WO2014037527A1 - Maturation of mammalian cells by modulation of protein phosphorylation - Google Patents

Maturation of mammalian cells by modulation of protein phosphorylation Download PDF

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WO2014037527A1
WO2014037527A1 PCT/EP2013/068511 EP2013068511W WO2014037527A1 WO 2014037527 A1 WO2014037527 A1 WO 2014037527A1 EP 2013068511 W EP2013068511 W EP 2013068511W WO 2014037527 A1 WO2014037527 A1 WO 2014037527A1
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cells
population
mature
phosphorylation
mammalian cells
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Anna PHILPOTT
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Cambridge Enterprise Limited
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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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
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    • C12N5/0676—Pancreatic cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
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    • C12N2510/00—Genetically modified cells

Definitions

  • This invention relates to compositions and methods for the in vitro maturation of mammalian cells.
  • islet beta cells have been proposed to ameliorate type 1 diabetes , and is also likely to be an effective treatment for many sufferers of Type 2 diabetes ( Pearl , E . J. & Horb, M. E. Clin Genet 74, 316-324 (2008) ) . Indeed, successful islet transplantation has been shown to result in relief from a requirement for insulin therapy and relief from diabetes-related symptoms (Daneman, D . Lancet 367 847-858 (2006) ) .
  • Stem cells could act as a source for B cell replacement ( Furth, M. E. & Atala, A . J Cell Biochem 106, 507-511 (2009) ) .
  • B cell replacement Furth, M. E. & Atala, A . J Cell Biochem 106, 507-511 (2009) .
  • a maj or obstacle remains in that stem cells must be induced to adopt beta cell fate and maintain that fate
  • stem cells indefinitely when transplanted back into the patient, retaining functionality and avoiding the risk of teratoma formation . It is widely acknowledged that for this to occur, stem cells must be taken down the developmental pathway that beta cells usually take when undergoing specification and differentiation in the developing embryo (Pearl et al (2008) ) . Based on these developmental events
  • Ngn3 is also required for islet cell maturation and function in both embryos and adults (Wang, S . et al . Proc Natl Acad Sci U S A 106, 9715-9720, (2009) ) .
  • trans-differentiation of exocrine pancreas cells into functional beta cells can be driven by ectopic expression of only 3 transcription factors , Pdxl , Mafa and Ngn3 ( Zhou, Q . , et al . Nature 455 , 627-632 (2008) ) , and these factors need only be
  • Ngn3 overexpression alone is capable of up regulating insulin, glucagon and somatostatin along with other markers of beta cell functionality (Treff , N. R. et al . Stem Cells 24, 2529-2537, (2006) ; Serafimidis , I., et al . Stem Cells 26 , 3-16 (2008) )
  • the present invention relates to the promotion of maturation of immature mammalian cells by manipulation of the phosphorylation of serine/proline and threonine/proline phosphorylation sites of basic helix-loop-helix (bHLH) transcription factors .
  • Reducing, inhibiting or preventing phosphorylation of bHLH transcription factors such as neurogenin-3 , may be useful in the production of mature cell populations , for example for use in therapy and disease modelling .
  • An aspect of the invention provides a method of producing mature mammalian cells comprising;
  • bHLH basic helix loop helix
  • phosphorylated bHLH protein in the mammalian cells promotes maturation and produces mature mammalian cells .
  • Phosphorylation may be reduced by reducing the number of
  • phosphorylation sites such as Ser-Pro (SP) or Thr-Pro (TP) sites
  • SP Ser-Pro
  • TP Thr-Pro
  • Suitable bHLH proteins include proneural transcription factors , such as neurogenin-2 (Ngn2), neurogenin-3 (Ngn3) , Achaete- scute homolog 1 (Ascll ) , Pancreas specific transcription factor la (Ptfla) and
  • Suitable mammalian cells are fully differentiated and include endocrine cells , such as intestinal and pancreatic endocrine cells , for example pancreatic beta cells, and neurons , including
  • dopaminergic cortical , cerebellar, spinal , and peripheral neurons .
  • suitable mammalian cells include intestinal , prostate and lung cells.
  • Figure 1 shows in vitro translated S35-Met labelled mNGN3 or S- Am G 3 incubated in interphase (I) or mitotic (M) Xenopus egg extract with or without phosphatase treatment and analysed by SDS- PAGE.
  • Figure 2 shows mNGN3-HA transfected into PI 9 cells and harvested for western blotting at increasing times , lysates treated with
  • Figure 3 shows the results of qPCR for the expression of NeuroD, Delta ( and neural b-tubulin in Xenopus embryos inj ected (side to right ) with GFP ( control ) , phosphomutant 6S-A Ngn3 and wild type Ngn3. Error bars show the error of the mean of 3 biological
  • Figure 4 shows the results of ChIP from transfected PI 9 cells showing the occupancy of mNgn3 WT and 6S-A on promoters . Error bars show the standard deviation of three qPCR technical replicates .
  • Figure 5 shows qPCR measurement of insulin expression in Xenopus embryos inj ected with mRNA to wild-type or 6S-A (phosphomutant ) Ngn3.
  • Figure 6 shows qPCR measurement of Ngn3 and NeuroD expres sion heterozygous mouse ES cells that express either WT or 6S-A
  • Figure 7 shows qPCR measurement of Insulin expres sion in
  • heterozygous wt or 6S-A (phosphomutant ) Ngn3 knock-in mouse ES cells (where WT or phosphomutant Ngn3 are expressed from the endogenous promoter) subj ected to a 3 stage endo I , II I II protocol of
  • Figure 8 shows confocal microscopy images of homozygous wt or 6S-A (phosphomutant) Ngn3 knock-in mouse ES cells subj ected to a 3 stage endo I , I I III protocol of differentiation into definitive endoderm, following 32 days in endo III .
  • Figure 9 shows the phosphorylation of Ascll by recombinant Cdks .
  • WT and 6S-A (phosphomutant) Ascll 35-S labelled IVT proteins were incubated with the indicated cyclin/ cdks in kinase buffer
  • Figure 10 shows quantitative measurements of morphological
  • Figure 11 shows quantitative measurements of morphological
  • Figure 12 shows a comparison of noraderinergic and seratonergic markers conducted by qPCR at tailed stage following inj ection of Xenopus embryos with mRNA for WT, or phosphomutant forms of Ascll with mutation of predicted cdk sites (Ascll Cdk) , GSk3beta sites (Ascll Gsk3B) or cdk and Gsk3B sites simultaneously (Ascll Cdk Gsk3B) .
  • the methods described herein relate to the maturation of
  • phosphorylation of a bHLH proneural protein in the cells This may be useful in the production of mature mammalian cells .
  • the amount of phosphorylation of the bHLH protein in the immature mammalian cells may be reduced relative to the amount of
  • the ability of the bHLH protein to drive both differentiation and maturation of mammalian cells is reduced by phosphorylation at multiple phosphorylation sites , such as Ser-Pro and Thr-Pro (SP/TP) sites , within the bHLH protein . Reducing the phosphorylation of these sites in the immature mammalian cells increases the amount of bHLH protein in the immature mammalian cells which lacks
  • This un (der) phosphorylated bHLH protein has increased activity and promotes maturation of the immature mammalian cells .
  • the increase in activity upon reduced phosphorylation may be semiquantitative and maximal maturation may be achieved when the bHLH protein is devoid of phosphorylated phosphorylation sites, such as Ser-Pro and Thr-Pro (SP/TP) sites .
  • SP/TP Ser-Pro and Thr-Pro
  • phosphorylation of the bHLH protein in the immature mammalian cells may be reduced by expression in the cells of a modified bHLH protein which lacks some or all of the phosphorylation sites present in the wild type bHLH protein, for example Ser-Pro and Thr-Pro (SP/TP) sites , or by exposure of the cells to a kinase inhibitor which inhibits phosphorylation of the phosphorylation sites present in the endogenous bHLH protein, for example Ser-Pro and Thr-Pro (SP/TP) sites .
  • phosphorylation of the bHLH protein may be reduced by reducing the number of Ser-Pro (SP) or Thr-Pro (TP) phosphorylation sites in the bHLH protein that are
  • Phosphorylation sites in a bHLH protein are sites that are subj ect to phosphorylation by Cdk, GSK3 ⁇ and/or other cellular kinases , preferably sites that are phosphorylated by Cdk, GSK3 ⁇ or Cdk and GSK3 .
  • Phosphorylation sites within bHLH proteins are well-known in the art and may include Ser-Pro and Thr-Pro (SP/TP) sites . Other phosphorylation sites may be identified by routine sequence
  • phosphorylation sites in human Ascll may include the SP sites Ser93 , Serl 90 , Serl94, Ser207, and Ser223 and the GSK3 sites Ser203, Ser210 and Ser214.
  • Phosphorylation sites in human Ngn3 may include the SP sites Ser38 , Serl 61 , Serl 65 , Serl74 , Serl83, and Ser204, and the non-ST/TP 6 ⁇ 3 ⁇ sites Ser69 and Serl77.
  • the mammalian cells may be maintained in culture following the reduction in bHLH protein phosphorylation until they develop the mature cell phenotype .
  • human cells may be maintained in culture for 21 days or more, or 28 days or more , following the reduction in bHLH protein phosphorylation .
  • Suitable culture conditions for maintenance of di fferent mammalian cell types such as islet beta cells and neurons , are well known in the art.
  • the efficiency of maturation may depend on the nature of the cell population . For example, after reducing the amount of bHLH protein phosphorylation, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more , 40% or more , 50% or more, 60% or more, 70% or more, 80% or more , 85% or more , 90% or more, 95% or more or 98% or more of the immature mammalian cells in the population may adopt a mature phenotype .
  • the methods described herein may be in vitro, ex vivo or in vivo, preferably in vitro or ex vivo.
  • the immature mammalian cells may be from any mammalian species , preferably mouse or human cells.
  • Immature mammalian cells display a stable immature phenotype which differs from the fully functional phenotype of mature cells .
  • the immature cells may lack one or more functions or features of the mature cell or may display one or more functions or features which the mature cell lacks .
  • the immature cells may lack one or more functions or features of the mature cell or may display one or more functions or features which the mature cell lacks .
  • immature mammalian cells may display the ability to proliferate, whilst mature mammalian cells do not retain display the ability to proliferate .
  • the immature cells acquire the mature phenotype through maturation as described herein .
  • Immature mammalian cells produced as described herein may be characterised by the formation of teratomas following 1 S 1 1 1 l lltO ITOCLsrit ⁇ O ⁇ — h1J171cl ⁇ .1
  • the immature mammalian cells may be of any cell type, including neurons , such as dopaminergic, cortical , spinal , and peripheral neurons , and endocrine cells , such as intestinal , pancreatic, lung and prostate endocrine cells .
  • neurons such as dopaminergic, cortical , spinal , and peripheral neurons
  • endocrine cells such as intestinal , pancreatic, lung and prostate endocrine cells .
  • the immature mammalian cells are pancreatic endocrine cells or precursors thereto, most preferably pancreatic islet beta cells or precursors thereto .
  • Islet beta cells secrete insulin in response to glucose .
  • immature islet beta cells may display a phenotype which includes one or more of : the absence of insulin or urocortin-3 expression; the absence of glucose responsive insulin secretion; low threshold glucose responsive insulin secretion; absence of beta islet morphology; and inability to engraft in host models .
  • an immature islet beta cell phenotype may also include one or more of; expression of inactive phosphorylated Ngn3 ; the absence of expression of direct or indirect downstream Ngn3 targets , such as neuroD, MyTl and insulin; and the absence of other
  • the immature mammalian cells are neurons .
  • immature neurons cells may display a phenotype which includes one or more of : short neurites ; unbranched or substantially unbranched neurites ; ability to proliferate ; NCAM expression; SOX2 expression; absence of TuJl and MAP2 expression; and reduction, dysregulation or absence of electrical excitability .
  • the methods described herein may be useful in the production of mature mammalian cells .
  • Mature mammalian cells display a mature phenotype which differs from the phenotype of the immature cells and which includes phenotypic characteristics of maturation or maturity, such as insulin- expression in beta islet cells and extended neurite outgrowth in neurons .
  • Mature mammalian cells may be identified by determining the presence of these phenotypic characteristics of maturity .
  • Mature mammalian cells produce as described herein may be any mammalian cells produced as described herein.
  • Mature islet beta cells may display a phenotype which includes one or more of : neuroD, MyTl , insulin and/ or urocortin 3 expression; glucose-responsive insulin secretion; physiological thresholds of glucose responsiveness ; beta islet morphology; and ability to engraft in host models .
  • Immature islet beta cells may acquire 1, 2, 3, more than 3 , or all of the above phenotypic characteristics of maturity as they progress through maturation as described herein .
  • the pancreatic endocrine subtype that is developed by immature pancreatic endocrine cells during maturation may be determined by the phosphorylation sites that are
  • Reducing phosphorylation at one or both of Cdk and GSK3 ⁇ phosphorylation sites may be useful in producing specific pancreatic endocrinal sub-types , such as islet alpha, islet beta, islet delta or islet PP cells, or increasing the proportion of specific pancreatic endocrinal sub-types in a mature population of pancreatic endocrine cells .
  • Mature neurons may display a phenotype which includes one or more of : absence of SOX2 expression; TuJl expression; MAP2 expression; extended and branched neurites ; neurites able to connect to adj acent neurites and form neural networks ; electrical excitability; and improved engraftment in animal model s .
  • immature neurons may acquire 1, 2, 3 or more or all of the above phenotypic characteristics of maturity, as they progress through maturation as described herein.
  • Neurons may also acquire one or more markers associated with subtype specificity, such as Thl , Lmxla, Foxa2 , Nurrl, Pitx3 and Enl, as they progress through maturation as described herein .
  • markers associated with subtype specificity such as Thl , Lmxla, Foxa2 , Nurrl, Pitx3 and Enl, as they progress through maturation as described herein .
  • one or more factors that specify neuronal subtype may be expressed or introduced into the cells when the phosphorylation of the bHLH protein is reduced as described herein .
  • the neuronal subtype that is developed by the immature neurons during maturation may be determined by the phosphorylation sites that are unphosphorylated in the bHLH protein . Reducing
  • phosphorylation at one or both of Cdk and GSK3 ⁇ phosphorylation sites may be useful in producing specific neuronal sub-types , such as noradrenergic, serotonergic , glutaminergic , GABAergic, TH+ve dopaminergic, and cholinergic neurons , or increasing the proportion of specific neuronal sub-types in a mature population of neurons .
  • a bHLH protein such as Ascll
  • reduction or absence of phosphorylation at both Cdk and GSK3 phosphorylation sites in a bHLH protein, such as Ascll within a population of immature mammalian cells may increase the amount of noradrenergic neurons produced relative to serotonergic neurons , compared to reduction or absence of phosphorylation at either Cdk or GSK3 phosphorylation sites alone .
  • Consensus phosphorylation sites for Cdk and GSK3 phosphorylation are well known in the art and suitable sites may include Ser-Pro and Thr-Pro (SP/TP) sites .
  • bHLH protein phosphorylation may be reduced as described herein in the immature mammalian cells after
  • the immature mammalian cells may be fully differentiated i.e. they are committed to a single cellular fate and have no further differentiation potential .
  • Immature mammalian cells which are differentiated may express markers characteristic of a fully differentiated cell and may lack expression of markers characteristic of pluripotent or
  • bHLH protein phosphorylation may be reduced as described herein in the immature mammalian cells before
  • mammalian cells may be undifferentiated or partially differentiated (i.e. not fully differentiated) when bHLH protein phosphorylation is reduced, or may be committed to a cellular fate but have yet to adopt the mature characteristics of that fate .
  • the cells may be pluripotent cells or partly differentiated progenitor or precursor cells which are not yet committed to a single cellular fate and possess further differentiation potential .
  • Reduction in bHLH protein phosphorylation may drive the immature cells through both differentiation and maturation .
  • Suitable immature mammalian cells for the maturation of islet beta cells may include definitive endoderm, primitive gut tube , posterior foregut , and pancreatic endoderm and endocrine precursor cells .
  • Suitable immature mammalian cells for the maturation of neurons may include neuroectoderm cells, neural stem cells , induced neural stem cells and neural progenitor cells .
  • Immature mammalian cells for maturation as described herein may be isolated cells .
  • Suitable isolated immature mammalian cells may be maintained in vitro using standard cell culture techniques .
  • Immature mammalian cells for maturation as described herein may be produced in vitro from antecedent cells .
  • antecedent cell may be a pluripotent cell .
  • a pluripotent cell exhibits an undifferentiated phenotype and is capable of giving rise to any cell type in an individual.
  • Pluripotent cells include embryonic and non-embryonic (e.g. foetal or adult) stem cells and induced pluripotent stem (iPS ) cells .
  • An embryonic stem cell is a pluripotent cell derived from an early stage embryo .
  • An ESC may be a cell obtained from an embryo or may be a descendent of such a cell .
  • Suitable hESCs cells may be obtained from a cultured hESC cell line, such as H9 or hSF-6.
  • iPS cells are pluripotent cells which are derived from non- pluripotent , fully differentiated ancestor cells .
  • Suitable cells include adult fibroblasts and peripheral blood cells .
  • Ancestor cells are typically reprogrammed by the introduction of pluripotency genes or proteins , such as Oct4 , Sox2 and Soxl into the cell .
  • the genes or proteins may be introduced into the differentiated cells by any suitable technique , including viral or plasmid transfection or direct protein delivery .
  • Other genes for example Kif genes , such as Kif-1, -2 , -4 and -5; Myc genes such as C-myc , L-myc and N-myc ;
  • nanog; and Lin28 may also be introduced into the cell to increase induction efficiency .
  • Techniques for the production of iPS cells are well known in the art . (Yamanaka et al Nature 2007; 448:313-7; Yamanaka 6 2007 Jun 7; 1 (1) : 39-49. Kim et al Nature. 2008 Jul 31;
  • the antecedent cell may be an iPS cell obtained from an individual , for example an individual with a di sease condition, such as a neural or diabetic condition .
  • a mammalian pluripotent cell for example, a human or mouse pluripotent cell, may express one or more of the following
  • pluripotency associated markers 0ct4, Sox2 , Alkaline Phosphatase, SSEA-3 , Nanog, SSEA-4 and Tra-1-60.
  • a human pluripotent cell may lack markers associated with specific differentiative fates , such as Bra, Soxl7, FoxA2 , FP, Soxl, NCAM, GATA6, GATA4 , Handl and CDX2.
  • Suitable methods for the directed differentiation or forward programming of pluripotent cells into various differentiated cell types including pancreatic beta cells , are well-known in the art
  • antecedent pluripotent cells are cultured with one or more growth factors and/or growth factor inhibitors to direct differentiation into the appropriate cell type .
  • pluripotent antecedent cells such as mouse or human iPS or ES cells may be used to produce populations of immature endocrine pancreas cells , islet beta cells, neural cells and neurons for maturation as described above .
  • Suitable methods are well-known in the art (see for example Zhang et al (2009) Cell Res 19(4) 429- 438 ; Hosoya et al Int . J. Dev . Biol . 56 : 313-323 (2012) ; Guo et al Endocrine Reviews May 1 , 2009 vol . 30 no . 3 214-227; Tateshi et al JBC 283 46 31601-31607; Furth, M. E. et al J Cell Biochem 106, 507- 511 (2009); Pearl, E. J. & et al Clin Genet 74, 316-324 (2008);
  • the antecedent cell may be a differentiated somatic cell .
  • the antecedent cell may be a different cell type to the immature mammalian cell.
  • Suitable antecedent cells include fibroblasts and other mammalian cell types .
  • an immature beta cell or neuron for maturation as described herein may be produced by trans- differentiation of fibroblasts , such as dermal fibroblasts .
  • Suitable methods for the trans-differentiation of differentiated cells into various differentiated cell types is well-known in the art (Caiazzo et al . Nature 476, 224-227 and Pang et al , Nature 476, 220-223) .
  • Antecedent cells may be reprogrammed into differentiated cells through the introduction of reprogramming factors into the cells .
  • Reprogramming factors may be introduced in the form of nucleic acids (Warren L et al . Cell Stem Cell . 2010 Nov 5; 7(5) : 618-30) or proteins (Zhou H, et al Cell Stem Cell . 2009 May 8; 4 (5) :381-4) by any suitable technique , including plasmid or more preferably, viral transfection, direct protein delivery or direct delivery of nucleic acid, such as mRNA, for example by RNA mediated transfection .
  • the population of antecedent cells may be cultured to allow reprogramming to occur .
  • Suitable techniques for reprogramming cells though expression of nucleic acid encoding one or more reprogramming factors are well- known in the art (Takahashi et al 2007; Takahashi et al 2007; Seki et al 2010; Loh et al 2010; Staerk et al 2010) .
  • reprogramming factors which a re employed depends on the type of reprogrammed cell which is desired For example, iPS cells or fibroblast antecedent cells may be used to produce
  • Immature neurons may be produced from antecedent fibroblast cells by expression of Brn2, MyTl and optionally NeuroD, in addition to Ascll , Ngn2 , Ngn3 , Ptfla and/or atonal , depending on the type of neuron .
  • Additional subtype specifiers for instance Lmxla, Foxa2 , Nurrl , Pitx3 and/or Enl may also be expressed .
  • Antecedent cells for the production of immature mammalian cells for use as described herein may be obtained from an individual or may be derived from an individual .
  • differentiated somatic cells such as fibroblasts
  • iPS cells may be derived from cells obtained from an individual .
  • the individual may be a mammal , preferably a human .
  • the individual may be healthy (i.e. without any disease condition) or may have a disease condition .
  • the antecedent cells may be healthy cells i.e. cells without a disease-associated genotype or may display a genotype associated with a disease condition .
  • Mature differentiated cells produced from antecedent cells obtained from an individual as described herein may useful in disease modelling or therapy .
  • mature differentiated cells produced from antecedent cells with a disease-associated genotype may be useful in modelling the disease which is associated with the genotype .
  • differentiation and maturation as described herein may be carried out in a single step .
  • phosphorylation may be reduced in the immature mammalian cells before , during or after reprograming or differentiation .
  • bHLH protein phosphorylation may be reduced in the antecedent cells .
  • bHLH protein phosphorylation is reduced in the immature mammalian cells in vitro or ex vivo i.e. the immature mammalian cells are not be part of a human or animal body when bHLH protein phosphorylation is reduced.
  • Un (der) phosphorylated bHLH protein remains in the cells after reprograming or differentiation and drives maturation to produce cells with a mature differentiated cell phenotype .
  • Basic helix-loop-helix (bHLH) proneural proteins are a well- characterised family of proneural transcription factors which are characterised by the presence of two alpha helices linked by a loop .
  • Basic helix-loop-helix (bHLH) proteins include neurogenin-2 , neurogenin-3 , Ascll , Ptfla and Atonal .
  • sequences of bHLH proteins suitable for use in accordance with the methods described herein may include multiple phosphorylation sites located on either side of the bHLH domain .
  • phosphorylation sites may be Serine-Proline (SP) sites , Threonine- Proline (TP) sites or other sites that are phosphorylated by Cdk, GSK3 or other cellular kinases .
  • the phosphorylation sites are phosphorylated by Cdk and/or GSK3 ⁇ .
  • the sequence of a bHLH protein may include 1 , 2 , 3, 4 , 5, 6, 7 , 8 , 9 , 10 or more phosphorylation sites , for example SP and/or TP sites or non-SP/TP sites phosphorylated by GSK .
  • mouse Ngn2 contains 9 SP sites and 1 TP site
  • mouse Ngn3 contains 5 SP and 1 TP site
  • human Ngn3 contains 6 SP sites
  • human and mouse Ascll contain 5 SP sites .
  • These SP and/or TP phosphorylation sites are located on either side of the bHLH domain and are easily recognised in the amino acid sequence of the bHLH protein .
  • the phosphorylation sites in the bHLH protein may be phosphorylated by a mammalian cell kinase , for example a cyclin dependent kinase, such as cdkl , cdk2 and cdk4 , Glycogen synthase kinase 3 beta (GSK3 ) , Mitogen Activation Protein Kinase (MAPK) , such as MAPK1 and MAPK3, or another proline-directed kinase .
  • a mammalian cell kinase for example a cyclin dependent kinase, such as cdkl , cdk2 and cdk4 , Glycogen synthase kinase 3 beta (GSK3 ) , Mitogen Activation Protein Kinase (MAPK) , such as MAPK1 and MAPK3, or another proline-directed kinase .
  • Phosphorylation of SP/TP phosphorylation sites and/or other sites phosphorylated by ⁇ inhibits both the differentiation activity and the maturation activity of the bHLH protein .
  • endogenous bHLH protein in immature mammalian cells may be phosphorylated at multiple SP/TP phosphorylation sites and is inactive or has both reduced differentiation activity and reduced maturation activity .
  • 1 , 2 , 3, 4 , 5 , 6, 7, 8 , 9 , 10 or more or all SP/TP phosphorylation sites may be phosphorylated in the endogenous bHLH protein .
  • the phosphorylation sites of a bHLH protein may be readily identified and phosphorylation sites for human Ascll and human Ngn3 are set out above .
  • Reducing phosphorylation of the SP/TP phosphorylation sites and/or other sites phosphorylated by GSK results in a semi-quantitative enhancement of both the differentiation and maturation activities of bHLH proteins .
  • additive removal of phosphate groups for example by progressively mutating the SP and TP sites and/or other sites phosphorylated by ⁇ , leads to incremental increases in transcription of targets that drive differentiation and
  • bHLH protein may still retain maturation activity if some phosphorylated SP and TP sites are present , maturation activity is maximal in the absence of
  • the phosphorylation of SP and TP sites and optionally other ⁇ phosphorylation sites in the bHLH protein is reduced in the immature mammalian cells i.e. the number of phosphorylated SP and TP sites decreased or reduced compared to the number of phosphorylated SP and TP sites in the bHLH protein in the mammalian cells before being subj ected to the methods described herein or in control mammalian cells which are not subj ected to the methods described herein .
  • a bHLH protein with reduced phosphorylation may be entirely devoid of phosphorylated SP and TP phosphorylation sites or may have fewer phosphorylated SP and TP phosphorylation sites that the bHLH protein in untreated control cells.
  • the bHLH protein may have 1 , 2, 3, 4 , 5 , 6, 7 , 8 , 9 , 10 or more than 10 fewer phosphorylated SP and TP phosphorylation sites .
  • phosphorylation of the bHLH protein at SP and TP phosphorylation sites and optionally other GSKfi phosphorylation sites may be reduced by 5% or more, 10% or more, 20% or more, 30% or more , 40% or more , 50% or more , 60% or more , 70% or more, 80% or more, or 90% or more following treatment as described herein .
  • phosphorylation of the bHLH protein may be greater in the immature mammalian cells by 5% or more, 10% or more , 20% or more, 30% or more , 40% or more , 50% or more , 60% or more, 70% or more, 80% or more , or 90% or more compared to the mature cell .
  • the phosphorylation of a bHLH protein by one or more specific mammalian cell kinases may be reduced .
  • the phosphorylation of a bHLH protein by a cyclin dependent kinase (Cdk) , GSK3 ⁇ or both may be reduced . This may also be useful in directing the cell into specific mature cell sub-types .
  • Reducing phosphorylation at one or both of Cdk and GSK3 ⁇ phosphorylation sites may be useful in producing specific neuronal sub-types , such as noradrenergic, serotonergic , glutaminergic , GABAergic, TH+ve dopaminergic, and cholinergic neurons , or increasing the proportion of specific neuronal sub-types in a mature population of neurons .
  • specific neuronal sub-types such as noradrenergic, serotonergic , glutaminergic , GABAergic, TH+ve dopaminergic, and cholinergic neurons
  • reducing the phosphorylation of both Cdk2 and GSK3 ⁇ sites in Ascll may enhance neuronal differentiation and promote the maturation of noradrenergic neurons or other specific subtypes of neurons .
  • Reducing the phosphorylation of either Cdk2 or GSK3 ⁇ sites in Ascll may promote the maturation of serotonergic neurons or other specific subtypes of neurons . Reducing phosphorylation at one or both of Cdk and GSK3 ⁇ phosphorylation sites in an immature
  • pancreatic endocrine cell may be useful in producing specific subtypes of mature pancreatic cells , such as islet alpha, islet beta, islet delta or islet PP cells .
  • the identity of the bHLH protein which contains a reduced number of phosphorylated SP and TP sites as described herein depends on the type of differentiated cells which are to be matured.
  • Ascll may be used to mature cortical or peripheral neurons ;
  • Ptfla may be used to mature cortical neurons or pancreatic endocrine precursors ;
  • Atonal may be used to mature intestinal endocrine cells, lung cells or cerebellum cells ;
  • Ngn2 may be used to mature cortical or spinal neurons ;
  • Ngn3 may be used to mature pancreatic endocrine cells , intestinal endocrine cells , or hypothalamic neurons .
  • Ngn3 with a reduced number of phosphorylated SP and TP sites may be used to mature pancreatic endocrine cells , such as islet beta cells .
  • amino acid sequences of mammalian bHLH proteins are readily available on public databases .
  • Ascl 1 (achaete-scute homolog 1 ; also known as ASH1 ; HASHI; MASHI; bHLHa46 ; Gene ID 429) may be human Ascl 1 having the sequence of NCBI database entry NP___004307.2 GI : 55743094 or a naturally occurring variant or allele thereof .
  • Ptfla pancreas specific transcription factor, la; also known as bHLHa29 ; PTFl-p48 ; Gene ID 256297) may be human Ptfla having the sequence of database entry NP__835455.1 GI: 30039710 or a naturally occurring variant or allele thereof .
  • Atohl (atonal homolog 1; also known as ATHI; HATHI ; MATH- 1 ; bHLHal 4 ; Gene ID 474 ) may be human Aohl having the sequence of database entry NP___005163.1 GI : 4885075 or a naturally occurring variant or allele thereof
  • Ngn2 neuroogenin-2 ; also known as Atoh4 , Math4A, neurog2 , bHLHa8 , ngn-2 ; Gene ID 63973
  • Ngn3 neuroogenin-3 ; also known as Atoh5 , ath4B, NGN-3 , bHLHa7 , neurog3 ; Gene ID 63973
  • Ngn3 neurogenin-3 ; also known as Atoh5 , ath4B, NGN-3 , bHLHa7 , neurog3 ; Gene ID 63973
  • NP___066279.2 GI : 68989258 or a naturally occurring variant or allele thereof may be human NGN3 having the sequence of database entry NP___066279.2 GI : 68989258 or a naturally occurring variant or allele thereof .
  • a naturally occurring variant or allele of a reference bHLH sequence listed above is easily identified by a skilled person and may have at least 80%, at least 85% , at least 90%, at least 95% , or at least 98% sequence identify to the reference bHLH sequence listed above .
  • GAP GCG Wisconsin Package 1 ", Accelrys , San Diego CA
  • GAP uses the Needleman & Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps .
  • Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al .
  • Particular naturally occurring amino acid sequence alleles or variants may differ from a reference bHLH sequence by insertion, addition, substitution or deletion of 1 , 2 , 3 , 4 , 5 , 6, 7 , 8 , 9 , or 10 or more amino acids .
  • the phosphorylation of a single bHLH protein may be reduced in the immature mammalian cells as described herein .
  • the phosphorylation of more than one bHLH protein for example two , three, four or more different bHLH proteins , may be reduced.
  • some neuronal cell types may co-express or sequentially express the bHLH proteins atonal , Ascll and/or Ngn2.
  • phosphorylation of all of these bHLH proteins may potentiate the transition of neurons along both the differentiation and maturation pathways and/or modulate the neurona1 sub-type so generated.
  • the phosphorylation of the bHLH protein Ngn3 and optionally Ptfla may be reduced in order to mature differentiated pancreatic endocrine cells, preferably pancreatic islet beta cells .
  • a method of producing mature pancreatic endocrine cells, such as mature islet beta cells may comprise;
  • the number of phosphorylated SP/TP sites in Ngn3 may be reduced in the population of cells .
  • the subtype of pancreatic endocrine cell that is developed by the immature pancreatic endocrine cells during maturation may be determined by the phosphorylation sites that are unphosphorylated in the Ngn3 protein . Reducing phosphorylation at one or both of Cdk and GSK3 phosphorylation sites may be useful in producing specific endocrinal sub-types , such as islet alpha, islet beta, islet delta and and islet PP cells, or increasing the proportion of specific endocrine pancreatic sub-types in a mature population of pancreatic endocrine cells . Phosphorylation sites for Cdk and GSK3 ⁇
  • the pancreatic endocrine cells are islet beta cells .
  • Suitable immature pancreatic endocrine cells may include immature islet beta cells and pancreatic endocrine precursor cells .
  • reducing the phosphorylation of Ngn3 SP/TP sites in the immature islet beta cells may induce or promote the onset of a mature islet beta cell phenotype.
  • a mature islet beta cell phenotype may be characterised by
  • mature islet beta cells may display a high and physiological threshold level of glucose
  • phosphorylation sites in the bHLH protein may be reduced in the immature mammalian cells as described herein by any suitable technique .
  • phosphorylation may be reduced by expressing a modified bHLH protein in the immature mammalian cells .
  • the modified bHLH protein may retain the activity of the wild-type bHLH protein but may be devoid of phosphorylated sites in the immature mammalian cells or may contain fewer phosphorylated sites than wild-type bHLH protein (i.e. the modified bHLH protein may be phosphorylation-deficient) .
  • the modified bHLH protein may be devoid of phosphorylated SP/TP sites in the immature
  • mammalian cells may contain fewer phosphorylated SP/TP sites than wild-type bHLH protein .
  • a suitable modified bHLH protein may include fewer SP/TP
  • modified bHLH protein may have 1 , 2 , 3 , 4, 5 , 6, 7 , 8 , 9, 10 or more than 10 fewer SP/TP sites than the unmodified bHLH protein .
  • modified bHLH protein may not contain any SP/TP phosphorylation sites at all . Since its sequence contains fewer SP/TP sites, fewer phosphorylated SP/TP sites are present in the modified bHLH protein relative to the wild-type bHLH protein following expression in the immature
  • modified bHLH protein may not contain any phosphorylated SP/TP sites at all .
  • a modified bHLH protein may be producing by modifying the amino acid sequence to remove 1 , 2, 3, 4 , 5 , 6, 7 , 8, 9 , 10 or more than 10 SP/TP phosphorylation sites .
  • An SP/TP phosphorylation site may be removed by inserting, substituting or deleting 1 , 2 or more amino acids in the amino acid sequence which remove or replace the Serine- Proline or Threonine-Proline motif of the phosphorylation site with a sequence which is not phosphorylated by mammalian cell kinases .
  • the Serine or Threonine residue of an SP/TP phosphorylation site may be substituted for an Alanine or other amino residue .
  • the SP or TP motif in the wild-type bHLH protein may be altered to AP in the modified bHLH protein .
  • AP is not a kinase substrate .
  • suitable phosphorylation deficient human Ascll and Ngn3 sequences are shown in SEQ ID NOS : 2 and 4 and examples of suitable phosphorylation deficient mouse Ngn3 sequences are shown in SEQ ID NOS : 6 and 7.
  • Other phosphorylation deficient bHLH proteins may be produced using standard techniques .
  • the serine/threonine residue , proline residue or both of an SP/TP phosphorylation site may be substituted for a glutamic acid or aspartic acid residue . This may be useful in mimicking fully phosphorylated bHLH protein and potentiating progenitor cell maintenance .
  • a modified bHLH protein may be encoded by an exogenous nucleic acid.
  • Nucleic acid which encodes a modified bHLH protein may be produced and manipulated using standard techniques . Suitable techniques and protocols for mutagenesis, manipulation, cloning and expression of recombinant nucleic acids are well known in the art ( see for example Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (Mar 1997) Humana Press Inc , Molecular Cloning: a Laboratory- Manual : 3rd edition, Russell et al . , 2001, Cold Spring Harbor
  • a nucleic acid sequence encoding a modified bHLH protein may be comprised within an expression vector .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences , including promoter sequences , terminator fragments , polyadenylation sequences , enhancer sequences , marker genes and other sequences as appropriate .
  • the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in the mammalian cells cell .
  • constitutive promoters for example viral promoters such as CMV or SV40 , and inducible promoters , such as Tet-on controlled promoters .
  • Preferred vectors for use in the present methods include lentiviral and retroviral vectors .
  • Expression vectors containing the nucleic acid may be transfected into the immature mammalian cells . Any convenient technique for the transfection may be employed . Following transfection, the modified bHLH protein is expressed in the immature mammalian cells and causes them to mature . In other embodiments , transposon-mediated or other random integration transgenesis techniques may be employed .
  • the nucleic acid encoding the modified bHLH protein may then be expressed in the immature mammalian cells .
  • the immature mammalian cells may be matured as described herein with minimal or no genetic modification of the cells .
  • Suitable techniques are known in the art and include the use of excisable lentiviral and transposon vectors ; repeated application of transient plasmid, episomal and adenovirus vectors or; the use of small molecules , synthetic mRNA and/or microRNAs (Sidhu KS. Expert Opin Biol Ther . (2011) 11(5) : 569-79; Woltjen K et al (2009) Nature 458 (7239) : 766-70; Chou BK et al . Cell Res. 2011 21 (3) :518-29) .
  • one or more modified bHLH proteins or encoding nucleic acids may be introduced directly into the immature mammalian cells .
  • Suitable techniques are known in the art for the introduction of nucleic acids (Warren L et al . Cell Stem Cell . 2010 Nov 5 ; 7(5) : 618- 30) or proteins (Zhou H, et al Cell Stem Cell . 2009 May 8; 4(5) : 381- 4 ) .
  • the immature mammalian cells may be cultured in the presence of the modified bHLH protein or nucleic acid under
  • modified bHLH proteins or encoding nucleic acid may be introduced into immature mammalian cells by traditional methods such as lipofection, electroporation, calcium phosphate precipitation, particle bombardment and/or microinj ection, or may be delivered into cells by a protein delivery agent .
  • Nucleic acids for direct delivery into immature mammalian cells may be translatable by endogenous translation factors within the cell .
  • a suitable synthetic mRNA may be modified.
  • 5- methylcytidine may be substituted for cytidine, and pseudouridine for uridine , followed by phosphatase treatment (Warren, et al 2010 ) .
  • the cells may be cultured for 5 or more days , 10 or more , 15 or more, 20 or more or 25 or more days .
  • immature islet beta cells may be matured by introducing modified Ngn3 into the cells .
  • a modified Ngn3 is phosphorylation deficient and may be devoid of ST/TP phosphorylation sites or may contain fewer ST/TP phosphorylation sites than the wild type .
  • Suitable modified Ngn3 proteins include SEQ ID NOS : 4 , 6 and 7.
  • a method may comprise;
  • phosphorylation deficient Ngn3 protein may be introduced into the immature mammalian cells by RNA-mediated transfection in accordance with known techniques and expressed.
  • a phosphorylation-deficient bHLH protein such as Ngn3
  • expression or introduction of a phosphorylation-deficient bHLH protein, such as Ngn3 increases the amount of bHLH protein with reduced phosphorylation in the cells and thereby promotes maturation of the cells .
  • phosphorylation of ST/TP sites in the bHLH protein may be reduced in the immature mammalian cells by reducing the amount of phosphorylation of endogenously expressed bHLH protein in the cells .
  • the amount of phosphorylation may be reduced by inhibiting the activity of one or more protein kinases which target SP and TP phosphorylation sites in the immature mammalian cells .
  • the activity of one or more of a cyclin dependent kinase (Cdk) , GSK3 and MAPK may be inhibited.
  • the activity of one or more protein kinases may be inhibited by treating or exposing the population of cells to a protein kinase inhibitor .
  • the cells may be cultured in a nutrient medium which comprises a protein kinase inhibitor .
  • a protein kinase inhibitor Many suitable kinase inhibitors are known in the art, including GSK3beta, MAP kinase and eye1in-dependent kinase (cdk) inhibitors (Cicenas J et la Cancer Res Clin Oncol . 2011 Oct; 137 (10) : 1409-18; Trujillo JI . Expert Opin Ther Pat. 2011 Jul; 21 (7) : 1045-69; and agman AS et al Curr Pharm Des . 2004; 10 (10) :1105-37) .
  • Cdk inhibitors such as roscovitine, N- [ 5- [ [ [ [ 5- ( 1 , 1- dimethylethyl ) -2-oxazolyl ] methyl ] thio] -2-thiazolyl ] -4 - piperidinecarboxamide (BMS 387032; SNS-032 ) , flavopiridol ,
  • olomucine , dinaciclib, N- ( 4-piperidinyl ) -4- (2, 6- dichlorobenzoylamino ) -lH-pyrazole-3-carboxamide (AT7519) , and palbociclib may be employed, most preferably olomucine , roscovitine or palbociclib .
  • kinase inhibitor to reduce the phosphorylation of bHLH proteins and increase maturation activity may be less than the concentration required to bring about cell cycle arrest or other toxic effects .
  • concentrations of kinase inhibitor for maturation of any particular cell type may readily be determined empirically by a skilled person . In some embodiments , an initial concentration of about 40 ⁇ may be employed.
  • the kinase inhibitor may be added to culture medium continually or withdrawn, once maturation has commenced . Culture medium conditions will vary depending on the cell type which is to undergo maturation .
  • the phenotype of the cells may be monitored to determine the onset of a mature phenotype .
  • Phenotype may be monitored by determining the expression of one or more of mature cell markers and/or immature cell markers by the population of cells This allows the extent of maturation of the population to be determined. Cells which express one or more mature cell markers and/or do not express one or more immature cell markers may be identified as mature cells.
  • Suitable mature beta cell markers include insulin, MafA and Ucn3.
  • Suitable mature neuronal markers may include TuJl, MAP2 and Thl .
  • Cell markers expressed by a cell may be identified using standard techniques , such as PGR, western blotting, immunocytochemistry and in situ hybridisation .
  • Phenotype may be monitored by determining the presence of one or more morphological features of mature cells .
  • Morphological features of mature neurons include extended and/or branched neurites , such as axons and dendrites .
  • the length and branch structure of neurites in a population of neurons may be determined .
  • Phenotype may be monitored by determining the presence of electrical properties of the mature cells . For example , the response of a population of neurons to an electrical stimulus and/or the ability of a population of neurons to produce a spontaneous action potential may be determined .
  • Phenotype may be monitored by determining the production of one or more other features of a mature phenotype . For example, for
  • maturation of islet beta cells one or more of the following may be determined; glucose threshold for insulin secretion; ability to survive and function in mouse kidney capsule transplantation models; and ability to rescue rodent models of diabetes , where beta cell mass has been reduced and ability to reverse or partially ameliorate symptoms of type II diabetes in rodent models .
  • glucose threshold for insulin secretion For maturation of neurons , ability of neurons to form neural networks and/or innervate the striatum in rodent models may be determined .
  • a population of mature differentiated mammalian cells produced by the present methods may be heterogeneous and may contain immature cells or cells or other cell types.
  • mature cells such as mature neurons
  • mature cells may be separated from other cells in the population, as described below.
  • the population may be substantially free from other cell types .
  • the population of cells may contain 80% or more, 85% or more , 90% or more , or 95% or more mature differentiated mammalian cells of the relevant cell type , following reduction in bHLH phosphorylation .
  • the population of mature differentiated mammalian cells may be sufficiently free of other cell types that no purification is required .
  • a population of mature beta cells may comprise 50% or more, 60% or more, 70% or more , 80% or more , 85% or more , 90% or more, 95% or more, or 99% or more insulin expressing cells which display a physiological glucose response .
  • a method may further comprise isolating and/or purifying the mature mammalian cells .
  • Cells may be separated from other cell types in the population using any technique known to those skilled in the art , including those based on the recognition of extracellular epitopes by antibodies and/or magnetic beads or fluorescence activated cell sorting (FACS ) , including the use of antibodies against extracellular regions of characteristic markers .
  • FACS fluorescence activated cell sorting
  • the mature mammalian cells may be maintained in culture , stored, for example frozen using conventional techniques , or used in therapeutic or other
  • Mature mammalian cells may be cultured to maintain a homogenous or substantially homogenous population .
  • the population of mature cells may maintained in culture for at least 40 , at least 60 , at least 80 , at least 100, or more than 100 days .
  • the culture of immature and mature mammalian cells is well-known in the art ( see, for example, Basic Cell Culture Protocols , C.
  • Europabioproducts , R&D Systems for example, standard mammalian cell culture conditions may be employed, for example 37°C, 21% Oxygen, 5% Carbon Dioxide . Media is preferably changed every two days and cells allowed to settle by gravity .
  • mature differentiated cells produced as described herein for therapeutic applications are clinical grade .
  • Another aspect of the invention provides a population of isolated mature mammalian cells which have been produced by a method as described above .
  • Mature beta cells produced as described above may be capable of functionality in in vivo models without teratoma formation (Kroon et al (2008) Nature Biotechnology 26, 443-452) .
  • Mature neurons matured produced as described above may be capable of faithfully reproducing the functionality of mature neurons in vivo compared to neurons produced by previous in vitro methods .
  • Mammalian cells matured as described herein may be stable in vivo .
  • the cells may retain a functional mature phenotype indefinitely in vivo following implantation .
  • the rate of teratoma formation following implantation of mammalian cells matured as described herein may be reduced relative to the immature mammalian cells not subj ected to maturation .
  • the rate of teratoma formation following implantation of the mature mammalian cells is zero or substantially zero .
  • the population of mature cells are pancreatic endocrine cells.
  • Mature pancreatic endocrine cells may have a mature pancreatic endocrine cell phenotype .
  • the mature cells may express a pancreatic hormone selected from insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin and preferably insulin .
  • the mature cells do not express more than one pancreatic hormone and do not, for example, express both glucagon and insulin or both insulin and somatostatin .
  • Mature pancreatic endocrine cells may be capable of forming mature secretory granules in vivo
  • the population of mature cells are mature islet beta cells .
  • Mature beta cells may express insulin and C-peptide . Mature beta cells may also express PCSK1, PCSK2, NKX6-1, MAFA, PDX1 , amylin (islet amyloid polypeptide ) and Unc3.
  • Mature beta cells may be glucose-responsive .
  • the mature beta cells may exhibit physiological thresholds for the expression and secretion of insulin in response to glucose .
  • the population of mature cells may be mature neurons .
  • the mature neurons may have a mature neuronal phenotype , for example a mature cortical , peripheral , cerebellar, hypothalamic or spinal neuronal phenotype .
  • Mature neurons may have extended and branched axons and dendrites , display electrical excitability, be capable of forming neuronal network and may engraft and restore functionality in engraftment models of neurological diseases , such as Parkinson' s di sease .
  • a population of mature cells produced as described herein may useful in disease modelling.
  • antecedent cells with a genotype associated with a disease condition may be used to produce mature mammalian cells with the disease associated genotype in vitro. These cells may be tested in vitro for physiological differences compared to matched healthy control cells . For example , response to stress, electrical activity, response to toxins , mitochondrial function and/or resistance to cell death may be tested.
  • fibroblasts derived from a patient with a genetic predisposition to a neural condition may be trans-differentiated to mature neurons in vitro and those neurons tested for physiological differences compared to matched healthy controls neurons .
  • a population of mature cells produced as described herein, or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein has been reduced as described herein and which are undergoing maturation, may be useful in therapy, for example cell-replacement therapy .
  • bHLH basic helix loop helix
  • the population may form part of a therapeutic composition .
  • a population of mature cells produced as described above may be formulated with a pharmaceutically acceptable carrier and/or a tissue scaffold .
  • a therapeutic composition, medicament, or other composition may comprise a population of mature differentiated cells, along with a pharmaceutically acceptable excipient, carrier, buffer,
  • a therapeutic composition may be produced by admixing a population of mature mammalian cells , for example mature beta cells , with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients .
  • composition may be administered to a patient, e.g. for treatment (which may include preventative treatment ) of dysfunctional tissue, as described above .
  • a composition comprising mature beta cells may be administered to a patient for treatment (which may include preventative treatment ) of damaged or dysfunctional
  • pancreatic tissue and in particular for the treatment of a diabetic condition, such as type 1 or type 2 diabetes .
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil .
  • a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil .
  • Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included.
  • the composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability .
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability .
  • isotonic vehicles such as Sodium Chloride , Ringer ' s Inj ection, or Lactated Ringer ' s Inj ection .
  • a composition may be prepared using artificial cerebrospinal fluid.
  • Cells may be implanted into a patient by any technique known in the art (e.g. Lindvall , 0. (1998) Mov . Disord . 13, Suppl . 1:83-7; Freed, C.R., et al . , (1997) Cell Transplant , 6, 201-202; Kordower , et al . , (1995) New England Journal of Medicine , 332, 1118-1124; Freed, C.R., (1992) New England Journal of Medicine , 327 , 1549-1555, Le Blanc et al, Lancet 2004 May 1;363 (9419) : 1439-41) .
  • any technique known in the art e.g. Lindvall , 0. (1998) Mov . Disord . 13, Suppl . 1:83-7; Freed, C.R., et al . , (1997) Cell Transplant , 6, 201-202; Kordower , et al . , (1995) New England Journal of Medicine
  • Administration of a composition in accordance with the present invention is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a “prophylactically effective amount” or a “therapeutically effective amount” as the case may be, although prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors .
  • a composition may be administered alone or in combination with other treatments , either simultaneously or sequentially dependent upon the condition to be treated.
  • aspects of the invention provide a method of treatment comprising admini stering a population of mature mammalian cells produced as described above to an individual in need thereof; the use of population of mature cells produced as described above in the manufacture of a medicament for use in a method of treatment; and a population of mature cells produced as described above for use in a method of treatment .
  • the mature mammalian cells may be immunologically compatible with the individual .
  • the mature mammalian cells may be immunologically compatible with the individual .
  • the mature mammalian cells may be produced from antecedent cells obtained or derived from the same individual or an immunologically compatible individual .
  • the mature mammalian cells may be produced from fibroblasts or ES cells obtained from the individual or iPS cells derived from fibroblasts or other cells obtained from the individual ; or the mature mammalian cells may be produced from stored or banked fibroblast, ES or iPS cells which are immunologically compatible with the individual .
  • the nature of the method treatment depends on the cell type .
  • a method of treatment of a diabetic condition may comprise administering a population of mature pancreatic beta cells produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn3 , has been reduced as described herein and which are undergoing maturation into mature pancreatic beta cells, to an individual in need thereof; the use of population of mature pancreatic beta cells produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn3 , has been reduced as described herein and which are undergoing maturation into mature pancreatic beta cells in the manufacture of a medicament for use in a method of treatment of a diabetic condition; and a population of mature pancreatic beta cells produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as
  • Diabetic conditions include type 1 and type 2 diabetes .
  • Treatment may ameliorate or relieve diabetes-related symptoms in the individual or reduce or remove the need for insulin therapy .
  • a method of treatment of a neural condition may comprise administering a population of mature neurons produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as gn2 or Ascll , has been reduced as described herein and which are undergoing maturation into neurons to an individual in need thereof; the use of population of mature neurons produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn2 or Ascll , has been reduced as described herein and which are undergoing maturation into neurons , in the manufacture of a medicament for use in a method of treatment of a neural condition; and a population of mature neurons produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn2 or Ascll , has been reduced
  • Neural conditions may include neurodegenerative conditions , such as Parkinson' s Disease, and conditions associated with damaged or dysfunctional neural tissue, for example stroke or spinal cord or peripheral nerve inj ury .
  • mature midbrain dopaminergic TH+ neurons produced as described above may be used to treat Parkinson' s disease .
  • phosphorylation deficient Ascll may be used to treat Parkinson' s disease .
  • a population of mature cells produced as described herein or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein has been reduced as described herein and which are undergoing maturation may also be useful in screening for compounds with modulatory effects .
  • bHLH basic helix loop helix
  • Screening may include drug or small molecule screening .
  • the isolated mature differentiated cells may be contacted with a test compound and the effect of the test compound on the cells is determined. Screening may also include functional genomic
  • a gene may be suppressed, knocked out or otherwise inactivated in the isolated mature differentiated cells and the effect of the inactivation on the cells determined.
  • a method of screening a compound may comprise;
  • test compound o cell death/ survival growth, proliferation, condition, aggregatio function, hormone production, glucose responsiveness and/or ge expression may be determined.
  • a method of screening a compound may comprise;
  • test compound on morphological features , such as extended and/or branched neuritesm, or on the electrical properties of the neurons may be determined .
  • mature differentiated cells may be produced from iPS cells as described herein .
  • the iPS cells may be from a normal individual or an individual with a disease condition .
  • the iPS cells may be differentiated into a cell type that is affected in the disease condition and subj ected to maturation as described herein .
  • the mature differentiated cells may express a detectable reporter or display an observable cellular phenotype which differs between disease affected cells and normal cells .
  • the mature differentiated cells may be exposed to test compounds and the effect of the test compound on the reporter expression or observable cellular phenotype determined. Compounds which cause the mature cells to revert from disease cell state to the normal state may be identified.
  • the one or more genes in the mature differentiated cells may be inactivated, for example by targeted mutation or RNAi suppression, and the effect of the inactivation on the reporter expression or observable mature cellular phenotype determined.
  • Genes whose inactivation causes the cells to revert from disease cell state to the normal state may be identified.
  • Screening may include toxicology screening .
  • the isolated mature cells may be contacted with a test compound at various concentrations that mimic abnormal/normal concentrations in vivo.
  • the effect of the test compound on the cells may be determined and toxic effects identified.
  • Toxicology screening is well known in the art (see for example Barbaric I et al . Biochem Soc Trans . 2010 Aug; 38 (4) : 1046-50) .
  • Phosphorylation assays in Xenopus extract were performed as for degradation assays (Vosper et al . , 2007; Vosper et al . , 2009) , except that extract was supplemented with 0.2 mM MG132 (Biomol ) and samples were incubated for 45 min . Where indicated, following the 45 min incubation in extract , 400 u of ⁇ phosphatase (NEB) were incubated with the sample at 30 °C for 30 min (Ali et al , (2011) ) .
  • PI 9 cells were cultured in a-MEM (Gibco) with 7.5% newborn calf serum and 2.5% fetal bovine serum (HyClone) , 1% Glutamax (Gibco) , and 100 units/ml penicillin/ 100 streptomycin (Sigma) .
  • Cells were transfected with Lipofeetamine 2000 ( Invitrogen) , and extracts western blotted with anti-HA-peroxidase (3F10 ) [Roche ] or anti-c-Myc ( 9E10 ) [Santa-Cruz] . Where indicated, cells were treated with 400 u of ⁇ -phosphatase (NEB) (Ali et al, (2011) ) .
  • cDNA was generated from stage 15 or stage 26 Xenopus embryos , using 50 ng per qPCR reaction, in the Light-cycler 480 PGR system (Roche, Wales, UK) with SYBR Green mix (Roche) , ⁇ -actin, GAPDH and EFl were used as housekeeping genes .
  • Thermal cycling conditions 95 ° C for 5 min, 45 cycles of 95 ° C for 10 sec, 60 ° C for 10 sec, and 72°C for 10 sec (All et al, (2011) ; Hindley et al (2012) ) .
  • mNgn2 / 9S-A mNgn2 expression were cross-linked with 1% formaldehyde .
  • 5 ⁇ g of rat monoclonal anti-HA antibody (Roche) or anti- IgG (Abeam) (as a control ) were used per ChIP reaction and quantified using SYBR Green mix .
  • Signal over background ' normalisation method was used to quantify immunoprecipitated DNA (Ali et al , (2011) ; Hindley et al (2012) ) .
  • Lenti-X 293T cells (Clontech) were grown to 90% confluency in Tet- free media and transfected using the Lenti-X Tet-On 3G Inducible Expression System (Clontech) . The media was replaced the following day . 48 hours after transfection, viruses were harvested from the culture supernatant using the Lenti-X Concentrator (Clontech) .
  • Lentiviral aliquots are snap frozen in dry ice and stored in -80 °C .
  • Viral titrations were obtained using the Lenti-X qRT-PCR Titration Kit (Clontech) .
  • Target cells were plated into 24-well plates at a concentration of 20,000 cells/well and infected the following day with lentivurses in DMEM supplemented with 5]ig/ml polybrene .
  • MOI Tet3G activator
  • the media was replaced the following day with fresh Tet- free media, and target cells were induced using DMEM supplemented with 2 g/ ⁇ doxycycline on day 3 following infection .
  • DMEM media was replaced by NDiff N2B27 Neuronal Differentiation Media (StemCells) supplemented with doxycycline .
  • Media was replaced every 2-3 days until day 12 for mouse fibroblasts and day 21 for human fibroblasts .
  • cells were fixed and stained for neuronal differentiation using 1:500 anti-Tuj 1 (Covance ) and 1:200 anti-MAP2 (Sigma) .
  • Embryos were inj ected into 1 ventral vegetal cell at the 8 cell stage with mRNA encoding either WT Ngn3 (WT) or 6S-A Ngn3 (named SP here) , both fused to the glucocorticoid receptor (GR) .
  • WT WT Ngn3
  • 6S-A Ngn3 named SP here
  • GR glucocorticoid receptor
  • Ngn3 has 6 SP sites .
  • Ngn3 is multiply phosphorylated in both interphase (I) and mitosis (M) ( Figure 1), and Ngn3 is also multiply phosphorylated in mouse PI 9 embryonal carcinoma cells ( Figure 2 ) .
  • I interphase
  • M mitosis
  • Figure 2 mouse PI 9 embryonal carcinoma cells
  • hypothalamic neurogenesis as well an endocrine pancreas and gut cell formation in the mouse embryo drives ectopic neurogenesis .
  • We generated a phosphomutant mNgn3 6S-A Ngn3 , serines of SP sites mutated to alanines ) .
  • Xenopus embryos were inj ected with the phosphomutant 6S-A Ngn3 mRNA and wild type Ngn3 mRNA and subj ected to in situ hybridisation for the neuronal marker neural beta tubulin .
  • 6S-A Ngn3 was observed to be significantly more active that the wild-type protein in driving neurogenesis in this in vivo setting, indicating similar regulation to Ngn2.
  • RNA encoding GFP, mNgn3 WT and mNgn3 6S-A was inj ected into Xenopus embryos at the 1-cell stage .
  • RNA was extracted from stage 19 embryos and the expression of selected target genes quantified by qPCR, normalised to expression of the housekeeping gene EFla and expressed relative to the expression level in GFP-inj ected embryosm ( Figure 3 ) .
  • Significant increases were observed in the expression of NeuroD (approx 5 fold) and Neural- ⁇ -tubulin (Npt ) (approx 2.5 fold) in mNgn3 6S-A-inj ected embryos compared to mNgn3 WT-inj ected embryos .
  • PI 9 cells were transfected 24 hours after plating with mNgn3 WT HA or mNgn3 6S-A HA and fixed and cross-linked after 24 hours .
  • the resulting chromatin was normalised for mNgn3 protein levels and immunoprecipitated with either an anti-HA antibody or IgG .
  • the promoter occupancy of mNgn3 was assessed by qPCR for the i ) NeuroDl and ii ) Dill promoters , expressed as the fold change of anti-HA immunoprecipitated samples against IgG ( Figure 4 ) .
  • mNgn3 WT HA was found to be enriched at both promoters , and in both cases the binding of mNgn3 6S-A HA was greater than that of the wild-type form of the protein .
  • the phosphomutant gn3 was observed to bind NeuroD and Delta promoters approx . 2-fold more efficiently than wild-type Ngn3.
  • Ngn3 is required for generation of entero- endocrine cells of the adult gut , and we analysed these cells first .
  • the phosphomutant Ngn3 adult mice maintained half the number of Chromagranin A-expressing entero-endocrine cells compared to those of the wild-type control .
  • Ngn3 expression generally drive cell cycle exit in vivo and in vitro (Miyatsuka, T . , et al . Proc Natl Acad Sci U S A 108 , 185-190, (2011) ) but Ngn3 expression does not always result in cell cycle exit and differentiation per se; low levels of Ngn3 are found in first transition proliferating pancreatic progenitors and very low levels that persist in adult islets may contribute to proper expansion of these endocrine cells (Miyatsuka, T . , et al . (2011 ) ) . Under some circumstances and levels of expression, dephosphorylated Ngn3 may also enhance cell cycling prior to promoting differentiation.
  • Ngn3 phospho- status of endogenously expressed Ngn3 affected its ability to regulate entero- endocrine cell differentiation .
  • preventing Ngn3 phosphorylation may significantly enhance its ability to drive endocrine differentiation and maturation in the pancreas when Ngn3 is over-expressed .
  • the phospho- status of Ngn3 may affect its ability to drive pancreatic beta cell differentiation and maturation in vitro from ES-derived cultures and may be manipulated to enhance beta cell generation for potential use in islet transplantation therapy .
  • Fertilised Xenopus eggs were left uninj ected or inj ected with mRNA for encoding wild-type or phosphomutant 6S-A Ngn3 into cells destined to form endoderm. Embryos were allowed to develop until swimming tadpole stages , when insulin would be expressed after ectopic expression of Ngn3. Insulin expression was then measured by qPCR. Phosphomutant Ngn3 resulted in enhanced transdifferentiation of embryonic endodermal tissue into ectopic insulin expressing pancreatic islet tissue compared to the wild-type Ngn3 ( Figure 5 ) .
  • mouse ES cells expressing phosphomutant Ngn3 in place of the wild-type Ngn2 heterozygously were observed to show enhanced NeuroD (an early marker of beta cell maturation) expression when induced to differentiate towards endocrine pancreas by culture media conditions .
  • Mouse ES cells were engineered to express one copy of either Wt Ngn3-eYFP or 6S-A Ngn3-eYFP from the endogenous locus , then
  • Ngn3 and NeuroD were measured by qPCR in heterozygous mouse ES cells expressing either WT or 6S-A (phosphomutant ) Ngn3 , taken down definitive endoderm differentiation pathway through endol , endol I and endoIII media ( Figure 6) .
  • phosphomutant Ngn3 was found to enhance expression of endogenous Ngn3 and neuroD and promote differentiation compared to cells expressing WT Ngn3. Inhibition of phosphorylation of endogenous Ngn3 during endoderm/pancreatic islet differentiation would be found to enhance expression of endogenous Ngn3 and neuroD and promote differentiation compared to cells expressing WT Ngn3. Inhibition of phosphorylation of endogenous Ngn3 during endoderm/pancreatic islet differentiation would
  • Ngn3 neuroD and therefore pancreatic endoderm and beta cell differentiation .
  • Heterozygous wt or 6S-A (phosphomutant ) Ngn3 knock-in mouse ES cells in which the WT or phosphomutant Ngn3 was expressed from the endogenous promoter were subj ected to a 3 stage endo 1 , 11 III protocol of differentiation into definitive endoderm. After 2 days in endo III , phosphomutant Ngn3 ES cells were found to express insulin while wild-type ES cell , do not, indicating that even when expressed at endogenous levels , inhibiting phosphorylation of Ngn3 enhances insulin expression in vitro ( Figure 7 ) .
  • Ngn3 knock-in mouse ES cells (where WT or phosphomutant Ngn3 is expressed from the endogenous promoter) were subj ected to a 3 stage endo 1 , 11 III protocol of differentiation into definitive endoderm. After 32 days in endo III , phosphomutant Ngn3 ES cells were observed to be morphologically distinct from wt Ngn3-expressing cells ( Figure 8 ) and showed enhanced maturation and organisation, forming potentially islet-like clusters . These results indicate that, even when expressed at endogenous levels , inhibiting phosphorylation of Ngn3 enhances morphological maturation in vitro . Manipulating phosphorylation of the endogenous protein may therefore be useful in achieving cell maturation .
  • the basic helix-loop-helix (bHLH) transcription factor Ascll (also known as Mashl) is a key regulator of stem cell maintenance and differentiation in both the central and peripheral nervous systems (CNS and PNS ) , and multiple direct downstream transcriptional targets have been identified.
  • Xenopus egg extracts were used to investigate cell cycle-dependent post-translational protein modifications of in vitro translated proteins , visualized by slowed migration on SDS PAGE .
  • Ascll is modified in both interphase (I) extracts , and to a greater extent in mitotic (M) extracts , and phosphatase treatment confirmed that phosphorylation has occurred .
  • Ascll contains 6 serine-proline pairs , potential sites of cdk-dependent phosphorylation . Mutation of these serines to alanine to generate 6S-A Ascll restored SDS PAGE mobility (although some residual phosphorylation in mitosis appears to remain, indicating phosphorylation on additional sites ) . Further mutational analysis revealed that multiple SP sites on both sides of the bHLH domain were phosphorylated.
  • Ascll is phosphorylated on multiple SP sites by cdks .
  • Ascll may also be phosphorylated by other kinases such as GSK3 and MAP kinases .
  • 6S-A Ascll gives a 7-fold enhancement of expression of the direct pro-differentiation targets MyTl and neural beta-tubulin compared to Ascll . Delta expression is enhanced 2-fold. In contrast, overexpression of Ascll or 6S-A Ascll has little effect on expression of the pro-proliferative genes E2F1 , cdk2 and Skp2.
  • Phosphomutant Ascll is significantly more active in driving
  • Notch intracellular domain was found to inhibit both endogenous neurogenesis, and neurogenesis driven by Ascll mRNA inj ection in Xenopus embryos .
  • phosphomutant Ascll was found to still efficiently drive neuronal differentiation in the presence of NICD .
  • reducing or preventing phosphorylation of proneural proteins on SP/TP sites may confer resistance to Notch mediated inhibition, which is a common characteristic of differentiation driven by proneural protein activity .
  • Transdifferentiation of mouse fibroblasts can be achieved by expression of Brn2, Ascll and MyTlL, while addition of NeuroD to these "BAM factors" is required for differentiation of human fibroblasts .
  • BAM factors Basal Activated Growth Factors
  • these neurons Although capable of firing action potentials , these neurons generally appear immature with relatively short axons and dendrites .
  • Our results provide indication that phosphomutant Ascll may be more efficient at up-regulation of down-stream targets associated with differentiation than wild-type Ascll , including Ascll .
  • 6S-A Ascll may potentiate neuronal transdifferent!ation with and without MyTlL, Brn2 and NeuroD in human and mouse fibroblasts .
  • BAM factors induced functional neurons but with short axonal and dendritic outgrowths , which is likely to affect their ability to make connections and innervate .
  • BAM using phosphomutant Ascll resulted in neurons which displayed increased maturity as evidenced by multiple extended neurites .
  • Overexpression of the "BAM" transcription factors Ascll , Brn2 and MyTIL can drive transdifferentiation of fibroblasts into functional neurons ( iN cells ; Pang, N . , et al . Cell Stem Cell 9, 517-525, (2011 ) , and these have been proposed as a cell source for
  • lentviruses expressing neurogenic conversion factors as labelled . 21 days after transduction with T or S-A hAscll in BAM (Brn2, Ascll and MyTi ) or BAM + NeuroD protocols, cells were stained for DNA and the marker of neuronal differentiation Tuj 1 to neuronal show morphology . We saw that, in human lung fibroblasts , BAM factors plus phosphomutant Ascll induced twice as many neurons as BAM factors plus wild-type Ascll and the neurons so generated showed
  • BAM factors plus NeuroD also led to the induction of neurons with short axonal and dendritic outgrowths .
  • BAM plus NeuroD using phosphomutant Ascll resulted in neurons , which displayed increased maturity as evidenced by multiple extended neurites
  • FIG. 10 Wild type Ascll with NeuroD produced no neurons while phosphomutant Ascll with NeuroD did produce neurons , although these have shorter neurites than the BAM plus NeuroD combination, and neuronal differentiation occurred with low efficiency ( Figure 10 ) . Moreover, neurons generated using the phosphomutant Ascll showed significantly enhanced electrophysiological maturity compared to those generated with wild-type Ascll . Our data show that substituting phospho-mutant Ascll for wild-type Ascll in human fibroblast transdifferentiation significantly enhances the trans-differentiation of human fibroblasts into mature neurons compared to those generated using wild-type Ascll ( Figure 10 ) .
  • Phosphomutant Ascll promotes conversion efficiency and neuronal complexity and maturity of induced neuron (iN) cells . Furthermore, our preliminary data indicates that phosphomutant Ascll alone, without Brn2 and MyTl, may be sufficient to generate some neurons in the transdifferentiation assay, while wild-type Ascll is not. These cells do not undergo extensive proliferation post BAM factor addition, yet the phosphomutant Ascll still promotes more mature neurons , indicating that phosphorylation on SP sites by kinases other than cdks may usually be occurring and regulating activity . Thus , multi-site phosphorylation controls the ability of Ascll to drive cell di fferentiation and maturation in vitro.
  • Phosphomutant Ascll was observed to promote conversion efficiency and neuronal maturity of the iN cells when cultured with the glycogen synthase kinase-3b inhibitor .
  • the iN cells generated from the human fibroblasts (using BAM factors , Brn2 , Ascll , MyTl , with or without NeuroD) and small molecule inhibitors were observed to be more mature and complex when using the phosphomutant protein
  • a partially retinoic acid resistant neuroblastoma cell line , SKNDZ , that expresses endogenous Ascll was treated with either 5 ⁇ retinoic acid (RA) , 10 ⁇ palbociclib (a CDK inhibitor currently in clinical trials ) or in combination for 24 , 48 or 96 hours .
  • RA retinoic acid
  • palbociclib a CDK inhibitor currently in clinical trials
  • SKNAS retinoic acid resistant neuroblastoma cell line
  • RA retinoic acid
  • CDKi 20 ⁇ roscovitine
  • mRNA for WT or phosphomutant forms of Ascll with mutation of predicted cdk sites (Ascll Cdk) , GSk3beta sites (Ascll Gsk3B) or cdk and Gsk3B sites simultaneously (Ascll Cdk Gsk3B) were inj ected into fertilsed Xenopus eggs and a comparison of noraderinergic and seratonergic markers conducted by qPCR at the tailbud stage ( Figure 12 ) . Overall neurogenesis was assayed by neural beta tubulin expression .
  • Serotonergic and noradrenergic subtype markers were found to be differentially influenced by the availability of phosphorylation sites targeted by Cdks and GSk3beta .
  • mutation of both Cdk and GSK3B sites increased noradrenergic marker expression relative to serotonergic marker expression compared to mutation of either Cdk or GSK3B sites alone .
  • a mutant with SP sites and additional potential target sites for GSK3 beta was found to be more active at driving neuronal differentiation (as assayed by neural beta tubulin expression) compared to a mutant of SP sites alone , demonstrating that further phosphosites may contribute to inhibition of the ability of proneural proteins to drive
  • Human Ngn3 sequence ( Seq ID NO : 3 ) : mtpqpsgapt vqvtreters fprasedevt cptsappspt rtrgncaeae eggcrgaprk Irarrggrsr pkselalskq rrsrrkkand rernrmhnln saldalrgvl ptfpddaklt kietlrfahn yiwaltqtlr iadhslyale ppaphcgelg spggspgdwg slyspvsqag slspaaslee rpgllgatfs aclspgslaf sdf1
  • 6S-A hNgn3 sequence (Seq ID NO: 4) : (modified residues highlighted) mtpqpsgapt vqvtreters fprasedevt cptsappapt rtrgncaeae eggcrgaprk Irarrggrsr pkselalskq rrsrrkkand rernrmhnln saldalrgvl ptfpddaklt kietlrfahn yiwaltqtlr iadhslyale ppaphcgelg apggapgdwg slyspvsqag s lapaas lee rpgllgatfs aclapgslaf sdf1
  • LRARRGGRNRP SELALS QRRSRRK ANDRERNRMHNL SALDALRGVLPTFPDDAKLT 120

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Abstract

This invention relates to the manipulation of the phosphorylation of basic helix-loop-helix (bHLH) transcription factors, such as neurogenin-3 (Ngn-3), neurogenin-2 (Ngn-2) and Achaete-scute homolog 1 (Ascl1), to promote the maturation of immature mammalian cells. This may be useful in the production of mature mammalian cell populations, for example, for use in therapy and disease modelling.

Description

Maturation of Mammalian Cells by Modulation of Protein
Phosphorylation
Field of Invention
This invention relates to compositions and methods for the in vitro maturation of mammalian cells.
Background of Invention
Many illnesses of middle and old age result from a progressive loss of tissue function including Alzheimer' s disease, Parkinson' s disease , heart failure and notably diabetes . These chronic
conditions generally require long-term medication, but recent spectacular advances mostly grounded in basic biology have raised the possibility that defective cells could be directly replaced. An increase in the mass of islet beta cells has been proposed to ameliorate type 1 diabetes , and is also likely to be an effective treatment for many sufferers of Type 2 diabetes ( Pearl , E . J. & Horb, M. E. Clin Genet 74, 316-324 (2008) ) . Indeed, successful islet transplantation has been shown to result in relief from a requirement for insulin therapy and relief from diabetes-related symptoms (Daneman, D . Lancet 367 847-858 (2006) ) .
Stem cells (either ES or iPS ) could act as a source for B cell replacement ( Furth, M. E. & Atala, A . J Cell Biochem 106, 507-511 (2009) ) . However, a maj or obstacle remains in that stem cells must be induced to adopt beta cell fate and maintain that fate
indefinitely when transplanted back into the patient, retaining functionality and avoiding the risk of teratoma formation . It is widely acknowledged that for this to occur, stem cells must be taken down the developmental pathway that beta cells usually take when undergoing specification and differentiation in the developing embryo (Pearl et al (2008) ) . Based on these developmental
pathways , protocols have been published where human ES cells can be induced to form relatively immature hormone-expressing endocrine cells (D 'Amour, K. A. et al . Nat Biotechnol 24, 1392-1401, (2006) ) , while in vivo implantation results in further maturation and beta cell functionality (Kroon, E . et al . Nat Biotechnol 26 , 443-452 , (2008)) . However, a caveat to this approach is that teratoma formation was also occasionally observed, indicating the need for transplantation of islet cells that are differentiated to a more mature state . This is currently a road-block to generating more mature islet cells in vitro .
During foetal development, all endocrine cells in the pancreas are derived from Ngn3+ progenitors , which is expres sed in two waves during pancreas development (Rukstalis, J. M . & Habener, J . F.177- 184, (2009); Schwitzgebel , V. M. et al . Development 127, 3533-3542 (2000 ) ) . Overexpression of Ngn3 from the early pancreatic Pdxl promoter induces early and ectopic differentiation of islet cells (Johansson, K . A. et al . Dev Cell 12 , 457-465 (2007) ) , while further in vivo mouse experiments show that upregulation of Ngn3 at a defined developmental stage results in cell cycle exit and promotes formation of insulin-expressing cells, at the expense of exocrine pancreas (Johansson, K. A. et al . (2007) , Ectopic expression of Ngn3 in pancreatic duct cells promotes formation of insulin- expressing cells (Heremans , Y. et al . J Cell Biol 159, 303-312 (2002 ) ) and expression of Ngn3 in cultured pancreatic explants promotes endocrine differentiation (Dominguez-Bendala, J . et al .
Diabetes 54 , 720-726 (2005) ) . In the Xenopus embryo , transient expression of Ngn3 in embryonic endoderm is sufficient to bring about early and ectopic formation of pancreatic endocrine cells in the liver, stomach and duodenum (Oropez , D . & Horb, M . Genesis 50 , 271-285, (2012) ) . In addition to a requirement for endocrine and beta cell specification, Ngn3 is also required for islet cell maturation and function in both embryos and adults (Wang, S . et al . Proc Natl Acad Sci U S A 106, 9715-9720, (2009) ) . Furthermore, in the adult mouse , trans-differentiation of exocrine pancreas cells into functional beta cells can be driven by ectopic expression of only 3 transcription factors , Pdxl , Mafa and Ngn3 ( Zhou, Q . , et al . Nature 455 , 627-632 (2008) ) , and these factors need only be
expressed transiently for cells to stably adopt beta cell fate , making the possibility of their use in gene therapy more accessible . Manipulation of NGN3 activity to promote stem cells to adopt an endocrine pancreatic fate ex vivo is also currently an intense focus of interest for researchers (Samson, S . L . & Chan, L . Trends
Endocrinol Metab 17 , 92-100 (2006) ) . For instance, in mES cells , Ngn3 overexpression alone is capable of up regulating insulin, glucagon and somatostatin along with other markers of beta cell functionality (Treff , N. R. et al . Stem Cells 24, 2529-2537, (2006) ; Serafimidis , I., et al . Stem Cells 26 , 3-16 (2008) )
All the in vivo and in vi tro studies described above have focussed on manipulation of the level of Ngn3 transcripts .
However, more nuanced methods of regulating activity of Ngn3 are needed to enhance the functionality of beta and other islet cells for clinical applications . Summary of Invention
The present invention relates to the promotion of maturation of immature mammalian cells by manipulation of the phosphorylation of serine/proline and threonine/proline phosphorylation sites of basic helix-loop-helix (bHLH) transcription factors . Reducing, inhibiting or preventing phosphorylation of bHLH transcription factors , such as neurogenin-3 , may be useful in the production of mature cell populations , for example for use in therapy and disease modelling .
An aspect of the invention provides a method of producing mature mammalian cells comprising;
providing a population of immature mammalian cells; and;
reducing the phosphorylation of a basic helix loop helix (bHLH) protein in said population . The presence of bHLH protein with reduced phosphorylation or unphosphorylated bHLH protein (collectively termed "un(der)
phosphorylated bHLH protein" ) in the mammalian cells promotes maturation and produces mature mammalian cells . Phosphorylation may be reduced by reducing the number of
phosphorylation sites , such as Ser-Pro (SP) or Thr-Pro (TP) sites , in the bHLH protein that are phosphorylated, for example by inhibiting phosphorylation of these sites or removing one or more phosphorylation sites , such as SP or TP sites , from the sequence of the bHLH protein . Suitable bHLH proteins include proneural transcription factors , such as neurogenin-2 (Ngn2), neurogenin-3 (Ngn3) , Achaete- scute homolog 1 (Ascll ) , Pancreas specific transcription factor la (Ptfla) and
Atonal . Suitable mammalian cells are fully differentiated and include endocrine cells , such as intestinal and pancreatic endocrine cells , for example pancreatic beta cells, and neurons , including
dopaminergic, cortical , cerebellar, spinal , and peripheral neurons . Other suitable mammalian cells include intestinal , prostate and lung cells.
Brief Description of the Figures
Figure 1 shows in vitro translated S35-Met labelled mNGN3 or S- Am G 3 incubated in interphase (I) or mitotic (M) Xenopus egg extract with or without phosphatase treatment and analysed by SDS- PAGE.
Figure 2 shows mNGN3-HA transfected into PI 9 cells and harvested for western blotting at increasing times , lysates treated with
phosphatase as indicated .
Figure 3 shows the results of qPCR for the expression of NeuroD, Delta ( and neural b-tubulin in Xenopus embryos inj ected (side to right ) with GFP ( control ) , phosphomutant 6S-A Ngn3 and wild type Ngn3. Error bars show the error of the mean of 3 biological
replicates , each with 5 embryos per condition .
Figure 4 shows the results of ChIP from transfected PI 9 cells showing the occupancy of mNgn3 WT and 6S-A on promoters . Error bars show the standard deviation of three qPCR technical replicates . Figure 5 shows qPCR measurement of insulin expression in Xenopus embryos inj ected with mRNA to wild-type or 6S-A (phosphomutant ) Ngn3.
Figure 6 shows qPCR measurement of Ngn3 and NeuroD expres sion heterozygous mouse ES cells that express either WT or 6S-A
(phosphomutant) Ngn3 and are differentiated into definitive
endoderm.
Figure 7 shows qPCR measurement of Insulin expres sion in
heterozygous wt or 6S-A (phosphomutant ) Ngn3 knock-in mouse ES cells (where WT or phosphomutant Ngn3 are expressed from the endogenous promoter) subj ected to a 3 stage endo I , II I II protocol of
differentiation into definitive endoderm .
Figure 8 shows confocal microscopy images of homozygous wt or 6S-A (phosphomutant) Ngn3 knock-in mouse ES cells subj ected to a 3 stage endo I , I I III protocol of differentiation into definitive endoderm, following 32 days in endo III .
Figure 9 shows the phosphorylation of Ascll by recombinant Cdks . WT and 6S-A (phosphomutant) Ascll 35-S labelled IVT proteins were incubated with the indicated cyclin/ cdks in kinase buffer
supplemented with 87.5 μΜ ATP for 1 hour prior to separation on SDS- PAGE.
Figure 10 shows quantitative measurements of morphological
maturation of iN cells generated from human fibroblasts using BAM factors , Brn2 , Ascll , MyTl, with or without NeuroD. 60 randomly selected lOx visual fields of fluorescent microscopy were counted to determine the percentage of Hoescht positive cells that are also Tuj 1 positive . ≥49 neurons were measured to determine quantitative measurements of morphological maturation . Data are presented as mean +/- s.e.m. for a three independent experiment ; * p≤0.05; *** , p<0.005.
Figure 11 shows quantitative measurements of morphological
maturation (neurite length and branchpoints ) of iN cells generated from human fibroblasts (using BAM factors , Brn2 , Ascll , MyTl , with or without NeuroD) and small molecule Smad pathway and GSk3beta inhibitors . 60 randomly selected 1 Ox visual fields of fluorescent microscopy were counted to determine the percentage of Hoescht positive cells that are also Tuj 1 positive (conversion efficiency) . ≥49 neurons were measured to determine .
Figure 12 shows a comparison of noraderinergic and seratonergic markers conducted by qPCR at tailed stage following inj ection of Xenopus embryos with mRNA for WT, or phosphomutant forms of Ascll with mutation of predicted cdk sites (Ascll Cdk) , GSk3beta sites (Ascll Gsk3B) or cdk and Gsk3B sites simultaneously (Ascll Cdk Gsk3B) .
Detailed Description of Invention
The methods described herein relate to the maturation of
phenotypically immature mammalian cells by reducing the
phosphorylation of a bHLH proneural protein in the cells . This may be useful in the production of mature mammalian cells .
The amount of phosphorylation of the bHLH protein in the immature mammalian cells may be reduced relative to the amount of
phosphorylation of endogenous bHLH protein in untreated control cells and/or the amount of phosphorylation of the endogenous bHLH protein in the immature mammalian cells before treatment .
The ability of the bHLH protein to drive both differentiation and maturation of mammalian cells is reduced by phosphorylation at multiple phosphorylation sites , such as Ser-Pro and Thr-Pro (SP/TP) sites , within the bHLH protein . Reducing the phosphorylation of these sites in the immature mammalian cells increases the amount of bHLH protein in the immature mammalian cells which lacks
phosphorylated phosphorylation sites or which has fewer
phosphorylated phosphorylation sites than the bHLH protein in untreated control cells . This un (der) phosphorylated bHLH protein has increased activity and promotes maturation of the immature mammalian cells . The increase in activity upon reduced phosphorylation may be semiquantitative and maximal maturation may be achieved when the bHLH protein is devoid of phosphorylated phosphorylation sites, such as Ser-Pro and Thr-Pro (SP/TP) sites .
As described in more detail below, phosphorylation of the bHLH protein in the immature mammalian cells may be reduced by expression in the cells of a modified bHLH protein which lacks some or all of the phosphorylation sites present in the wild type bHLH protein, for example Ser-Pro and Thr-Pro (SP/TP) sites , or by exposure of the cells to a kinase inhibitor which inhibits phosphorylation of the phosphorylation sites present in the endogenous bHLH protein, for example Ser-Pro and Thr-Pro (SP/TP) sites . In some preferred embodiments , phosphorylation of the bHLH protein may be reduced by reducing the number of Ser-Pro (SP) or Thr-Pro (TP) phosphorylation sites in the bHLH protein that are
phosphorylated. Phosphorylation sites in a bHLH protein are sites that are subj ect to phosphorylation by Cdk, GSK3 β and/or other cellular kinases , preferably sites that are phosphorylated by Cdk, GSK3 β or Cdk and GSK3 . Phosphorylation sites within bHLH proteins are well-known in the art and may include Ser-Pro and Thr-Pro (SP/TP) sites . Other phosphorylation sites may be identified by routine sequence
analysis, for example using tools such as Kinase Phos 2.0 and
NetPhos 2.0.
For example , phosphorylation sites in human Ascll ( SEQ ID NO: 1 ) may include the SP sites Ser93 , Serl 90 , Serl94, Ser207, and Ser223 and the GSK3 sites Ser203, Ser210 and Ser214.
Phosphorylation sites in human Ngn3 (SEQ ID NO: 3 ) may include the SP sites Ser38 , Serl 61 , Serl 65 , Serl74 , Serl83, and Ser204, and the non-ST/TP 6ΞΚ3β sites Ser69 and Serl77. The mammalian cells may be maintained in culture following the reduction in bHLH protein phosphorylation until they develop the mature cell phenotype . For example , human cells may be maintained in culture for 21 days or more, or 28 days or more , following the reduction in bHLH protein phosphorylation .
Suitable culture conditions for maintenance of di fferent mammalian cell types , such as islet beta cells and neurons , are well known in the art.
The efficiency of maturation may depend on the nature of the cell population . For example , after reducing the amount of bHLH protein phosphorylation, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more , 40% or more , 50% or more, 60% or more, 70% or more, 80% or more , 85% or more , 90% or more, 95% or more or 98% or more of the immature mammalian cells in the population may adopt a mature phenotype .
The methods described herein may be in vitro, ex vivo or in vivo, preferably in vitro or ex vivo.
The immature mammalian cells may be from any mammalian species , preferably mouse or human cells.
Immature mammalian cells display a stable immature phenotype which differs from the fully functional phenotype of mature cells . For example , the immature cells may lack one or more functions or features of the mature cell or may display one or more functions or features which the mature cell lacks . For example , in some
embodiments , immature mammalian cells may display the ability to proliferate, whilst mature mammalian cells do not retain display the ability to proliferate . The immature cells acquire the mature phenotype through maturation as described herein .
Immature mammalian cells produced as described herein may be characterised by the formation of teratomas following 1 S 1 1 1 l lltO ITOCLsrit ΤΊO ΓΊ— h1J171clΠ .1
The immature mammalian cells may be of any cell type, including neurons , such as dopaminergic, cortical , spinal , and peripheral neurons , and endocrine cells , such as intestinal , pancreatic, lung and prostate endocrine cells .
In some preferred embodiments , the immature mammalian cells are pancreatic endocrine cells or precursors thereto, most preferably pancreatic islet beta cells or precursors thereto . Islet beta cells secrete insulin in response to glucose .
For example , immature islet beta cells may display a phenotype which includes one or more of : the absence of insulin or urocortin-3 expression; the absence of glucose responsive insulin secretion; low threshold glucose responsive insulin secretion; absence of beta islet morphology; and inability to engraft in host models . In addition, an immature islet beta cell phenotype may also include one or more of; expression of inactive phosphorylated Ngn3 ; the absence of expression of direct or indirect downstream Ngn3 targets , such as neuroD, MyTl and insulin; and the absence of other
phenotypic manifestations of maturation .
In other preferred embodiments , the immature mammalian cells are neurons .
For example , immature neurons cells may display a phenotype which includes one or more of : short neurites ; unbranched or substantially unbranched neurites ; ability to proliferate ; NCAM expression; SOX2 expression; absence of TuJl and MAP2 expression; and reduction, dysregulation or absence of electrical excitability . The methods described herein may be useful in the production of mature mammalian cells . Mature mammalian cells display a mature phenotype which differs from the phenotype of the immature cells and which includes phenotypic characteristics of maturation or maturity, such as insulin- expression in beta islet cells and extended neurite outgrowth in neurons . Mature mammalian cells may be identified by determining the presence of these phenotypic characteristics of maturity .
Mature mammalian cells produce as described herein may be
characterised by the absence o
into irscipisnt irodont GIT OthΘIT m m Inmm m cim __m t1 moc!^^1 s «
Mature islet beta cells may display a phenotype which includes one or more of : neuroD, MyTl , insulin and/ or urocortin 3 expression; glucose-responsive insulin secretion; physiological thresholds of glucose responsiveness ; beta islet morphology; and ability to engraft in host models .
Immature islet beta cells may acquire 1, 2, 3, more than 3 , or all of the above phenotypic characteristics of maturity as they progress through maturation as described herein .
In some embodiments , the pancreatic endocrine subtype that is developed by immature pancreatic endocrine cells during maturation may be determined by the phosphorylation sites that are
unphosphorylated in the bHLH protein . Reducing phosphorylation at one or both of Cdk and GSK3 β phosphorylation sites may be useful in producing specific pancreatic endocrinal sub-types , such as islet alpha, islet beta, islet delta or islet PP cells, or increasing the proportion of specific pancreatic endocrinal sub-types in a mature population of pancreatic endocrine cells .
Mature neurons may display a phenotype which includes one or more of : absence of SOX2 expression; TuJl expression; MAP2 expression; extended and branched neurites ; neurites able to connect to adj acent neurites and form neural networks ; electrical excitability; and improved engraftment in animal model s . For example, immature neurons may acquire 1, 2, 3 or more or all of the above phenotypic characteristics of maturity, as they progress through maturation as described herein.
Neurons may also acquire one or more markers associated with subtype specificity, such as Thl , Lmxla, Foxa2 , Nurrl, Pitx3 and Enl, as they progress through maturation as described herein .
In some embodiments , one or more factors that specify neuronal subtype , may be expressed or introduced into the cells when the phosphorylation of the bHLH protein is reduced as described herein .
The neuronal subtype that is developed by the immature neurons during maturation may be determined by the phosphorylation sites that are unphosphorylated in the bHLH protein . Reducing
phosphorylation at one or both of Cdk and GSK3 β phosphorylation sites may be useful in producing specific neuronal sub-types , such as noradrenergic, serotonergic , glutaminergic , GABAergic, TH+ve dopaminergic, and cholinergic neurons , or increasing the proportion of specific neuronal sub-types in a mature population of neurons . For example , reduction or absence of phosphorylation at both Cdk and GSK3 phosphorylation sites in a bHLH protein, such as Ascll , within a population of immature mammalian cells may increase the amount of noradrenergic neurons produced relative to serotonergic neurons , compared to reduction or absence of phosphorylation at either Cdk or GSK3 phosphorylation sites alone . Consensus phosphorylation sites for Cdk and GSK3 phosphorylation are well known in the art and suitable sites may include Ser-Pro and Thr-Pro (SP/TP) sites .
In some embodiments , bHLH protein phosphorylation may be reduced as described herein in the immature mammalian cells after
differentiation is completed . In other words , the immature mammalian cells may be fully differentiated i.e. they are committed to a single cellular fate and have no further differentiation potential . Immature mammalian cells which are differentiated may express markers characteristic of a fully differentiated cell and may lack expression of markers characteristic of pluripotent or
undifferentiated or partly differentiated cells.
In other embodiments, bHLH protein phosphorylation may be reduced as described herein in the immature mammalian cells before
differentiation is completed . In other words , the immature
mammalian cells may be undifferentiated or partially differentiated (i.e. not fully differentiated) when bHLH protein phosphorylation is reduced, or may be committed to a cellular fate but have yet to adopt the mature characteristics of that fate . For example, the cells may be pluripotent cells or partly differentiated progenitor or precursor cells which are not yet committed to a single cellular fate and possess further differentiation potential . Reduction in bHLH protein phosphorylation may drive the immature cells through both differentiation and maturation . Suitable immature mammalian cells for the maturation of islet beta cells may include definitive endoderm, primitive gut tube , posterior foregut , and pancreatic endoderm and endocrine precursor cells . Suitable immature mammalian cells for the maturation of neurons may include neuroectoderm cells, neural stem cells , induced neural stem cells and neural progenitor cells .
Immature mammalian cells for maturation as described herein may be isolated cells . Suitable isolated immature mammalian cells may be maintained in vitro using standard cell culture techniques .
Immature mammalian cells for maturation as described herein may be produced in vitro from antecedent cells .
Various approaches for the production of differentiated mammalian cells from antecedent cells into are available in the art , including directed differentiation, forward programming and trans- differentiation . Suitable techniques are well-known in the art .
In some embodiments , antecedent cell may be a pluripotent cell . A pluripotent cell exhibits an undifferentiated phenotype and is capable of giving rise to any cell type in an individual.
Pluripotent cells include embryonic and non-embryonic (e.g. foetal or adult) stem cells and induced pluripotent stem ( iPS ) cells .
An embryonic stem cell (ESC) is a pluripotent cell derived from an early stage embryo . An ESC may be a cell obtained from an embryo or may be a descendent of such a cell . Suitable hESCs cells may be obtained from a cultured hESC cell line, such as H9 or hSF-6.
Further examples of suitable human embryonic stem cells are
described in (Thomson JA et al Science 282 : 1145-1147 (1998) ;
Reubinoff et al . Nat Biotechnol 18 : 399-404 (2000); Cowan, C . A. et al. N. Engl . J. Med. 350, 1353-1356 (2004) , Gage, F.H. , et al . Ann. Rev. Neurosci . 18 159-192 (1995) ; Gotlieb (2002) Annu . Rev. Neurosci 25 381-407) ; and Carpenter et al . (2003) Stem Cells . 5(1) : 79-88 see also : the NIH stem cell registry) . iPS cells are pluripotent cells which are derived from non- pluripotent , fully differentiated ancestor cells . Suitable cells include adult fibroblasts and peripheral blood cells . Ancestor cells are typically reprogrammed by the introduction of pluripotency genes or proteins , such as Oct4 , Sox2 and Soxl into the cell . The genes or proteins may be introduced into the differentiated cells by any suitable technique , including viral or plasmid transfection or direct protein delivery . Other genes , for example Kif genes , such as Kif-1, -2 , -4 and -5; Myc genes such as C-myc , L-myc and N-myc ;
nanog; and Lin28 may also be introduced into the cell to increase induction efficiency . Techniques for the production of iPS cells are well known in the art . (Yamanaka et al Nature 2007; 448:313-7; Yamanaka 6 2007 Jun 7; 1 (1) : 39-49. Kim et al Nature. 2008 Jul 31;
454 (7204) : 646-50; Takahashi Cell . 2007 Nov 30; 131 (5) : 861-72. Park et al Nature. 2008 Jan 10; 451 (7175) : 141-6; Kimet al Cell Stem Cell . 2009 Jun 5; 4 (6) : 472-6. ) In some embodiments , the antecedent cell may be an iPS cell obtained from an individual , for example an individual with a di sease condition, such as a neural or diabetic condition . A mammalian pluripotent cell, for example, a human or mouse pluripotent cell, may express one or more of the following
pluripotency associated markers : 0ct4, Sox2 , Alkaline Phosphatase, SSEA-3 , Nanog, SSEA-4 and Tra-1-60. A human pluripotent cell may lack markers associated with specific differentiative fates , such as Bra, Soxl7, FoxA2 , FP, Soxl, NCAM, GATA6, GATA4 , Handl and CDX2.
Suitable methods for the directed differentiation or forward programming of pluripotent cells into various differentiated cell types , including pancreatic beta cells , are well-known in the art
(Furth, M . E. et al J Cell Biochem 106, 507-511 (2009); Pearl, E. J. et al Clin Genet 74, 316-324 (2008); Daneman, D. Lancet 367, 847- 858, (2006); D 'Amour, K. A. et al . Nat Biotechnol 24, 1392-1401, (2006); Kroon, E. et al . Nat Biotechnol 26, 443-452, (2008); Treff, N. R. et al . Stem Cells 24, 2529-2537, (2006); Serafimidis, I., et al . Stem Cells 26 , 3-16 (2008) ; ; Kim et al Nature 418, 50-56; Kim et al PNAS 108, 14169-14174; Kim et al Cell stem cell 9, 413-419) .
Typically, antecedent pluripotent cells are cultured with one or more growth factors and/or growth factor inhibitors to direct differentiation into the appropriate cell type .
For example , pluripotent antecedent cells , such as mouse or human iPS or ES cells may be used to produce populations of immature endocrine pancreas cells , islet beta cells, neural cells and neurons for maturation as described above . Suitable methods are well-known in the art (see for example Zhang et al (2009) Cell Res 19(4) 429- 438 ; Hosoya et al Int . J. Dev . Biol . 56 : 313-323 (2012) ; Guo et al Endocrine Reviews May 1 , 2009 vol . 30 no . 3 214-227; Tateshi et al JBC 283 46 31601-31607; Furth, M. E. et al J Cell Biochem 106, 507- 511 (2009); Pearl, E. J. & et al Clin Genet 74, 316-324 (2008);
Daneman, D. Lancet 367, 847-858, (2006) ; D 'Amour, K. A. et al . Nat Biotechnol 24, 1392-1401, (2006); Kroon, E. et al . Nat Biotechnol 26, 443-452, (2008); Pang, N. et al . Cell Stem Cell 9, 517-525, (2011) ; Ladewig et al , 2012 Nat Methods .9(6) : 575-8) ; Zhang et al (2013) Neuron 78, 785-798; Meng-Lu et al Nat. Commun. 4:2183 doi : 10.1038/ncomms3183 (2013) . In other embodiments, the antecedent cell may be a differentiated somatic cell .
The antecedent cell may be a different cell type to the immature mammalian cell. Suitable antecedent cells include fibroblasts and other mammalian cell types . For example, an immature beta cell or neuron for maturation as described herein may be produced by trans- differentiation of fibroblasts , such as dermal fibroblasts . Suitable methods for the trans-differentiation of differentiated cells into various differentiated cell types is well-known in the art (Caiazzo et al . Nature 476, 224-227 and Pang et al , Nature 476, 220-223) . Antecedent cells may be reprogrammed into differentiated cells through the introduction of reprogramming factors into the cells . Reprogramming factors may be introduced in the form of nucleic acids (Warren L et al . Cell Stem Cell . 2010 Nov 5; 7(5) : 618-30) or proteins (Zhou H, et al Cell Stem Cell . 2009 May 8; 4 (5) :381-4) by any suitable technique , including plasmid or more preferably, viral transfection, direct protein delivery or direct delivery of nucleic acid, such as mRNA, for example by RNA mediated transfection .
Following introduction of the reprogramming nucleic acids or proteins , the population of antecedent cells may be cultured to allow reprogramming to occur .
Suitable techniques for reprogramming cells though expression of nucleic acid encoding one or more reprogramming factors are well- known in the art (Takahashi et al 2007; Takahashi et al 2007; Seki et al 2010; Loh et al 2010; Staerk et al 2010) .
The choice of reprogramming factors which a re employed depends on the type of reprogrammed cell which is desired For example, iPS cells or fibroblast antecedent cells may be used to produce
populations of immature neurons for maturation as described herein . Immature neurons may be produced from antecedent fibroblast cells by expression of Brn2, MyTl and optionally NeuroD, in addition to Ascll , Ngn2 , Ngn3 , Ptfla and/or atonal , depending on the type of neuron . Additional subtype specifiers , for instance Lmxla, Foxa2 , Nurrl , Pitx3 and/or Enl may also be expressed .
Antecedent cells for the production of immature mammalian cells for use as described herein may be obtained from an individual or may be derived from an individual . For example, differentiated somatic cells , such as fibroblasts , may be obtained from an individual or iPS cells may be derived from cells obtained from an individual .
The individual may be a mammal , preferably a human .
The individual may be healthy (i.e. without any disease condition) or may have a disease condition .
The antecedent cells may be healthy cells i.e. cells without a disease-associated genotype or may display a genotype associated with a disease condition .
Mature differentiated cells produced from antecedent cells obtained from an individual as described herein may useful in disease modelling or therapy . For example, mature differentiated cells produced from antecedent cells with a disease-associated genotype may be useful in modelling the disease which is associated with the genotype .
In some embodiments , differentiation and maturation as described herein may be carried out in a single step . bHLH protein
phosphorylation may be reduced in the immature mammalian cells before , during or after reprograming or differentiation . In other words, bHLH protein phosphorylation may be reduced in the antecedent cells .
Preferably, bHLH protein phosphorylation is reduced in the immature mammalian cells in vitro or ex vivo i.e. the immature mammalian cells are not be part of a human or animal body when bHLH protein phosphorylation is reduced.
Un (der) phosphorylated bHLH protein remains in the cells after reprograming or differentiation and drives maturation to produce cells with a mature differentiated cell phenotype .
Basic helix-loop-helix (bHLH) proneural proteins are a well- characterised family of proneural transcription factors which are characterised by the presence of two alpha helices linked by a loop . Basic helix-loop-helix (bHLH) proteins include neurogenin-2 , neurogenin-3 , Ascll , Ptfla and Atonal .
The sequences of bHLH proteins suitable for use in accordance with the methods described herein may include multiple phosphorylation sites located on either side of the bHLH domain . These
phosphorylation sites may be Serine-Proline (SP) sites , Threonine- Proline (TP) sites or other sites that are phosphorylated by Cdk, GSK3 or other cellular kinases . In some preferred embodiments , the phosphorylation sites are phosphorylated by Cdk and/or GSK3 β .
The sequence of a bHLH protein may include 1 , 2 , 3, 4 , 5, 6, 7 , 8 , 9 , 10 or more phosphorylation sites , for example SP and/or TP sites or non-SP/TP sites phosphorylated by GSK . For example , mouse Ngn2 contains 9 SP sites and 1 TP site; mouse Ngn3 contains 5 SP and 1 TP site; human Ngn3 contains 6 SP sites ; and human and mouse Ascll contain 5 SP sites . These SP and/or TP phosphorylation sites are located on either side of the bHLH domain and are easily recognised in the amino acid sequence of the bHLH protein .
The phosphorylation sites in the bHLH protein may be phosphorylated by a mammalian cell kinase , for example a cyclin dependent kinase, such as cdkl , cdk2 and cdk4 , Glycogen synthase kinase 3 beta (GSK3 ) , Mitogen Activation Protein Kinase (MAPK) , such as MAPK1 and MAPK3, or another proline-directed kinase . For example , the Serine or Threonine residues of SP/TP phosphorylation sites in the bHLH protein may be phosphorylated by the kinase . Phosphorylation of SP/TP phosphorylation sites and/or other sites phosphorylated by ΘΞΚβ, inhibits both the differentiation activity and the maturation activity of the bHLH protein . For example , endogenous bHLH protein in immature mammalian cells may be phosphorylated at multiple SP/TP phosphorylation sites and is inactive or has both reduced differentiation activity and reduced maturation activity . For example , 1 , 2 , 3, 4 , 5 , 6, 7, 8 , 9 , 10 or more or all SP/TP phosphorylation sites may be phosphorylated in the endogenous bHLH protein . The phosphorylation sites of a bHLH protein may be readily identified and phosphorylation sites for human Ascll and human Ngn3 are set out above .
Reducing phosphorylation of the SP/TP phosphorylation sites and/or other sites phosphorylated by GSK , results in a semi-quantitative enhancement of both the differentiation and maturation activities of bHLH proteins . For example, additive removal of phosphate groups , for example by progressively mutating the SP and TP sites and/or other sites phosphorylated by ΘΞΚβ , leads to incremental increases in transcription of targets that drive differentiation and
maturation and an increase in the expression of associated
phenotypic markers . Although the bHLH protein may still retain maturation activity if some phosphorylated SP and TP sites are present , maturation activity is maximal in the absence of
phosphorylated SP and TP sites .
In accordance with the methods described herein, the phosphorylation of SP and TP sites and optionally other ΘΞΚβ phosphorylation sites in the bHLH protein is reduced in the immature mammalian cells i.e. the number of phosphorylated SP and TP sites decreased or reduced compared to the number of phosphorylated SP and TP sites in the bHLH protein in the mammalian cells before being subj ected to the methods described herein or in control mammalian cells which are not subj ected to the methods described herein .
A bHLH protein with reduced phosphorylation may be entirely devoid of phosphorylated SP and TP phosphorylation sites or may have fewer phosphorylated SP and TP phosphorylation sites that the bHLH protein in untreated control cells. For example, the bHLH protein may have 1 , 2, 3, 4 , 5 , 6, 7 , 8 , 9 , 10 or more than 10 fewer phosphorylated SP and TP phosphorylation sites .
For example , phosphorylation of the bHLH protein at SP and TP phosphorylation sites and optionally other GSKfi phosphorylation sites , may be reduced by 5% or more, 10% or more, 20% or more, 30% or more , 40% or more , 50% or more , 60% or more , 70% or more, 80% or more, or 90% or more following treatment as described herein . In other words , phosphorylation of the bHLH protein may be greater in the immature mammalian cells by 5% or more, 10% or more , 20% or more, 30% or more , 40% or more , 50% or more , 60% or more, 70% or more, 80% or more , or 90% or more compared to the mature cell .
In some embodiments , the phosphorylation of a bHLH protein by one or more specific mammalian cell kinases may be reduced . For example , the phosphorylation of a bHLH protein by a cyclin dependent kinase (Cdk) , GSK3 β or both, may be reduced . This may also be useful in directing the cell into specific mature cell sub-types . Reducing phosphorylation at one or both of Cdk and GSK3 β phosphorylation sites may be useful in producing specific neuronal sub-types , such as noradrenergic, serotonergic , glutaminergic , GABAergic, TH+ve dopaminergic, and cholinergic neurons , or increasing the proportion of specific neuronal sub-types in a mature population of neurons . For example , reducing the phosphorylation of both Cdk2 and GSK3 β sites in Ascll may enhance neuronal differentiation and promote the maturation of noradrenergic neurons or other specific subtypes of neurons . Reducing the phosphorylation of either Cdk2 or GSK3 β sites in Ascll may promote the maturation of serotonergic neurons or other specific subtypes of neurons . Reducing phosphorylation at one or both of Cdk and GSK3 β phosphorylation sites in an immature
pancreatic endocrine cell may be useful in producing specific subtypes of mature pancreatic cells , such as islet alpha, islet beta, islet delta or islet PP cells . The identity of the bHLH protein which contains a reduced number of phosphorylated SP and TP sites as described herein depends on the type of differentiated cells which are to be matured. For example, Ascll may be used to mature cortical or peripheral neurons ; Ptfla may be used to mature cortical neurons or pancreatic endocrine precursors ; Atonal may be used to mature intestinal endocrine cells, lung cells or cerebellum cells ; Ngn2 may be used to mature cortical or spinal neurons ; and Ngn3 may be used to mature pancreatic endocrine cells , intestinal endocrine cells , or hypothalamic neurons . In preferred embodiments , Ngn3 with a reduced number of phosphorylated SP and TP sites may be used to mature pancreatic endocrine cells , such as islet beta cells .
The amino acid sequences of mammalian bHLH proteins are readily available on public databases .
For example , Ascl 1 (achaete-scute homolog 1 ; also known as ASH1 ; HASHI; MASHI; bHLHa46 ; Gene ID 429) may be human Ascl 1 having the sequence of NCBI database entry NP___004307.2 GI : 55743094 or a naturally occurring variant or allele thereof .
Ptfla (pancreas specific transcription factor, la; also known as bHLHa29 ; PTFl-p48 ; Gene ID 256297) may be human Ptfla having the sequence of database entry NP__835455.1 GI: 30039710 or a naturally occurring variant or allele thereof .
Atohl (atonal homolog 1; also known as ATHI; HATHI ; MATH- 1 ; bHLHal 4 ; Gene ID 474 ) may be human Aohl having the sequence of database entry NP___005163.1 GI : 4885075 or a naturally occurring variant or allele thereof
Ngn2 (neurogenin-2 ; also known as Atoh4 , Math4A, neurog2 , bHLHa8 , ngn-2 ; Gene ID 63973) may be human NGN2 having the sequence of database entry NP_076924.1 GI : 31077092 or a naturally occurring variant or allele thereof Ngn3 (neurogenin-3 ; also known as Atoh5 , ath4B, NGN-3 , bHLHa7 , neurog3 ; Gene ID 63973 ) may be human NGN3 having the sequence of database entry NP___066279.2 GI : 68989258 or a naturally occurring variant or allele thereof .
A naturally occurring variant or allele of a reference bHLH sequence listed above is easily identified by a skilled person and may have at least 80%, at least 85% , at least 90%, at least 95% , or at least 98% sequence identify to the reference bHLH sequence listed above .
Amino acid similarity and identity a re generally defined with reference to the algorithm GAP (GCG Wisconsin Package1", Accelrys , San Diego CA) . GAP uses the Needleman & Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps . Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al .
(1990) J. Mol. Biol. 215 : 405-410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85 : 2444-2448) , or the Smith- Waterman algorithm ( Smith and Waterman (1981) J . Mol Biol . 147 : 195- 197), generally employing default parameters .
Particular naturally occurring amino acid sequence alleles or variants may differ from a reference bHLH sequence by insertion, addition, substitution or deletion of 1 , 2 , 3 , 4 , 5 , 6, 7 , 8 , 9 , or 10 or more amino acids .
In some embodiments , the phosphorylation of a single bHLH protein, such as neurogenin-3 , may be reduced in the immature mammalian cells as described herein . In other embodiments , the phosphorylation of more than one bHLH protein, for example two , three, four or more different bHLH proteins , may be reduced. For instance, some neuronal cell types may co-express or sequentially express the bHLH proteins atonal , Ascll and/or Ngn2. Manipulation of the
phosphorylation of all of these bHLH proteins may potentiate the transition of neurons along both the differentiation and maturation pathways and/or modulate the neurona1 sub-type so generated.
In preferred embodiments, the phosphorylation of the bHLH protein Ngn3 and optionally Ptfla, may be reduced in order to mature differentiated pancreatic endocrine cells, preferably pancreatic islet beta cells .
A method of producing mature pancreatic endocrine cells, such as mature islet beta cells, may comprise;
providing a population of immature pancreatic endocrine cells; and
reducing the phosphorylation of Ngn3 in the population of cells .
For example , the number of phosphorylated SP/TP sites in Ngn3 may be reduced in the population of cells .
Reducing Ngn3 phosphorylation by reducing the number of
phosphorylated SP/TP sites promotes the maturation of the immature pancreatic endocrine cells .
The subtype of pancreatic endocrine cell that is developed by the immature pancreatic endocrine cells during maturation may be determined by the phosphorylation sites that are unphosphorylated in the Ngn3 protein . Reducing phosphorylation at one or both of Cdk and GSK3 phosphorylation sites may be useful in producing specific endocrinal sub-types , such as islet alpha, islet beta, islet delta and and islet PP cells, or increasing the proportion of specific endocrine pancreatic sub-types in a mature population of pancreatic endocrine cells . Phosphorylation sites for Cdk and GSK3 β
phosphorylation in the Ngn3 are well known in the art and described elsewhere herein .
Preferably, the pancreatic endocrine cells are islet beta cells . Suitable immature pancreatic endocrine cells may include immature islet beta cells and pancreatic endocrine precursor cells . In some embodiments, reducing the phosphorylation of Ngn3 SP/TP sites in the immature islet beta cells may induce or promote the onset of a mature islet beta cell phenotype. A mature islet beta cell phenotype may be characterised by
expression of urocortin-3 and insulin, glucose responsiveness and the absence of teratomas wh transplanted back into recipient rodent models. In some embodiments, mature islet beta cells may display a high and physiological threshold level of glucose
(Blum, B. et al . Nat Biotechnol 30, 261-264, (2012) ) . Phosphorylation of phosphorylation sites, preferably SP/TP
phosphorylation sites , in the bHLH protein may be reduced in the immature mammalian cells as described herein by any suitable technique . In some embodiments , phosphorylation may be reduced by expressing a modified bHLH protein in the immature mammalian cells .
The modified bHLH protein may retain the activity of the wild-type bHLH protein but may be devoid of phosphorylated sites in the immature mammalian cells or may contain fewer phosphorylated sites than wild-type bHLH protein (i.e. the modified bHLH protein may be phosphorylation-deficient) . For example, the modified bHLH protein may be devoid of phosphorylated SP/TP sites in the immature
mammalian cells or may contain fewer phosphorylated SP/TP sites than wild-type bHLH protein .
A suitable modified bHLH protein may include fewer SP/TP
phosphorylation sites than the wild-type (i.e. unmodified) bHLH protein . For example , the modified bHLH protein may have 1 , 2 , 3 , 4, 5 , 6, 7 , 8 , 9, 10 or more than 10 fewer SP/TP sites than the unmodified bHLH protein . In some preferred embodiments , modified bHLH protein may not contain any SP/TP phosphorylation sites at all . Since its sequence contains fewer SP/TP sites, fewer phosphorylated SP/TP sites are present in the modified bHLH protein relative to the wild-type bHLH protein following expression in the immature
mammalian cells . In some preferred embodiments , modified bHLH protein may not contain any phosphorylated SP/TP sites at all .
A modified bHLH protein may be producing by modifying the amino acid sequence to remove 1 , 2, 3, 4 , 5 , 6, 7 , 8, 9 , 10 or more than 10 SP/TP phosphorylation sites . An SP/TP phosphorylation site may be removed by inserting, substituting or deleting 1 , 2 or more amino acids in the amino acid sequence which remove or replace the Serine- Proline or Threonine-Proline motif of the phosphorylation site with a sequence which is not phosphorylated by mammalian cell kinases . In some embodiments , the Serine or Threonine residue of an SP/TP phosphorylation site may be substituted for an Alanine or other amino residue . For example , the SP or TP motif in the wild-type bHLH protein may be altered to AP in the modified bHLH protein . AP is not a kinase substrate . Examples of suitable phosphorylation deficient human Ascll and Ngn3 sequences are shown in SEQ ID NOS : 2 and 4 and examples of suitable phosphorylation deficient mouse Ngn3 sequences are shown in SEQ ID NOS : 6 and 7. Other phosphorylation deficient bHLH proteins may be produced using standard techniques . In some preferred embodiments , the serine/threonine residue , proline residue or both of an SP/TP phosphorylation site may be substituted for a glutamic acid or aspartic acid residue . This may be useful in mimicking fully phosphorylated bHLH protein and potentiating progenitor cell maintenance .
A modified bHLH protein may be encoded by an exogenous nucleic acid.
Nucleic acid which encodes a modified bHLH protein may be produced and manipulated using standard techniques . Suitable techniques and protocols for mutagenesis, manipulation, cloning and expression of recombinant nucleic acids are well known in the art ( see for example Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (Mar 1997) Humana Press Inc , Molecular Cloning: a Laboratory- Manual : 3rd edition, Russell et al . , 2001, Cold Spring Harbor
Laboratory Press) .
A nucleic acid sequence encoding a modified bHLH protein may be comprised within an expression vector . Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences , including promoter sequences , terminator fragments , polyadenylation sequences , enhancer sequences , marker genes and other sequences as appropriate . Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in the mammalian cells cell . Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in
expression systems are well-known in the art and include
constitutive promoters , for example viral promoters such as CMV or SV40 , and inducible promoters , such as Tet-on controlled promoters . Preferred vectors for use in the present methods include lentiviral and retroviral vectors .
Expression vectors containing the nucleic acid may be transfected into the immature mammalian cells . Any convenient technique for the transfection may be employed . Following transfection, the modified bHLH protein is expressed in the immature mammalian cells and causes them to mature . In other embodiments , transposon-mediated or other random integration transgenesis techniques may be employed .
The nucleic acid encoding the modified bHLH protein may then be expressed in the immature mammalian cells .
In some preferred embodiments , the immature mammalian cells may be matured as described herein with minimal or no genetic modification of the cells . Suitable techniques are known in the art and include the use of excisable lentiviral and transposon vectors ; repeated application of transient plasmid, episomal and adenovirus vectors or; the use of small molecules , synthetic mRNA and/or microRNAs (Sidhu KS. Expert Opin Biol Ther . (2011) 11(5) : 569-79; Woltjen K et al (2009) Nature 458 (7239) : 766-70; Chou BK et al . Cell Res. 2011 21 (3) :518-29) .
In some embodiments, one or more modified bHLH proteins or encoding nucleic acids , such as mR A, may be introduced directly into the immature mammalian cells .
Suitable techniques are known in the art for the introduction of nucleic acids (Warren L et al . Cell Stem Cell . 2010 Nov 5 ; 7(5) : 618- 30) or proteins (Zhou H, et al Cell Stem Cell . 2009 May 8; 4(5) : 381- 4 ) . For example , the immature mammalian cells may be cultured in the presence of the modified bHLH protein or nucleic acid under
conditions which allow for entry of the proteins or nucleic acid into the cells . In some embodiments , entry of modified bHLH proteins into the cells may be facilitated by a membrane penetrating peptide or poly-arginine tail , which may be linked or attached to the modified bHLH protein . Modified bHLH proteins or encoding nucleic acid may be introduced into immature mammalian cells by traditional methods such as lipofection, electroporation, calcium phosphate precipitation, particle bombardment and/or microinj ection, or may be delivered into cells by a protein delivery agent .
Nucleic acids for direct delivery into immature mammalian cells may be translatable by endogenous translation factors within the cell . A suitable synthetic mRNA may be modified. For example, 5- methylcytidine may be substituted for cytidine, and pseudouridine for uridine , followed by phosphatase treatment (Warren, et al 2010 ) .
Following the introduction of modified bHLH proteins or nucleic acids into the immature mammalian cells, the cells may be cultured for 5 or more days , 10 or more , 15 or more, 20 or more or 25 or more days .
Suitable culture conditions which are permissive for maturation will vary between cell types and may readily be determined by a skilled person . In some preferred embodiments, immature islet beta cells may be matured by introducing modified Ngn3 into the cells . As described above, a modified Ngn3 is phosphorylation deficient and may be devoid of ST/TP phosphorylation sites or may contain fewer ST/TP phosphorylation sites than the wild type . Suitable modified Ngn3 proteins include SEQ ID NOS : 4 , 6 and 7. For example , a method may comprise;
( i ) expressing nucleic acid encoding a phosphorylation deficient Ngn3 in the immature cells ; or,
ii ) introducing phosphorylation deficient Ngn3 protein or RNA encoding phosphorylation deficient Ngn3 protein directly into the immature cells .
In some preferred embodiments , RNA molecules encoding a
phosphorylation deficient Ngn3 protein may be introduced into the immature mammalian cells by RNA-mediated transfection in accordance with known techniques and expressed.
Expression or introduction of a phosphorylation-deficient bHLH protein, such as Ngn3 , in the immature mammalian cells increases the amount of bHLH protein with reduced phosphorylation in the cells and thereby promotes maturation of the cells .
In other embodiments , phosphorylation of ST/TP sites in the bHLH protein may be reduced in the immature mammalian cells by reducing the amount of phosphorylation of endogenously expressed bHLH protein in the cells .
The amount of phosphorylation may be reduced by inhibiting the activity of one or more protein kinases which target SP and TP phosphorylation sites in the immature mammalian cells . For example , the activity of one or more of a cyclin dependent kinase (Cdk) , GSK3 and MAPK may be inhibited.
The activity of one or more protein kinases may be inhibited by treating or exposing the population of cells to a protein kinase inhibitor . For example , the cells may be cultured in a nutrient medium which comprises a protein kinase inhibitor . Many suitable kinase inhibitors are known in the art, including GSK3beta, MAP kinase and eye1in-dependent kinase (cdk) inhibitors (Cicenas J et la Cancer Res Clin Oncol . 2011 Oct; 137 (10) : 1409-18; Trujillo JI . Expert Opin Ther Pat. 2011 Jul; 21 (7) : 1045-69; and agman AS et al Curr Pharm Des . 2004; 10 (10) :1105-37) .
For example , Cdk inhibitors , such as roscovitine, N- [ 5- [ [ [ 5- ( 1 , 1- dimethylethyl ) -2-oxazolyl ] methyl ] thio] -2-thiazolyl ] -4 - piperidinecarboxamide (BMS 387032; SNS-032 ) , flavopiridol ,
olomucine , dinaciclib, N- ( 4-piperidinyl ) -4- (2, 6- dichlorobenzoylamino ) -lH-pyrazole-3-carboxamide (AT7519) , and palbociclib, may be employed, most preferably olomucine , roscovitine or palbociclib . A suitable concentration of kinase inhibitor, such as a Cdk
inhibitor, to reduce the phosphorylation of bHLH proteins and increase maturation activity may be less than the concentration required to bring about cell cycle arrest or other toxic effects . Optimal concentrations of kinase inhibitor for maturation of any particular cell type may readily be determined empirically by a skilled person . In some embodiments , an initial concentration of about 40 μΜ may be employed. The kinase inhibitor may be added to culture medium continually or withdrawn, once maturation has commenced . Culture medium conditions will vary depending on the cell type which is to undergo maturation .
After phosphorylation of the bHLH protein has been reduced, for example by introducing a phosphorylation-deficient bHLH protein into the immature mammalian cells or by treating the immature mammalian cells with a kinase inhibitor, the phenotype of the cells may be monitored to determine the onset of a mature phenotype .
Phenotype may be monitored by determining the expression of one or more of mature cell markers and/or immature cell markers by the population of cells This allows the extent of maturation of the population to be determined. Cells which express one or more mature cell markers and/or do not express one or more immature cell markers may be identified as mature cells.
Suitable mature beta cell markers include insulin, MafA and Ucn3.
Suitable mature neuronal markers may include TuJl, MAP2 and Thl . Cell markers expressed by a cell may be identified using standard techniques , such as PGR, western blotting, immunocytochemistry and in situ hybridisation .
Phenotype may be monitored by determining the presence of one or more morphological features of mature cells .
Morphological features of mature neurons include extended and/or branched neurites , such as axons and dendrites . The length and branch structure of neurites in a population of neurons may be determined .
Phenotype may be monitored by determining the presence of electrical properties of the mature cells . For example , the response of a population of neurons to an electrical stimulus and/or the ability of a population of neurons to produce a spontaneous action potential may be determined .
Phenotype may be monitored by determining the production of one or more other features of a mature phenotype . For example, for
maturation of islet beta cells , one or more of the following may be determined; glucose threshold for insulin secretion; ability to survive and function in mouse kidney capsule transplantation models; and ability to rescue rodent models of diabetes , where beta cell mass has been reduced and ability to reverse or partially ameliorate symptoms of type II diabetes in rodent models . For maturation of neurons , ability of neurons to form neural networks and/or innervate the striatum in rodent models may be determined .
A population of mature differentiated mammalian cells produced by the present methods may be heterogeneous and may contain immature cells or cells or other cell types. In some embodiments, mature cells , such as mature neurons , may be easily distinguished from immature cells by morphological or other criteria and may therefore be useful for applications such as in vitro disease modelling .
Alternatively, mature cells may be separated from other cells in the population, as described below.
Alternatively, the population may be substantially free from other cell types . For example , the population of cells may contain 80% or more, 85% or more , 90% or more , or 95% or more mature differentiated mammalian cells of the relevant cell type , following reduction in bHLH phosphorylation . The population of mature differentiated mammalian cells may be sufficiently free of other cell types that no purification is required . For example, in some embodiments , a population of mature beta cells may comprise 50% or more, 60% or more, 70% or more , 80% or more , 85% or more , 90% or more, 95% or more, or 99% or more insulin expressing cells which display a physiological glucose response .
In other embodiments , a method may further comprise isolating and/or purifying the mature mammalian cells . Cells may be separated from other cell types in the population using any technique known to those skilled in the art , including those based on the recognition of extracellular epitopes by antibodies and/or magnetic beads or fluorescence activated cell sorting (FACS ) , including the use of antibodies against extracellular regions of characteristic markers .
Following maturation as described above, the mature mammalian cells may be maintained in culture , stored, for example frozen using conventional techniques , or used in therapeutic or other
applications as described below .
Mature mammalian cells may be cultured to maintain a homogenous or substantially homogenous population . For example, the population of mature cells may maintained in culture for at least 40 , at least 60 , at least 80 , at least 100, or more than 100 days . The culture of immature and mature mammalian cells is well-known in the art ( see, for example, Basic Cell Culture Protocols , C.
Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc. , U.S. (9 Dec 2004) ISBN: 1588292223; Culture of
Animal Cells : A Manual of Basic Technique , R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol
Methods . (2006) 310 : 40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) . Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma,
Europabioproducts , R&D Systems ) . For example, standard mammalian cell culture conditions may be employed, for example 37°C, 21% Oxygen, 5% Carbon Dioxide . Media is preferably changed every two days and cells allowed to settle by gravity .
Preferably, mature differentiated cells produced as described herein for therapeutic applications are clinical grade .
Another aspect of the invention provides a population of isolated mature mammalian cells which have been produced by a method as described above .
Mature beta cells produced as described above may be capable of functionality in in vivo models without teratoma formation (Kroon et al (2008) Nature Biotechnology 26, 443-452) .
Mature neurons matured produced as described above may be capable of faithfully reproducing the functionality of mature neurons in vivo compared to neurons produced by previous in vitro methods .
Mammalian cells matured as described herein may be stable in vivo . For example , the cells may retain a functional mature phenotype indefinitely in vivo following implantation . In some embodiments , the rate of teratoma formation following implantation of mammalian cells matured as described herein may be reduced relative to the immature mammalian cells not subj ected to maturation . Preferably, the rate of teratoma formation following implantation of the mature mammalian cells is zero or substantially zero .
In preferred embodiments, the population of mature cells are pancreatic endocrine cells.
Mature pancreatic endocrine cells may have a mature pancreatic endocrine cell phenotype . For example, the mature cells may express a pancreatic hormone selected from insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin and preferably insulin .
Preferably, the mature cells do not express more than one pancreatic hormone and do not, for example, express both glucagon and insulin or both insulin and somatostatin . Mature pancreatic endocrine cells may be capable of forming mature secretory granules in vivo
following engraftment or implantation .
In especially preferred embodiments , the population of mature cells are mature islet beta cells .
Mature beta cells may express insulin and C-peptide . Mature beta cells may also express PCSK1, PCSK2, NKX6-1, MAFA, PDX1 , amylin (islet amyloid polypeptide ) and Unc3.
Mature beta cells may be glucose-responsive . In some embodiments , the mature beta cells may exhibit physiological thresholds for the expression and secretion of insulin in response to glucose .
In other embodiments , the population of mature cells may be mature neurons . The mature neurons may have a mature neuronal phenotype , for example a mature cortical , peripheral , cerebellar, hypothalamic or spinal neuronal phenotype . Mature neurons may have extended and branched axons and dendrites , display electrical excitability, be capable of forming neuronal network and may engraft and restore functionality in engraftment models of neurological diseases , such as Parkinson' s di sease . A population of mature cells produced as described herein may useful in disease modelling.
For example, antecedent cells with a genotype associated with a disease condition may be used to produce mature mammalian cells with the disease associated genotype in vitro. These cells may be tested in vitro for physiological differences compared to matched healthy control cells . For example , response to stress, electrical activity, response to toxins , mitochondrial function and/or resistance to cell death may be tested.
In some embodiments , fibroblasts derived from a patient with a genetic predisposition to a neural condition, such as dementia, may be trans-differentiated to mature neurons in vitro and those neurons tested for physiological differences compared to matched healthy controls neurons .
A population of mature cells produced as described herein, or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein has been reduced as described herein and which are undergoing maturation, may be useful in therapy, for example cell-replacement therapy .
The population may form part of a therapeutic composition . For example , a population of mature cells produced as described above may be formulated with a pharmaceutically acceptable carrier and/or a tissue scaffold .
A therapeutic composition, medicament, or other composition may comprise a population of mature differentiated cells, along with a pharmaceutically acceptable excipient, carrier, buffer,
preservative, stabiliser, anti-oxidant or other material well known to those skilled in the art . The precise nature of the carrier or other material will depend on the route of administration . A therapeutic composition may be produced by admixing a population of mature mammalian cells , for example mature beta cells , with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients .
The composition may be administered to a patient, e.g. for treatment (which may include preventative treatment ) of dysfunctional tissue, as described above . For example , a composition comprising mature beta cells may be administered to a patient for treatment (which may include preventative treatment ) of damaged or dysfunctional
pancreatic tissue and in particular for the treatment of a diabetic condition, such as type 1 or type 2 diabetes .
Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil . Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included.
The composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability . Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride , Ringer ' s Inj ection, or Lactated Ringer ' s Inj ection . A composition may be prepared using artificial cerebrospinal fluid.
Cells may be implanted into a patient by any technique known in the art (e.g. Lindvall , 0. (1998) Mov . Disord . 13, Suppl . 1:83-7; Freed, C.R., et al . , (1997) Cell Transplant , 6, 201-202; Kordower , et al . , (1995) New England Journal of Medicine , 332, 1118-1124; Freed, C.R., (1992) New England Journal of Medicine , 327 , 1549-1555, Le Blanc et al, Lancet 2004 May 1;363 (9419) : 1439-41) .
Administration of a composition in accordance with the present invention is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors .
A composition may be administered alone or in combination with other treatments , either simultaneously or sequentially dependent upon the condition to be treated.
Related aspects of the invention provide a method of treatment comprising admini stering a population of mature mammalian cells produced as described above to an individual in need thereof; the use of population of mature cells produced as described above in the manufacture of a medicament for use in a method of treatment; and a population of mature cells produced as described above for use in a method of treatment .
To avoid unwanted immune responses , the mature mammalian cells may be immunologically compatible with the individual . In some
embodiments , the mature mammalian cells may be produced from antecedent cells obtained or derived from the same individual or an immunologically compatible individual . For example, the mature mammalian cells may be produced from fibroblasts or ES cells obtained from the individual or iPS cells derived from fibroblasts or other cells obtained from the individual ; or the mature mammalian cells may be produced from stored or banked fibroblast, ES or iPS cells which are immunologically compatible with the individual .
The nature of the method treatment depends on the cell type .
Preferably, a method of treatment of a diabetic condition may comprise administering a population of mature pancreatic beta cells produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn3 , has been reduced as described herein and which are undergoing maturation into mature pancreatic beta cells, to an individual in need thereof; the use of population of mature pancreatic beta cells produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn3 , has been reduced as described herein and which are undergoing maturation into mature pancreatic beta cells in the manufacture of a medicament for use in a method of treatment of a diabetic condition; and a population of mature pancreatic beta cells produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn3 , has been reduced as described herein and which are undergoing maturation into mature pancreatic beta cells for use in a method of treatment of a diabetic condition .
Diabetic conditions include type 1 and type 2 diabetes .
Treatment may ameliorate or relieve diabetes-related symptoms in the individual or reduce or remove the need for insulin therapy .
The treatment of diabetic conditions by cell replacement therapy is known in the art ( see for example Pearl and Horb (2008) Clinical Genetics 74, 4, 316-324) .
In other embodiments , a method of treatment of a neural condition may comprise administering a population of mature neurons produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as gn2 or Ascll , has been reduced as described herein and which are undergoing maturation into neurons to an individual in need thereof; the use of population of mature neurons produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn2 or Ascll , has been reduced as described herein and which are undergoing maturation into neurons , in the manufacture of a medicament for use in a method of treatment of a neural condition; and a population of mature neurons produced as described above or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein, such as Ngn2 or Ascll , has been reduced as described herein and which are undergoing maturation into neurons , for use in a method of treatment of a neural condition.
Neural conditions may include neurodegenerative conditions , such as Parkinson' s Disease, and conditions associated with damaged or dysfunctional neural tissue, for example stroke or spinal cord or peripheral nerve inj ury .
For example , mature midbrain dopaminergic TH+ neurons produced as described above , e.g. using phosphorylation deficient Ascll , may be used to treat Parkinson' s disease .
Cell replacement therapy for the treatment of neural conditions is established in the art ( Dyson SC , Barker RA . Expert Rev Neurother . 2011 Jun; 11(6) : 831-44) .
A population of mature cells produced as described herein or a population of immature cells in which the phosphorylation of a basic helix loop helix (bHLH) protein has been reduced as described herein and which are undergoing maturation may also be useful in screening for compounds with modulatory effects .
Screening may include drug or small molecule screening . For example, the isolated mature differentiated cells may be contacted with a test compound and the effect of the test compound on the cells is determined. Screening may also include functional genomic
screening . For example , a gene may be suppressed, knocked out or otherwise inactivated in the isolated mature differentiated cells and the effect of the inactivation on the cells determined.
A method of screening a compound may comprise;
contacting isolated mature cells produced by a method described above with a test compound, and;
determining the effect of the test compound on said cells and/or the effect of said cells on the test compound. For example, the effect of a test compound o cell death/ survival growth, proliferation, condition, aggregatio function, hormone production, glucose responsiveness and/or ge expression may be determined.
A method of screening a compound may comprise;
contacting isolated mature neurons produced by a method according with a test compound, and;
determining the effect of the test compound on said neurons and/or the effect of said neurons on the test compound.
The effect of the test compound on morphological features , such as extended and/or branched neuritesm, or on the electrical properties of the neurons may be determined .
In some embodiments , mature differentiated cells may be produced from iPS cells as described herein . The iPS cells may be from a normal individual or an individual with a disease condition . The iPS cells may be differentiated into a cell type that is affected in the disease condition and subj ected to maturation as described herein . The mature differentiated cells may express a detectable reporter or display an observable cellular phenotype which differs between disease affected cells and normal cells . The mature differentiated cells may be exposed to test compounds and the effect of the test compound on the reporter expression or observable cellular phenotype determined. Compounds which cause the mature cells to revert from disease cell state to the normal state may be identified.
Alternatively, the one or more genes in the mature differentiated cells may be inactivated, for example by targeted mutation or RNAi suppression, and the effect of the inactivation on the reporter expression or observable mature cellular phenotype determined.
Genes whose inactivation causes the cells to revert from disease cell state to the normal state may be identified.
Screening may include toxicology screening . For example , the isolated mature cells may be contacted with a test compound at various concentrations that mimic abnormal/normal concentrations in vivo. The effect of the test compound on the cells may be determined and toxic effects identified. Toxicology screening is well known in the art (see for example Barbaric I et al . Biochem Soc Trans . 2010 Aug; 38 (4) : 1046-50) .
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments
described above with the term "comprising" replaced by the term "consisting essentially of" .
Modifications of the above embodiments , further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such these are within the scope the present invention .
All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes .
"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other . For example "A and/or B" is to be taken as specific
disclosure of each of (i ) A, ( ii ) B and ( iii ) A and B, just as if each is set out individually herein .
It is to be understood that the application discloses all
combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise .
Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects , unless the context demands otherwise .
Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and described above . Experiments
Methods
Xenopus laevis extracts and embryos
Acquisition of Xenopus laevis eggs and embryos, preparation and inj ection of synthetic mRNA and DNA morpholinos , staging of embryos , in situ hybridisation and egg extracts preparation have been described previously (Vosper et al . , 2007 Biochem . J. 407 , 277-284.; Vosper et al. , 2009 J. Biol. Chem. 284, 15458-15468) . Embryos inj ected with Ngn3-GR were treated with dexamethasone at stage 12.5 to bring about nuclear entry (Hindley, C. et al . Development 139 , 1718-1723, (2012) ; Ali, F. et al . Development 138, 4267-4277,
(2011) ) .
In vi tro translated ( IVT ) protein synthesis and assays
Phosphorylation assays in Xenopus extract were performed as for degradation assays (Vosper et al . , 2007; Vosper et al . , 2009) , except that extract was supplemented with 0.2 mM MG132 (Biomol ) and samples were incubated for 45 min . Where indicated, following the 45 min incubation in extract , 400 u of λ phosphatase (NEB) were incubated with the sample at 30 °C for 30 min (Ali et al , (2011) ) .
Cell culture, transfections , immunocytochemistry and western blotting
PI 9 cells were cultured in a-MEM (Gibco) with 7.5% newborn calf serum and 2.5% fetal bovine serum (HyClone) , 1% Glutamax (Gibco) , and 100 units/ml penicillin/ 100
Figure imgf000041_0001
streptomycin (Sigma) . Cells were transfected with Lipofeetamine 2000 ( Invitrogen) , and extracts western blotted with anti-HA-peroxidase (3F10 ) [Roche ] or anti-c-Myc ( 9E10 ) [Santa-Cruz] . Where indicated, cells were treated with 400 u of λ-phosphatase (NEB) (Ali et al, (2011) ) .
Quantitative real-time PGR (qPCR)
cDNA was generated from stage 15 or stage 26 Xenopus embryos , using 50 ng per qPCR reaction, in the Light-cycler 480 PGR system (Roche, Sussex, UK) with SYBR Green mix (Roche) , β-actin, GAPDH and EFl were used as housekeeping genes . Thermal cycling conditions : 95 ° C for 5 min, 45 cycles of 95 ° C for 10 sec, 60 ° C for 10 sec, and 72°C for 10 sec (All et al, (2011) ; Hindley et al (2012) ) .
Chromatin Immunoprecipitation assays (ChIP)
DNA/protein complexes from PI 9 cells transfected with either HA- wiId-type mNgn2 or 9S-A mNgn2 for 24hrs, and normalised for
mNgn2 / 9S-A mNgn2 expression were cross-linked with 1% formaldehyde . 5 μg of rat monoclonal anti-HA antibody (Roche) or anti- IgG (Abeam) (as a control ) were used per ChIP reaction and quantified using SYBR Green mix . ' Signal over background ' normalisation method was used to quantify immunoprecipitated DNA (Ali et al , (2011) ; Hindley et al (2012) ) .
Transdifferentiation of fibroblasts
Lenti-X 293T cells (Clontech) were grown to 90% confluency in Tet- free media and transfected using the Lenti-X Tet-On 3G Inducible Expression System (Clontech) . The media was replaced the following day . 48 hours after transfection, viruses were harvested from the culture supernatant using the Lenti-X Concentrator (Clontech) .
Lentiviral aliquots are snap frozen in dry ice and stored in -80 °C . Viral titrations were obtained using the Lenti-X qRT-PCR Titration Kit (Clontech) .
Target cells were plated into 24-well plates at a concentration of 20,000 cells/well and infected the following day with lentivurses in DMEM supplemented with 5]ig/ml polybrene . A multiplicity of
infection (MOI ) of 3 was used for all viruses , including the Tet3G activator . The media was replaced the following day with fresh Tet- free media, and target cells were induced using DMEM supplemented with 2 g/μΐ doxycycline on day 3 following infection . On day 5 , DMEM media was replaced by NDiff N2B27 Neuronal Differentiation Media (StemCells) supplemented with
Figure imgf000042_0001
doxycycline . Media was replaced every 2-3 days until day 12 for mouse fibroblasts and day 21 for human fibroblasts . At the indicated time after infection, cells were fixed and stained for neuronal differentiation using 1:500 anti-Tuj 1 (Covance ) and 1:200 anti-MAP2 (Sigma) . Xenopus Expression
Embryos were inj ected into 1 ventral vegetal cell at the 8 cell stage with mRNA encoding either WT Ngn3 (WT) or 6S-A Ngn3 (named SP here) , both fused to the glucocorticoid receptor (GR) . At stage 12.5 of development, dexamethasone was added to the media to bring about Ngn3 nuclear entry then embryos were collected at stage 26 and processed for qPCR to detect insulin expression .
Results
1. NGN3
Ngn3 has 6 SP sites . Using Xenopus frog egg extracts , we observed that Ngn3 is multiply phosphorylated in both interphase (I) and mitosis (M) ( Figure 1), and Ngn3 is also multiply phosphorylated in mouse PI 9 embryonal carcinoma cells (Figure 2 ) . When expressed in Xenopus ectoderm, Ngn3 (which promotes
hypothalamic neurogenesis, as well an endocrine pancreas and gut cell formation in the mouse embryo ) drives ectopic neurogenesis . We generated a phosphomutant mNgn3 ( 6S-A Ngn3 , serines of SP sites mutated to alanines ) . Xenopus embryos were inj ected with the phosphomutant 6S-A Ngn3 mRNA and wild type Ngn3 mRNA and subj ected to in situ hybridisation for the neuronal marker neural beta tubulin . 6S-A Ngn3 was observed to be significantly more active that the wild-type protein in driving neurogenesis in this in vivo setting, indicating similar regulation to Ngn2.
5pg of RNA encoding GFP, mNgn3 WT and mNgn3 6S-A was inj ected into Xenopus embryos at the 1-cell stage . RNA was extracted from stage 19 embryos and the expression of selected target genes quantified by qPCR, normalised to expression of the housekeeping gene EFla and expressed relative to the expression level in GFP-inj ected embryosm ( Figure 3 ) . Significant increases were observed in the expression of NeuroD (approx 5 fold) and Neural- β-tubulin (Npt ) (approx 2.5 fold) in mNgn3 6S-A-inj ected embryos compared to mNgn3 WT-inj ected embryos .
PI 9 cells were transfected 24 hours after plating with mNgn3 WT HA or mNgn3 6S-A HA and fixed and cross-linked after 24 hours . The resulting chromatin was normalised for mNgn3 protein levels and immunoprecipitated with either an anti-HA antibody or IgG . The promoter occupancy of mNgn3 was assessed by qPCR for the i ) NeuroDl and ii ) Dill promoters , expressed as the fold change of anti-HA immunoprecipitated samples against IgG (Figure 4 ) . mNgn3 WT HA was found to be enriched at both promoters , and in both cases the binding of mNgn3 6S-A HA was greater than that of the wild-type form of the protein . The phosphomutant gn3 was observed to bind NeuroD and Delta promoters approx . 2-fold more efficiently than wild-type Ngn3.
We generated a mutant mouse line where the endogenous Ngn3 coding sequence has either been replaced by 6S-ANgn3 (phospho-mutant mice ) or with wild-type Ngn3 as a control , both also expressing eYFP from the Ngn3 locus .
As well its role in specification of endocrine pancreatic cells during development, Ngn3 is required for generation of entero- endocrine cells of the adult gut , and we analysed these cells first . We found that the phosphomutant Ngn3 adult mice maintained half the number of Chromagranin A-expressing entero-endocrine cells compared to those of the wild-type control . These results are consistent with a deficiency of progenitor maintenance in phosphomutant Ngn3- expressing cells and a propensity to premature differentiation as might be expected on expression of a form of Ngn3 that promotes differentiation more effectively than the wild-type protein .
High levels of Ngn3 expression generally drive cell cycle exit in vivo and in vitro (Miyatsuka, T . , et al . Proc Natl Acad Sci U S A 108 , 185-190, (2011) ) but Ngn3 expression does not always result in cell cycle exit and differentiation per se; low levels of Ngn3 are found in first transition proliferating pancreatic progenitors and very low levels that persist in adult islets may contribute to proper expansion of these endocrine cells (Miyatsuka, T . , et al . (2011 ) ) . Under some circumstances and levels of expression, dephosphorylated Ngn3 may also enhance cell cycling prior to promoting differentiation.
Our experiments in the mouse gut indicate that phospho- status of endogenously expressed Ngn3 affected its ability to regulate entero- endocrine cell differentiation . Building on this observation and our in vivo and in vi tro overexpression studies described above , preventing Ngn3 phosphorylation may significantly enhance its ability to drive endocrine differentiation and maturation in the pancreas when Ngn3 is over-expressed . In particular, the phospho- status of Ngn3 may affect its ability to drive pancreatic beta cell differentiation and maturation in vitro from ES-derived cultures and may be manipulated to enhance beta cell generation for potential use in islet transplantation therapy .
Targetted overexpression of phosphomutant Ngn3 was observed to promote transdifferentiation of gut and liver endoderm into
endocrine pancreas in Xenopus embryos , compared to wild-type Ngn3 , and qPCR on Xenopus embryos transfected with mRNA encoding WT Ngn3 (WT) or phosphorylation deficient Ngn3 ( 6S-A Ngn3 : SP) showed that expression of insulin was enhanced up to five-fold by 6S-A Ngn3 compared to WT Ngn3 (Figure 5 ) .
Fertilised Xenopus eggs were left uninj ected or inj ected with mRNA for encoding wild-type or phosphomutant 6S-A Ngn3 into cells destined to form endoderm. Embryos were allowed to develop until swimming tadpole stages , when insulin would be expressed after ectopic expression of Ngn3. Insulin expression was then measured by qPCR. Phosphomutant Ngn3 resulted in enhanced transdifferentiation of embryonic endodermal tissue into ectopic insulin expressing pancreatic islet tissue compared to the wild-type Ngn3 (Figure 5 ) .
Moreover, mouse ES cells expressing phosphomutant Ngn3 in place of the wild-type Ngn2 heterozygously were observed to show enhanced NeuroD (an early marker of beta cell maturation) expression when induced to differentiate towards endocrine pancreas by culture media conditions .
Mouse ES cells were engineered to express one copy of either Wt Ngn3-eYFP or 6S-A Ngn3-eYFP from the endogenous locus , then
subj ected to different culture conditions to promote differentiation into definitive endoderm and pancreas differentiation . Expression of Ngn3 and NeuroD was measured by qPCR in heterozygous mouse ES cells expressing either WT or 6S-A (phosphomutant ) Ngn3 , taken down definitive endoderm differentiation pathway through endol , endol I and endoIII media (Figure 6) . Expression of one allele of
phosphomutant Ngn3 was found to enhance expression of endogenous Ngn3 and neuroD and promote differentiation compared to cells expressing WT Ngn3. Inhibition of phosphorylation of endogenous Ngn3 during endoderm/pancreatic islet differentiation would
therefore enhance Ngn3 , neuroD and therefore pancreatic endoderm and beta cell differentiation .
Heterozygous wt or 6S-A (phosphomutant ) Ngn3 knock-in mouse ES cells in which the WT or phosphomutant Ngn3 was expressed from the endogenous promoter were subj ected to a 3 stage endo 1 , 11 III protocol of differentiation into definitive endoderm. After 2 days in endo III , phosphomutant Ngn3 ES cells were found to express insulin while wild-type ES cell , do not, indicating that even when expressed at endogenous levels , inhibiting phosphorylation of Ngn3 enhances insulin expression in vitro ( Figure 7 ) .
Homozygous wt or 6S-A (phosphomutant ) Ngn3 knock-in mouse ES cells (where WT or phosphomutant Ngn3 is expressed from the endogenous promoter) were subj ected to a 3 stage endo 1 , 11 III protocol of differentiation into definitive endoderm. After 32 days in endo III , phosphomutant Ngn3 ES cells were observed to be morphologically distinct from wt Ngn3-expressing cells (Figure 8 ) and showed enhanced maturation and organisation, forming potentially islet-like clusters . These results indicate that, even when expressed at endogenous levels , inhibiting phosphorylation of Ngn3 enhances morphological maturation in vitro . Manipulating phosphorylation of the endogenous protein may therefore be useful in achieving cell maturation .
2. Ascll
The basic helix-loop-helix (bHLH) transcription factor Ascll (also known as Mashl) is a key regulator of stem cell maintenance and differentiation in both the central and peripheral nervous systems (CNS and PNS ) , and multiple direct downstream transcriptional targets have been identified.
We tested whether preventing phosphorylation of the highly conserved serine-proline sites found either side of the bHLH domain might enhance the ability of Ascll to drive neuronal differentiation and maturation in vivo and in vitro .
Xenopus egg extracts were used to investigate cell cycle-dependent post-translational protein modifications of in vitro translated proteins , visualized by slowed migration on SDS PAGE . Ascll is modified in both interphase (I) extracts , and to a greater extent in mitotic (M) extracts , and phosphatase treatment confirmed that phosphorylation has occurred . Ascll contains 6 serine-proline pairs , potential sites of cdk-dependent phosphorylation . Mutation of these serines to alanine to generate 6S-A Ascll restored SDS PAGE mobility (although some residual phosphorylation in mitosis appears to remain, indicating phosphorylation on additional sites ) . Further mutational analysis revealed that multiple SP sites on both sides of the bHLH domain were phosphorylated.
In vi tro cdk kinase assays demonstrate that Ascll is phosphorylated predominantly by cdk2 (Figure 9) and phosphorylation in interphase extracts can be inhibited by addition of the Xenopus cdki Xicl .
Thus , we see that Ascll is phosphorylated on multiple SP sites by cdks . Ascll may also be phosphorylated by other kinases such as GSK3 and MAP kinases .
To assess that ability of Ascll to drive neurogenesis in vivo, we overexpressed Ascll and 6S-A Ascll by mRNA inj ection in Xenopus embryos. Mutation of SP sites was found to significantly enhance Ascll ' s ability to induce ectopic neurogenesis . Ascll directly transcriptionally controls numerous genes including pro- proliferative genes , effectors of differentiation and the Notch ligand Delta, which controls non cell-autonomous progenitor
maintenance . 6S-A Ascll gives a 7-fold enhancement of expression of the direct pro-differentiation targets MyTl and neural beta-tubulin compared to Ascll . Delta expression is enhanced 2-fold. In contrast, overexpression of Ascll or 6S-A Ascll has little effect on expression of the pro-proliferative genes E2F1 , cdk2 and Skp2.
Where a modest upregulation on Ascll expression was seen for E2F1 and Skp2, a difference between 6S-A Ascll and Ascll was not
observed. These results indicate that Ascll phospho- status has a direct influence on its ability to drive differentiation/maturation while having a much smaller, or no , influence on its ability to regulate progenitor maintenance and proliferation of neural stem cells .
Phosphorylation of Ascll in Interphase or Mitotic extracts had no effect on Ascll binding to a naked DNA template in electrophoretic mobility shift assay (EMSA) . However, chromatin
immunoprecipitation on promoters of downstream targets revealed that 6S-A Ascll bound at least 5 fold-more effectively to its cognate E- box containing promoters than the wild-type protein, including the E2F1 promoter, even though upregulation of expression was not detected.
Phosphomutant Ascll is significantly more active in driving
differentiation than the wild-type protein in vivo . cyclinA2/cdk2 also inhibits the ability of Ascll to drive ectopic neurogenesis , but that 6S-A Ascll is resistant to this inhibition . Ascll also requires cdki p27Xicl to induce ectopic neurons in Xenopus , but 6S-A Ascll does not . Thus , mutation of SP sites on Ascll renders the protein insensitive to cell cycle cues that would otherwise limit neuronal differentiation in vivo. Notch signalling limits neurogenesis and promotes progenitor maintenance by both transcriptiona1 repression of proneural genes but also by post-translational inhibition of proneural protein function . 6S-A Ascll upregulates both Delta as well as drivers of neuronal differentiation, yet resuIts in clearly enhanced neuronal differentiation . We investigated whether preventing Ascll
phosphorylation could confer resistance to inhibition by Notch signalling . Expression of constitutively active Notch intracellular domain (NICD) was found to inhibit both endogenous neurogenesis, and neurogenesis driven by Ascll mRNA inj ection in Xenopus embryos .
However, phosphomutant Ascll was found to still efficiently drive neuronal differentiation in the presence of NICD . Thus , reducing or preventing phosphorylation of proneural proteins on SP/TP sites may confer resistance to Notch mediated inhibition, which is a common characteristic of differentiation driven by proneural protein activity .
Transdifferentiation of mouse fibroblasts can be achieved by expression of Brn2, Ascll and MyTlL, while addition of NeuroD to these "BAM factors" is required for differentiation of human fibroblasts . Although capable of firing action potentials , these neurons generally appear immature with relatively short axons and dendrites . Our results provide indication that phosphomutant Ascll may be more efficient at up-regulation of down-stream targets associated with differentiation than wild-type Ascll , including Ascll . Hence, we investigated whether 6S-A Ascll may potentiate neuronal transdifferent!ation with and without MyTlL, Brn2 and NeuroD in human and mouse fibroblasts .
In mouse fibroblasts , BAM factors induced functional neurons but with short axonal and dendritic outgrowths , which is likely to affect their ability to make connections and innervate . We also saw that in mouse fibroblasts , BAM using phosphomutant Ascll resulted in neurons which displayed increased maturity as evidenced by multiple extended neurites . Overexpression of the "BAM" transcription factors Ascll , Brn2 and MyTIL, can drive transdifferentiation of fibroblasts into functional neurons ( iN cells ; Pang, N . , et al . Cell Stem Cell 9, 517-525, (2011 ) ) , and these have been proposed as a cell source for
transplantation in treatment of diseases such as Parkinsons .
However, this transdifferentiation is currently inefficient , and neurons so generated are generally phenotypically immature .
Human lung fibroblast-derived iN cells were infected with
lentviruses expressing neurogenic conversion factors as labelled . 21 days after transduction with T or S-A hAscll in BAM (Brn2, Ascll and MyTi ) or BAM + NeuroD protocols, cells were stained for DNA and the marker of neuronal differentiation Tuj 1 to neuronal show morphology . We saw that, in human lung fibroblasts , BAM factors plus phosphomutant Ascll induced twice as many neurons as BAM factors plus wild-type Ascll and the neurons so generated showed
significantly enhanced neurite outgrowth and branching . In
addition, BAM factors plus NeuroD also led to the induction of neurons with short axonal and dendritic outgrowths . BAM plus NeuroD using phosphomutant Ascll resulted in neurons , which displayed increased maturity as evidenced by multiple extended neurites
( Figure 10 ) . Wild type Ascll with NeuroD produced no neurons while phosphomutant Ascll with NeuroD did produce neurons , although these have shorter neurites than the BAM plus NeuroD combination, and neuronal differentiation occurred with low efficiency ( Figure 10 ) . Moreover, neurons generated using the phosphomutant Ascll showed significantly enhanced electrophysiological maturity compared to those generated with wild-type Ascll . Our data show that substituting phospho-mutant Ascll for wild-type Ascll in human fibroblast transdifferentiation significantly enhances the trans-differentiation of human fibroblasts into mature neurons compared to those generated using wild-type Ascll (Figure 10 ) . Phosphomutant Ascll promotes conversion efficiency and neuronal complexity and maturity of induced neuron ( iN) cells . Furthermore, our preliminary data indicates that phosphomutant Ascll alone, without Brn2 and MyTl, may be sufficient to generate some neurons in the transdifferentiation assay, while wild-type Ascll is not. These cells do not undergo extensive proliferation post BAM factor addition, yet the phosphomutant Ascll still promotes more mature neurons , indicating that phosphorylation on SP sites by kinases other than cdks may usually be occurring and regulating activity . Thus , multi-site phosphorylation controls the ability of Ascll to drive cell di fferentiation and maturation in vitro.
Human lung fibroblast-derived iN cells were infected with
lentiviruses expressing WT or S-A hAscll in BAM or BAM + NeuroD
(BAM ) with a small molecule glycogen synthase kinase-3b (CHI99021 ) and a SMAD signalling inhibitor (SB-431542 ) . 21 days later, cells were fixed and stained for DNA and the neuronal differentiation marker Tuj 1. The neuronal maturity of the Human lung fibroblast- derived iN cells was then determined by immunofluorescence
microscopy . Phosphomutant Ascll was observed to promote conversion efficiency and neuronal maturity of the iN cells when cultured with the glycogen synthase kinase-3b inhibitor . The iN cells generated from the human fibroblasts (using BAM factors , Brn2 , Ascll , MyTl , with or without NeuroD) and small molecule inhibitors were observed to be more mature and complex when using the phosphomutant protein
( Figure 11 ) . This provides indication that the phosphomutant protein may be useful in producing models for neurological disorders .
A partially retinoic acid resistant neuroblastoma cell line , SKNDZ , that expresses endogenous Ascll was treated with either 5 μΜ retinoic acid (RA) , 10 μΜ palbociclib (a CDK inhibitor currently in clinical trials ) or in combination for 24 , 48 or 96 hours .
Similarly, a retinoic acid resistant neuroblastoma cell line ( SKNAS ) that expresses endogenous Ascll was treated with either 5 μΜ retinoic acid (RA) , 20 μΜ roscovitine (CDKi ) or in combination for 24 , 48 or 96 hours . Cells were then analyzed for morphological differentiation by immunofluorescence against neural-β-tubulin and Hoechst . Combination treatment of CDKi and RA was observed to decrease cell number and induced morphological changes that are consistent with neurite formation, including significant neurite outgrowth, consistent with reduction of endogenous Ascll
phosphorylation enhancing its ability to drive neuronal
differentiation . Furthermore , CDK inhibitors and retinoic acid synergise to bring about differentiation of neuroblastoma cells that express endogenous Ascll . mRNA for WT, or phosphomutant forms of Ascll with mutation of predicted cdk sites (Ascll Cdk) , GSk3beta sites (Ascll Gsk3B) or cdk and Gsk3B sites simultaneously (Ascll Cdk Gsk3B) were inj ected into fertilsed Xenopus eggs and a comparison of noraderinergic and seratonergic markers conducted by qPCR at the tailbud stage (Figure 12 ) . Overall neurogenesis was assayed by neural beta tubulin expression . Serotonergic and noradrenergic subtype markers were found to be differentially influenced by the availability of phosphorylation sites targeted by Cdks and GSk3beta . For example , mutation of both Cdk and GSK3B sites increased noradrenergic marker expression relative to serotonergic marker expression compared to mutation of either Cdk or GSK3B sites alone . A mutant with SP sites and additional potential target sites for GSK3 beta was found to be more active at driving neuronal differentiation (as assayed by neural beta tubulin expression) compared to a mutant of SP sites alone , demonstrating that further phosphosites may contribute to inhibition of the ability of proneural proteins to drive
differentiation .
Sequences
Human Ascll protein sequence (Seq ID NO : 1 ) messakmesg gagqqpqpqp qqpflppaac ffataaaaaa aaaaaaaqsa qqqqqqqqqq qqapqlrpaa dgqpsggghk sapkqvkrqr ssspelmrck rrlnfsgfgy slpqqqpaav arrnerernr vklvnlgfat lrehvpngaa nkkmskvetl rsaveyiral qqlldehdav saa qagvls ptispnysnd lnsmagspvs syssdegsyd plspeeqell dftnwf
Human 5S~A Ascll sequence (Seq ID NO : 2 ) : (modified residues highlighted) messakmesg gagqqpqpqp qqpflppaac ffataaaaaa aaaaaaaqsa qqqqqqqqqq qqapqlrpaa dgqpsggghk sapkqvkrqr ssapelmrck rrlnfsgfgy slpqqqpaav arrnerernr vklvnlgfat lrehvpngaa nkkmskvetl rsaveyiral qqlldehdav saafqagvla ptiapnysnd lnsmagapvs syssdegsyd plapeeqell dftnwf
Human Ngn3 sequence ( Seq ID NO : 3 ) : mtpqpsgapt vqvtreters fprasedevt cptsappspt rtrgncaeae eggcrgaprk Irarrggrsr pkselalskq rrsrrkkand rernrmhnln saldalrgvl ptfpddaklt kietlrfahn yiwaltqtlr iadhslyale ppaphcgelg spggspgdwg slyspvsqag slspaaslee rpgllgatfs aclspgslaf sdf1
6S-A hNgn3 sequence (Seq ID NO: 4) : (modified residues highlighted) mtpqpsgapt vqvtreters fprasedevt cptsappapt rtrgncaeae eggcrgaprk Irarrggrsr pkselalskq rrsrrkkand rernrmhnln saldalrgvl ptfpddaklt kietlrfahn yiwaltqtlr iadhslyale ppaphcgelg apggapgdwg slyspvsqag s lapaas lee rpgllgatfs aclapgslaf sdf1
Mouse Ngn3 sequence (Seq ID NO: 5) : (modified residues highlighted)
MAPHPLDALTIQVSPETQQPFPGASDHEVLSSNSTPPSPTLI PRDCSEAEVGDCRGTSRK 60
LRARRGGRNRP SELALS QRRSRRK ANDRERNRMHNL SALDALRGVLPTFPDDAKLT 120
KIETLRFAHNYIWALTQTLRIADHSFYGPEPPVPCGELGSPGGGSNGDWGSIYSPVSQAG 180
NLSPTASLEEFPGLQVPSSPSYLLPGALVFSDFL 214
6S-A mNg 3 sequence (SED ID : 6 ) (modified residues highlighted)
MAPHPLDALTIQVAPETQQPFPGASDHEVLSSNSTPPAPTLIPRDCSEAEVGDCRGTSRK 60
LRARRGGRNRP SELALSKQRRSRRKKANDRERNRMHNLNSALDALRGVLPTFPDDAKLT 120
KIETLRFAHNYIWALTQTLRIADHSFYGPEPPVPCGELGAPGGGSNGDWGSIYAPVSQAG 180
LAPTASLEEFPGLQVPSAPSYLLPGALVFSDFL 214
T35A mNg 3 sequence (SED ID : 7 ) (modified residue highlighted)
MAPHPLDALTIQVSPETQQPFPGASDHEVLSSNSAPPSPTLIPRDCSEAEVGDCRGTSRK 60
LRARRGGRNRPKSELALSKQRRSRRKKANDRERNRMHNLNSALDALRGVLPTFPDDAKLT 120
KIETLRFAHNYIWALTQTLRIADHSFYGPEPPVPCGELGSPGGGSNGDWGSIYSPVSQAG 180
NLSPTASLEEFPGLQVPSSPSYLLPGALVFSDFL 214

Claims

Claims
1. A method of maturing mammalian cells comprising;
providing a population of immature mammalian cells; and;
reducing the phosphorylation of a basic helix loop helix (bHLH ) protein in said population .
2. A method according to claim 1 wherein said reduction in phosphorylation promotes maturation of the population of mammalian cells .
3. A method according to claim 1 or claim 2 wherein
phosphorylation is reduced by introducing a modified bHLH protein lacking one or more phosphorylation sites into the immature
mammalian cells .
4. A method according to claim 3 where the modified bHLH protein has at least one fewer cyclin-dependent kinase or GSK3 β
phosphorylation site than the wild type bHLH protein .
5. A method according to claim 3 or claim 4 wherein the modified bHLH protein is introduced into the immature mammalian cells by expression from an encoding nucleic acid .
6. A method according to claim 5 comprising transfecting the immature mammalian cells with an expression vector which comprises a nucleic acid encoding the modified bHLH protein operably linked to a regulatory element .
7. A method according to claim 6 wherein the vector is a
lentiviral or retroviral vector .
8. A method according to claim 5 comprising transfecting the immature mammalian cells with an RNA molecule which encodes the modified bHLH protein .
9. A method according to claim 1 or claim 2 wherein
phosphorylation is reduced by exposing the immature mammalian cells to a kinase inhibitor .
10. A method according to claim 9 wherein the kinase inhibitor is a cyclin dependent kinase inhibitor or a GSK3 β inhibitor .
11. A method according to claim 9 wherein the cyclin dependent kinase inhibitor is palbociclib, roscovitine or olomucine .
12. A method according to any one of claims 1 to 11 comprising monitoring the phenotype of the mammalian cells .
13. A method according to any one of claims 1 to 12 comprising determining the presence of one or more cells in the population which display a mature phenotype .
14. A method according to any one of claims 1 to 13 comprising isolating and/or purifying mature mammalian cells in the population .
15. A method according to any one of claims 1 to 14 comprising culturing, maintaining and/or storing the mature mammalian cells in the population .
16. A method according to any one of claims 1 to 14 wherein the immature mammalian cells are produced by directed differentiation of a pluripotent cell .
17. A method according to any one of claims 1 to 14 wherein the immature mammalian cells are produced by trans-differentiation of a differentiated cell .
18. A method according to any one of claims 1 to 17 wherein the bHLH protein is Ngn3 and the mammalian cells are pancreatic islet beta cells .
19. A method according to claim 18 wherein the mature islet beta cells express Insulin, C peptide and Uro3 and exhibit a
physiological glucose response.
20. A method according to any one of claims 1 to 17 wherein the bHLH protein is one or more of Ascll , Ngn2 and Atonal and the mammalian cells are neurons.
21. A method according to claim 19 wherein the mature neurons exhibit extended neurites .
22. A method according to claim 20 or claim 21 wherein the phosphorylation of Ascll by one of Cdk or GSK3 β is reduced, thereby altering proportions of neural sub-types in the population of mature neurons .
23. A method according to claim 20 or claim 21 wherein the phosphorylation of Ascll by both Cdk and GSK3 β is reduced, thereby altering proportions of neural sub-types in the population of mature neurons .
24. A method according to any one claims of 1 to 23 comprising formulating the mature mammalian cells with a pharmaceutically acceptable carrier and/or a tissue scaffold .
25. An isolated population of mature mammalian cells produced by a method according to any one of claims 1 to 23.
26. An isolated population of mature pancreatic islet beta cells produced by a method according to any one of claims 1 to 19.
27. An isolated population of mature neurons produced by a method according to any one of claims 1 to 17 or 20 to 23.
28. An isolated population of immature mammalian cells produced by a method according to any one of claims 1 to 23 , the phosphorylation of a basic helix loop helix (bHLH) protein being reduced in said population .
29. A method of treatment comprising administering a population of mature mammalian cells according to any one of claims 25 to 27 or an immature population according to claim 28 to an individual in need thereof
30. Use of population of mature cells according to any one of claims 25 to 27 or an immature population according to claim 28 in the manufacture of a medicament for use in a method of treatment .
31. A population of mature cells according to any one of claims 25 to 27 or an immature population according to claim 28 for use in a method of treatment .
32. A method, use or population according to any one of claims 28 to 30 wherein the mature mammalian cells are islet beta cells or the immature cells are immature islet beta cells, and the method of treatment is a method of treatment of a diabetic condition .
33. A method, use or population according to claim 32 wherein the diabetic condition is as type 1 or type 2 diabetes .
34. A method, use or population according to any one of claims 29 to 31 wherein the mature mammalian cells are neurons and the method of treatment is a method of treatment of a neural condition .
35. A method, use or population according to claim 34 wherein the neural condition is a neurodegenerative condition, stroke or neural damage .
36. A method of screening a compound comprising;
contacting a population of cells according to one of claims 25 to 28 with a test compound, and;
determining the effect of the test compound on said cells and/or the effect of said cells on the test compound .
37. A method of screening for a compound which activity useful in the treatment of a diabetic condition comprising;
contacting a population of cells according to claim 26 with a test compound, and;
determining the effect of the test compound on said cells and/or the effect of said cells on the test compound.
38. A method according to claim 37 wherein the effect of the test compound on cell death/ survival, growth, proliferation, condition, aggregation, function, insulin production, glucose-responsiveness and/or gene expression is determined .
39. A method of screening for a compound having activity useful in the treatment of a neural condition comprising;
contacting a population of neurons according to claim 27 with a test compound, and;
determining the effect of the test compound on said neurons and/or the effect of said neurons on the test compound .
40. A method according to claim 39 wherein the effect of the test compound on cell death/ survival, growth, proliferation, condition, aggregation, function, electrical excitability and/or gene
expression is determined .
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