS cells can tolerate activin/Tgf receptor inhibition in the presence of RARi plus ySi. Cultures could be passaged multiple times in AloX+RARi+ySi in the presence of A83-01 inhibitor at 0.5-1 . ⁇ . We confirmed diminished expression of Activin/Nodal target genes such as Nodal, Cripto and Lefty2 in these conditions by qRT- PCR. (Fig. 1 d). This suggests that established FS cells may not be absolutely dependent on continuous nodal/activin stimulation of the Smad2/3 pathway if lineage induction signaling is fully suppressed.
  • Example 3 - FS cells are distinct from mouse ESs and EpiSCs cells
  • FS cells Whether derived from embryos or from ES cells, FS cells do not survive if cultured in 2i or 2il_IF, demonstrating that they are distinct from ES cells and cannot spontaneously revert to ES cell status.
  • FS cells proliferate as flat monolayer colonies of epithelial-like cells (Fig. 1 a). They have a doubling time of 10-15 hours. FS differ from ES cells in absence of naive transcription factors such as Esrrb, Tfcp2l1 , Klf4 and Klf2. They differ from EpiSC in absence of early lineage markers such as Tbra, FoxA2 (Fig. 1 b) and Sox1 (Fig. 1 c). EpiS cells cultured in the presence of XAV (A FX) and feeder cells have been reported to show reduced expression of lineage markers (Sumi et al., 2013). However, we observed expression of lineage markers in EpiSC cultured A hl FX without feeders or undefined factors, (Fig.
  • Transcriptome analyses of early post-implantation embryos suggests the following additional markers which may distinguish formative cells from either naive or primed pluripotency.
  • Example 4 - FS cells are inefficiently derived directly from EpiS cells
  • EpiS cultures may be dedifferentiated to FS cells or may contain a sub-population of FS cells.
  • EpiS cells cultured in the presence of XAV (AhiFX) transferred directly into FS cell culture conditions (A i0 X) died or differentiated within several passages. If FGF was first withdrawn then activin reduced gradually at each passage, we observed extensive differentiation and cell death but a fraction of cells survived that could eventually be propagated similarly to FS cells.
  • EpiS cells (A h jF) transferred to FS cell conditions showed widespread differentiation and death with only rare cells surviving. In some cases, however, the surviving cells could be expanded. This suggests that EpiS cells may be converted at very low efficiency to FS cells by either adaptation or selection.
  • EpiS cells cultured in the presence of XAV (A h jFX) then transferred directly into A to X +Rari condition survived with some initial differentiation. After several passages, they could be propagated similarly to FS cells.
  • Example 5 - FS cells from later stage mouse epiblasts
  • FS cells could be established from later stage epiblasts, E6.5 and E7.5 (up to LS-EB stage) using AloX+RARi, although cell line derivation is at lower efficiency than from E5.5 epiblast (Table 3), this may indicate that the transition between formative and primed pluripotency is gradual in vivo and may not be complete or irreversible for all epiblast cells even at relatively late stages, consistent with a recent description of the cynomolgus macaque post-implantation embryo (Nakamura et al., 2016).
  • FS cells can be functionally distinguished from both ES cells and EpiS cells in their competence to respond directly to germ cell inductive cytokines and form primordial germ cell-like cells (PGCLC) (Fig. 1f).
  • PPCLC primordial germ cell-like cells
  • FS cells are collected by TrypLE Express and counted.
  • FS cells were cultured in non-adhesive U-bottom plate with hBMP2 (500 ng/ml), mSCF (100 ng/ml), hLIF (1 pg/ml) and Egf (50 ng/ml) for 4 days in the presence of Rock inhibitor (10 ⁇ Y-27632) and 15% KSR in GMEM (modified from (Hayashi et al., 201 1 )).
  • PGCLC were detected by expression of the surface marker genes (CD61 and SSEA1 ) by FACS, or by fixation and triple immuno-staining for Oct3/4, Stella and Blimpl .
  • PGCLC induction is a consistent property of FS cells cultured in Ai 0 X+RARi+ySi.
  • XAV epidermal growth factor
  • FS cells differentiated rapidly and efficiently into the three germ layers in vitro Fig. 1 h.
  • FS cells are directly plated in serum free medium (SF-03) or N2B27 in the presence of 10 - 20 ng/ml activin a and 3 ⁇ Chiron.
  • mesendoderm marker such as T/Bra, Foxa2 and Sox 17 by qRT-PCR.
  • FS cells were plated in N2B27 with Tgfb receptor inhibitor A83-01 (at 1 ⁇ ) on laminin coated plates.
  • Example 7 - FS cells can contribute to all lineages in chimaeras
  • formative pluripotent cells should have the potency to contribute to all lineages in chimaeras if they survive from the time of injection to early post-implantation.
  • Fig. 1j embryo-derived FS cells
  • Example 8 derivation of FS cells from human naive or primed pluripotent stem cells
  • naive markers KLF4, KLF17, TFCP2L1
  • OCT4 core pluripotency factor
  • TBRA, FOXA2 lineage priming markers
  • OCT6 POU3F1
  • OCT6 is of particular interest because this transcription factor is not normally expressed in conventional human pluripotent stem cells and thus provides a potential discriminating marker of the formative state.
  • Human FS cells were differentiated into definitive endoderm lineage according to (Loh et al., 2014). Cells were cultured in CDM2 medium supplemented with 100 ng/ml Activin A, 100 nM PI-103 (Bio-techne, 2930), 3 ⁇ CHI R99021 , 10 ng/ml FGF2, 3 ng/ml BMP4 (Peprotech) for one day. For the next 2 days the following supplements were applied: 100ng/ml Activin A, 100nM PI-103, 20ng/ml
  • FGF2, 250nM LDN 193189 After 3 days of differentiation, cells were fixed and stained for SOX17 or cells were analysed by either surface marker gene expression such as CXCR4 and c-KIT. We tested neural differentiation by dual SMAD inhibition (Chambers et al., 2009). Cells were fixed and stained for SOX1 at differentiation day 4. For lateral plate mesoderm differentiation, cells are differentiated by the protocol modified from (Loh et al., 2016) . Cells were treated with medium supplemented with Chiron (6uM), BMP-4
  • FS cells Human FS cells were harvested using TrypLE Express and counted. 3.000 FS cells were cultured in non-adhesive U-bottom plate with hBMP2 (500 ng/ml), mSCF (100 ng/ml), hLI F (1 pg/ml) and Egf (50 ng/ml) in the presence of Rock inhibitor (10 ⁇ Y-27632) and 15% KSR in GMEM (modified from (Hayashi et al., 201 1 )). By direct treatment with the inductive cytokine cocktail, FS cells showed expression of surface markers such as TNAP and CD38 between day 4 and 7. Immuno-staining for SOX17, BLIMP1 and OCT4 confirms PGCLC formation.
  • Human primed pluri potent stem cells can be transiently reset to naive status by HDAC inhibition, mRNA reprogramming, or other protocols, and short-term culture in t2il_tGo or other naive culture condition, then converted directly to FS cells.
  • Naive cells are directly transferred to A to X with/without RARi, or left initially for 6-10 days in basal medium (N2B27) to exit from the naive state, then transferred to Ai 0 X with/without RARi.
  • Human FS cells are passaged for every 4-6 days at 1 /10-20. For routine passaging, human FS cells are treated with either Accutase or TrypLE for 1 -2 min at room temperature.
  • Rock inhibitor (Y-27632, 10 ⁇ ) is added after passaging and medium changed on the following day.
  • Human primed cells cultured on matrigel in E8 medium may also be converted to FS cells by transfer into A
  • FS cells may be derived from human pre-implantation embryos either by culture of ICM explants directly in FS cell conditions or by initial expansion of naive epiblast cells in t2il_Go with or without XAV followed by transfer to FS cell conditions either at 1 st or 2 nd passage, or after establishment of stable naive cell lines.
  • mice and human FS cells are consistent with a distinct pluripotent stem cell type, transitional between previously characterised naive (ES) and primed (EpiSC) stem cell states and showing anticipated features of formative
  • Primed pluripotent stem cells are highly dependent on exogenous stimulation of the nodal/activin/TBF[5 signalling and FGF pathways (Brons et al., 2007; Tesar et al., 2007; Thomson et al., 1998; Vallier et al., 2005) through ligands provided by feeder cells (the concentration of activin A secreted by feeders is 6.2-15.0 ng/ml, (Kojima et al., 2014) ), serum or KSR, or as growth factor supplements.
  • FS cells expand in the presence of much lower concentrations of FGF and activin, and even in the presence of the A83-01 inhibitor.
  • FS cells depend on the tankyrase inhibitor XAV or other antagonists of Wnt signalling. Canonical Wnt pathway inhibition has previously been reported to reduce
  • EpiS cells cultured in the Ah,F plus XAV939 and 20% KSR on feeders are reported to colonise post-implantation embryos and contribute to alkaline phosphatase positive cells (Sumi et al, 2013).
  • EpiS cells have also been derived in Ah,F supplemented with the porcupine inhibitor IWP2, which blocks endogenous production of functional Wnt ligand, in the presence of serum (Kurek et al, 2015), or of KSR and feeders (Sugimoto et al., 2015). Kurek et al reported colonisation of blastocyst chimaeras, although not germ cell contribution. In the presence of serum, however, it is reported that post-implantation epiblast cells and EpiS cells can undergo epigenetic conversion to ES cells (Bao et al., 2009).
  • EpiS cells and human PSC have also been cultured using a combination of XAV and the GSK3 inhibitor CHIR99021 (CH) without added activin or FGF, but in the presence of serum (Kim et al, 2013). This condition is also reported to reduce heterogeneity but not to cause any fundamental change in identity. Additionally XAV has been deployed in combination with 2il_IF and KSR and reported to sustain a putative naive phenotype of human pluripotent stem cells (Zimmerlin et al, 2016). We have observed that XAV can be added to cultures of human naive pluripotent stem cells in t2il_Go (Takashima et al, 2014) and does not alter their phenotype or identity.
  • EPL early primitive ectoderm-like
  • EPL cells readily revert to ES cells, unlike FS cells or early post-implantation epiblast cells (Boroviak et al., 2014), therefore their developmental status is unclear. More recently it has been claimed that mouse ES cell differentiation in the presence of FGF, activin and CH (FAC) generates an "intermediate" pluripotent cell population that retains the ability to colonise chimaeras, including contribution to the developing germ line (Tsukiyama and Ohinata, 2014). However, FAC cultures retain high levels of naive markers, unlike FS cells, and also express endoderm lineage markers.
  • naive transcription factors such as Essrb and definitive endoderm markers
  • FAC cultures are either a mixture of ES cells and lineage specified cells or an in vitro artefactual cell type.
  • some EpiS cell derivations in the presence of KSR and feeders contain a minor fraction of cells with potency to colonise blastocyst chimaeras and revert to ES cells (Bernemann et al., 201 1 ; Han et al., 2010).
  • EpiS cell cultures may contain a sub-population of cells that either have unusually high plasticity, or may be representative of formative or naive phases of pi uri potency (Han et al., 2010).
  • somatic cells may be reprogrammed to induced pluripotent stem cell (iPSC) status by ectopic expression of transcription factors (Takahashi and Yamanaka, Cell, 2006; Takahashi et al. Cell 2007; Yu et al, Science, 2007) or chemical treatment (Hou et al, Science, 2013; Long et al, Cell Research, 2015).
  • iPSC formative induced pluripotent stem cells
  • the cells were then transferred to A5RX medium with low FGF2 (5ng/ml) until FS colonies formed and reached suitable size for picking. After colony picking cultures were expanded in FS conditions in A3RX medium without FGF2 on laminin/fibronectin. The results are shown in Fig. 2(j).
  • AFX medium N2B27 basal medium supplemented with Activin (5 ng/ml), FGF2 (5 ng/ml) and XAV939 (2 ⁇ );
  • A5RX medium N2B27 basal medium supplemented with Activin (5 ng/ml), BMS 493 (1 ⁇ ) and XAV939 (2 ⁇ ).
  • Example 1 Derivation of porcine formative pluripotent stem cells
  • Formative pluri potency is anticipated to be a generic feature of early mammalian development and establishment of formative pluripotent stem cells from various mammals opens up a range of biomedical and livestock applications.
  • Nanog is re-expressed in pre-gastrulation posterior epiblast in the mouse egg cylinder and in EpiSCs.
  • cynomolgus epiblast Nanog appears to be expressed continuously (Nakamura et al.. 2016)
  • b Factors such as Oct6 and Otx2 are up-regulated throughout the early post-implantation mouse epiblast but later become restricted to the anterior presumptive neuroectoderm c
  • a sub-set of primed cells in vitro are able to produce primordial germ cell-like cells (Irie et al., 2015; Sasaki et al., 2015)
  • FGF signaling inhibition in ESCs drives rapid genome- wide demethylation to the epigenetic ground state of piuripotency.
  • Klf4 reverts developmental ⁇ programmed restriction of ground state pluripotency.
  • Epiblast Stem Cell Subpopulations Represent Mouse Embryos of Distinct Pregastrulation Stages. Cell 143, 617-627.
  • Single-cell gene expression profiles define self-renewing, pluripotent, and lineage primed states of human pluripotent stem cells. Stem cell reports 2, 881-895.
  • SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253-268.
  • Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning. Development 133, 2497-2505.
  • Rathjen J., Lake, J. A., Bettess, M.D., Washington, J.M., Chapman, G., and Rathjen, P.D. (1999). Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 1 12 ( Pt 5), 601-612.
  • Epiblast ground state is controlled by canonical Wnt/beta-catenin signaling in the postimplantation mouse embryo and epiblast stem cells.
  • Tsakiridis A., Huang, Y., Blin, G., Skylaki, S., Wymeersch, F., Osorno, R., Economou, C, Karagianni, E., Zhao, S., Lowell, S., et al. (2014). Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors. Development 141, 1209-1221.
  • a modified EpiSC culture condition containing a GSK3 inhibitor can support germline-competent pluripotency in mice.
  • L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture.

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Abstract

L'invention concerne des procédés destinés à la production ou à la propagation d'une lignée de cellules souches formatives (SF), à partir d'une ou de plusieurs cellules souches pluripotentes précurseurs, ledit procédé comprenant les étapes consistant : (a) à fournir une ou plusieurs cellules souches pluripotentes précurseurs ; (b) à cultiver les cellules souches pluripotentes précurseurs dans des milieux de culture de cellules souches formatives. Le milieu de culture de cellules souches formatives comprend des quantités limitées d'activine et de facteur de croissance fibroblastique exogène, mais comporte un inhibiteur de Wnt, et généralement un inhibiteur du récepteur de l'acide rétinoïque. L'invention concerne en outre des matériaux et des procédés associés destinés à être utilisés dans la préparation et l'utilisation de lignées de cellules SF.
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CN117487743A (zh) * 2023-12-27 2024-02-02 广东省农业科学院动物科学研究所 一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法
EP4155387A4 (fr) * 2020-05-18 2024-07-31 Myoridge Co. Ltd. Procédé de production de cellules cibles, procédé de production de produit à l'aide de cellules cibles, et milieu sans sérum
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CN113728090A (zh) * 2019-04-08 2021-11-30 诺和诺德股份有限公司 从干细胞衍生的定形内胚层生成胰腺内胚层
JP7664856B2 (ja) 2019-05-22 2025-04-18 ハダシット メディカル リサーチ サービシーズ アンド ディベロップメント リミテッド ヒト多能性細胞を培養する方法
WO2020234888A1 (fr) * 2019-05-22 2020-11-26 Hadasit Medical Research Services And Development Ltd. Procédés de culture de cellules pluripotentes humaines
JP2022533745A (ja) * 2019-05-22 2022-07-25 ハダシット メディカル リサーチ サービシーズ アンド ディベロップメント リミテッド ヒト多能性細胞を培養する方法
WO2021102500A1 (fr) * 2019-11-26 2021-06-03 The University Of Western Australia Procédés de reprogrammation d'une cellule
JP2023511643A (ja) * 2019-11-26 2023-03-22 ザ・ユニヴァーシティ・オブ・ウェスタン・オーストラリア 細胞をリプログラミングする方法
EP4155387A4 (fr) * 2020-05-18 2024-07-31 Myoridge Co. Ltd. Procédé de production de cellules cibles, procédé de production de produit à l'aide de cellules cibles, et milieu sans sérum
WO2022063224A1 (fr) * 2020-09-24 2022-03-31 中国科学院动物研究所 Cellule souche pluripotente activée, son procédé de préparation et son utilisation
US20230332109A1 (en) * 2020-09-24 2023-10-19 Institute Of Zoology, Chinese Academy Of Sciences Activated pluripotent stem cell, and preparation method therefor and use thereof
EP4219684A4 (fr) * 2020-09-24 2024-05-15 Institute Of Zoology, Chinese Academy Of Sciences Cellule souche pluripotente activée, son procédé de préparation et son utilisation
CN114250195A (zh) * 2020-09-24 2022-03-29 中国科学院动物研究所 活化态多能干细胞及其制备方法和应用
AU2021350981B2 (en) * 2020-09-24 2025-06-05 Beijing Institute For Stem Cell And Regenerative Medicine Activated pluripotent stem cell, and preparation method therefor and use thereof
AU2021350981B9 (en) * 2020-09-24 2025-06-26 Beijing Institute For Stem Cell And Regenerative Medicine Activated pluripotent stem cell, and preparation method therefor and use thereof
EP4346395A4 (fr) * 2021-05-28 2025-04-23 ABS Global, Inc. Systèmes et procédés d'élevage in vitro dans le bétail
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CN117487743A (zh) * 2023-12-27 2024-02-02 广东省农业科学院动物科学研究所 一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法
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