WO2019197844A1 - Macrophage use - Google Patents

Macrophage use Download PDF

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Publication number
WO2019197844A1
WO2019197844A1 PCT/GB2019/051060 GB2019051060W WO2019197844A1 WO 2019197844 A1 WO2019197844 A1 WO 2019197844A1 GB 2019051060 W GB2019051060 W GB 2019051060W WO 2019197844 A1 WO2019197844 A1 WO 2019197844A1
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cells
klf1
pluripotent stem
stem cell
derived
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PCT/GB2019/051060
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French (fr)
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Lesley FORRESTER
Martha LOPEZ-YRIGOYEN
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The University Court Of The University Of Edinburgh
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/231—Interleukin-10 (IL-10)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2333—Interleukin-33 (IL-33)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1157—Monocytes, macrophages
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells

Definitions

  • This invention relates to methods of producing human pluripotent stem cell derived macrophages with an altered phenotype in response to KLF1 activation which have utility in increasing the efficiency of enucleation of erythroid cells, uses of such macrophages and the provision of an in vitro erythroblastic-island niche to study erythropoiesis.
  • Enucleation involves multiple molecular and cellular processing including histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions and vesicle trafficking and these all operate in a well-orchestrated signalling network within the erythroblastic island (El) niche [10, 11] Els consist of a central macrophage that has been proposed to act as a ‘nurse cell’ surrounded by 5-30 developing erythroblasts.
  • the macrophage-erythroblast interaction provides both positive and negative regulators of cell differentiation and development at early and late stages of erythroid maturation [12] Recreating the El niche in vitro would provide a model to study these molecular interactions in more detail and aid in the understanding of the later stages of erythropoiesis.
  • Human monocyte-derived macrophages can promote primary erythroblast proliferation and survival but studies have reported different effects on maturation and enucleation [13] [14] This discrepancy likely reflects the source and heterogeneous phenotype of the macrophage cell populations that were used.
  • monocyte-derived macrophage might not accurately reflect the El niche because they have a distinct developmental origin and phenotype to tissue resident macrophages [15-18]
  • the present invention is predicated on the surprising findings that KLF1 activation in differentiating iPSCs could be mediated, in part, by its action in non-erythroid support cells and the further surprising finding that a transcription factor KLF1 could alter the phenotype of human pluripotent stem cell derived macrophages and in a way to generate a more El- like phenotype.
  • KLF1 activation programmes induced pluripotent stem cell-derived macrophages (iPSC-DMs) into cells that have an El- like phenotype in terms of cell surface marker expression, an increase in phagocytic activity and were able to support the maturation and enucleation of differentiating erythroid cells.
  • the present invention provides an in vitro or ex vivo method of producing a population of human pluripotent stem cell derived macrophages with an altered phenotype, the method comprising modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population.
  • the method may comprise increasing the activity of KLF1.
  • the altered phenotype may comprise enhanced phagocytic activity and/or the altered phenotype may comprise an erythroblastic island macrophage phenotype.
  • the method may increase the level of a combination of two or more of, three or more of or all of: CD163, CD169, CD206 and CCR5 in the population.
  • the method may increase the level of such markers on the cell surface.
  • the method may increase the level of one or more markers selected from the group consisting of: CD1 1A, CD1 1 B, CD64, TNFa and PECAM 1.
  • the method may increase the RNA expression level of such markers.
  • the human pluripotent stem cell derived macrophages may be induced pluripotent stem cell (iPSC) derived macrophages.
  • iPSC induced pluripotent stem cell
  • the method may comprise the step of incorporating a transgene comprising a gene encoding KLF1 into the cells; optionally wherein the transgene is integrated into the safe harbour AAVS1 locus of the cell.
  • the transgene may be an inducible transgene and the method may further comprise the step of inducing the activity of KLF 1.
  • the transgene may be an inducible fusion transgene whereby KLF1 is fused to, for example, an ERT2 domain and the activity can be induced by addition of tamoxifen or similar agent.
  • the method may comprise CRISPR activation (CRISPRa).
  • the method comprises the step of providing the human pluripotent stem cell derived macrophages with IL-10.
  • the present invention further provides a human pluripotent stem cell derived macrophage comprising a KLF1 transgene.
  • the present invention provides the use of a human pluripotent stem cell derived erythroblastic island macrophage to increase enucleation of CD34+ HPC- derived erythroid cells in vitro or ex vi vo; optionally wherein the CD34+ H PC-derived erythroid cells are derived from the umbilical cord blood.
  • the human pluripotent stem cell derived erythroblastic island macrophage may be produced by a method of the invention.
  • the present invention provides an in vitro or ex vivo method for producing enucleated erythroid cells, said method comprising:
  • I L33 and, optionally one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL;
  • the CD34+ haematopoietic progenitor cells may be umbilical cord derived CD34+ haematopoietic progenitor cells.
  • the CD34+ haematopoietic progenitor cells may be derived from iPSCs, as shown by Lopez-Yrigoyen, et al. in Nature Communications 2018 Feb 20;10(1):881.
  • the population of human pluripotent stem cell derived macrophages may be human pluripotent stem cell derived macrophage produced a method of the invention or the population may comprise a human pluripotent stem cell derived macrophage of the invention.
  • the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone prior to step a.
  • the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone for at least 11 days.
  • the culturing step a may be carried out for at least 1 1 days.
  • the present invention further provides an in vitro or ex vivo model for studying erythropoiesis, said model comprising a combination of a human pluripotent stem cell derived erythroblastic island macrophage and umbilical cord blood derived CD34+ haematopoietic progenitor cells.
  • the human pluripotent stem cell derived erythroblastic island macrophage may be an induced pluripotent stem cell derived erythroblastic island macrophage.
  • the present invention provides IL-33 for use in the treatment of dyserythropoietic anaemia in a subject.
  • IL-33 may be used in combination with one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
  • the present invention provides a method of treating a subject having dyserythropoietic anaemia, said method comprising administering a therapeutically effective amount of IL33.
  • the method of treating a subject having dyserythropoietic anaemia may further comprising administering a therapeutically effective amount of one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
  • FIG. 1 shows that AAVS1 -targeted KLF1 transgene was expressed in iPSC- DM.
  • B Differentiation protocol used to generate macrophage from an iPSC cell line (iKLF1.2) carrying the AAVS1 -targeted KLF1- ER T2 transgene;
  • C Kwik-Diff stained cytospin preparations of iKLF1.2-derived macrophages stained with line (Scale bar, 20mhi); and
  • FIG. 2 shows that activation of KLF1 in iPSC-DM up-regulated El related markers and enhanced phagocytic activity.
  • D Images captured at 175 minutes after
  • FIG. 3 shows that maturation and enucleation of UCB CD34+ derived erythroid cells were enhanced when co-cultured with KLF1 -activated macrophages.
  • A/B Flow cytometry analyses of live CD235a + -gated cells (day 14) of UCB CD34+ -derived erythroid cells cultured alone or in co-culture with iPSC-DM in the presence and absence of tamoxifen (Tam) at day 14 (A) and day 21 (B) stained with anti-CD71 antibody and Hoechst dye (see Figure 6 for gating strategy and FMO controls);
  • D Cytospin of co-culture of KLF1 expressing iPSC-DM and UCB CD34+ cells showing close association and a fully mature erythroid cell with biconcave shape (arrow) (scale bar, 20mhi) and
  • FIG. 4 shows that KLF1 activation enhanced the effects of iPSC-DMs in a paracrine manner.
  • Figure 5 shows (A) Immunohistochemistry of iPSC-DM from control (SFCi55) or iKLF1.2 iPSCs in the presence and absence of tamoxifen using an anti-HA antibody that detects the HA-KLF1-ER 2 fusion protein.
  • Figure 6 shows the Gating strategy for analysis of erythroid maturation. Single, live cells that were CD235a+ were gated, then analysed for the expression of CD71 and Hoechst DNA stain (fluorescence minus one (FMO) controls are shown). The positive control is peripheral blood showing fully mature, CD7T, enucleated red blood cells.
  • FIG. 7 shows that maturation and enucleation of UCB derived CD34 + erythroid cells was enhanced when KLF 1 -activated macrophages were cultured in a transwell culture where contact was inhibited.
  • A/B Representative Flow cytometry analyses of live CD235a + - gated cells (day 14) of UCB CD34+ erythroid cells cultured alone or in co-culture with iPSC- DM in the presence and absence of tamoxifen (Tam) at day 14 (A) and day 21 (B) stained with anti-CD71 antibody and Hoechst dye (see Figure 6 for gating strategy and FMO controls and Figure 4A for quantification);
  • C Cytospins of UCB CD34+ cells (from left to right) cultured alone, alone plus tamoxifen, with iPSC-DM, with iPSC-DM and tamoxifen (KLF1 activation); arrows point to enucleated cells (E)
  • FIG. 8 shows that KLF1 activation in iPSC-DM up-regulates El related markers and enhances phagocytosis in a number of independently derived KLF1-ER T2 -expressing iPSC lines.
  • Quantitative RT-PCR analyses of El-macrophage related genes in macrophages derived from 3 independently generated iPSC-lines (iKLF 1.6, iKLF1.7 and iKLF 1.12) carrying the CAG-KLF1-ER 2 transgene in the AAVS1 locus in the presence and absence of tamoxifen (Tam)(n 4 biologically independent samples, non-parametric Wilcoxon Test)(A).
  • Tam tamoxifen
  • the present invention provides an in vitro or ex vivo method of producing a population of human pluripotent stem cell derived macrophages with an altered phenotype, the method comprising modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population.
  • Human pluripotent stem cell derived macrophages include macrophages produced from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The maturation of macrophages from such cells can be detected by the presence of mature macrophage specific markers. For example, a human pluripotent stem cell derived macrophages will be positive for the human macrophage specific 25F9 (human macrophage specific) and, optionally, CD11 b.
  • the method of generating human iPSCs may be that detailed in the Examples section of the patent.
  • ESC derived macrophages may be generated by culturing the ESCs in the presence of colony stimulating factor-1 (CSF-1) (also known as M-CSF) and IL-3 to form embryoid bodies (EB). Whilst EBs adhere to tissue culture plastic, macrophage progenitor cells are non-adherent and thus are released into the medium. The macrophage progenitor cells may then be harvested at various time points, for example after 10 or 20 days and plated onto non-treated Petri dishes and cultured in the presence of CSF-1 alone. This process can give rise to monocyte-like cells that adhere to the plastic forming a monolayer and mature into ESDM.
  • CSF-1 colony stimulating factor-1
  • IL-3 embryoid bodies
  • the maturation of the ESC into ESDM can be monitored by detecting the presence of mature macrophage specific markers F4/80 (mouse macrophage specific) or 25F9 (human macrophage specific) and CD1 1 b.
  • the method described yields a substantially homogenous population of ESDMs.
  • ESDMs for use in the invention may be human ESCs and hence the marker 25F9, optionally in combination with CD1 1 b can be used to determine maturation into ESDMs.
  • macrophages may be derived from iPSC.
  • the method for differentiation of iPSCs to macrophages may involve supplementing culture medium with a cytokine Mix 1 (comprising bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF) and stem cell factor (SCF)).
  • BMP4 bone morphogenetic protein
  • VEGF vascular endothelial growth factor
  • SCF stem cell factor
  • Cells may be cut, dislodged, divided and re cultured in fresh media supplemented with cytokine mix 1.
  • Cells may be cultured in suspension for 3 days with a cytokine top up on Day 2, to form EBs.
  • the EBs may then be transferred to media supplemented with cytokine Mix 2 (comprising M-CSF, IL3, Glutamax, Penicillin/Streptomycin and b-mercaptoethanol). EBs can be maintained in this medium for the remaining duration of the protocol, with spent medium being replaced with fresh medium every 3-4 days. After about 2 weeks, the EBs produced macrophage progenitors in the culture supernatant that were harvested and transferred to medium supplemented with cytokine Mix 3 (M-CSF, Glutamax, Penicillin/Streptomycin) and allowed to mature into iPSC- derived macrophages (iPSC-DM). Macrophage progenitors may continue to be harvested twice a week for approximately 2 months.
  • cytokine Mix 2 comprising M-CSF, IL3, Glutamax, Penicillin/Streptomycin and b-mercaptoethanol.
  • EBs can be maintained in this medium for the remaining duration of the protocol, with spent medium being replaced with fresh medium every 3
  • the iPSC-derived macrophages are from human iPSCs.
  • the resulting ESDMs or iPSC-DM may be subsequently polarised in vitro to adopt either a M 1 -like phenotype by treatment with LPS and IFNy.
  • ESDMs or iPSC-DM may be polarised to yield Alternatively Activated Macrophage with IL-4.
  • ESDMs may be polarized to yield AAMs with IL-4, IL-13 and CSF-1.
  • M1 -like polarised macrophages are also known as classically activated macrophages (CAM).
  • AAM derived from ESDMs may be characterised by their high expression of Chil3 (Ym1), Retnla (Fizz), Mrd (Mannose Receptor 1), and Arg1 (Arginase).
  • M1 -like polarised macrophages such as ESDM, BMDM or iPSC-DM
  • the human pluripotent stem cell derived macrophages may not be polarised, such macrophages may be referred to as“naive”.
  • phenotype it is meant an observable set of characteristics resulting from the interaction of the genotype of the cell with the environment.
  • modulating the activity of KLF1 will alter the phenotype of the population of human pluripotent stem cell derived macrophages.
  • the invention has surprising found that modulation of expression of the transcription factor KLF1 will alter the phenotype of human pluripotent stem cell derived macrophages.
  • activation i.e. increasing the level of activity of KLF1
  • activation can alter the phenotype of a human pluripotent stem cell derived macrophage to that of a human pluripotent stem cell derived erythroblastic island macrophage.
  • A“pluripotent stem cell derived erythroblastic island (El) macrophage” may be defined as a pluripotent stem cell derived macrophage in which one or more El markers are significantly increased.
  • a population of “pluripotent stem cell derived erythroblastic island (El) macrophages” may have increased levels of at least one of the following markers following activation of KLF1 : CD 163, CD 169, CD206 and CCR5.
  • Any known method may be used to increase the activity of KLF1.
  • one way is to increase the level of KLF1 present in the macrophages.
  • “increased levels” it is meant that at least one of the following is increased: the percentage of cells within the population expressing the marker of interest; the level of the marker on the surface of the cells within the population is significantly increased; or the mRNA expression of the marker is increased.
  • Any known method may be used to determine the percentage of cells within a sample of the population that express a desired marker such as flow cytometry analysis.
  • the presence, level or absence of a marker polypeptide or nucleic acid molecule (e.g. mRNA) in a population of macrophages can be determined by contacting the sample population with a compound or an agent capable of specifically detecting (e.g. specifically binding) the specific marker polypeptide or nucleic acid molecule.
  • a marker polypeptide or nucleic acid molecule e.g. mRNA
  • Routine methods may be used to obtain sample from a cell population. For example, by immersing the cell population in a buffer for extracting protein or mRNA.
  • the level of any specific marker in a cell population can be can be measured in a number of ways, including: measuring the mRNA that encodes the protein marker; measuring the amount of protein marker; or measuring the activity of the protein biomarker.
  • Any known mRNA detection method may be used to detect the level of mRNA of a marker of interest (e.g. CD163, CD169, CD206 or CCR5) in a sample.
  • a marker of interest e.g. CD163, CD169, CD206 or CCR5
  • the level of a specific mRNA in a sample can be determined both by in situ and by in vitro formats.
  • mRNA may be detected using Northern blot analysis, polymerase chain reaction, probe arrays or RNA sequencing.
  • a sample may be contacted with a nucleic acid molecule (i.e. a probe, such as a labeled probe) that can specifically hybridize to the specific mRNA of the marker of interest (e.g. e. CD163, CD169, CD206 or CCR5).
  • a nucleic acid molecule i.e. a probe, such as a labeled probe
  • the probe may be, for example, a complement to a full-length nucleic acid molecule, or a portion thereof, such as a nucleic acid molecule of at least 10, 15, 30, 50, 100, 250 or 350 nucleotides in length and which specifically hybridizes under stringent conditions to specific mRNA of interest.
  • hybridisation shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide binding complex, as taught in Berger and Immel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego CA), and confer a defined “stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.
  • the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g.
  • the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringency conditions (e.g. 65°C and O.lxSSC).
  • the level of a specific mRNA in a sample may be evaluated with nucleic acid amplification, for example by RT-PCR, ligase chain reaction, self-sustained sequence replication, transcriptional amplification or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art including RNA sequencing.
  • the level of at least one of CD163, CD169, CD206 and CCR5 may be measured by RT-PCR analysis.
  • the hPSCs following an increase in KLF1 , the hPSCs have an altered phenotype having a significant increase in at least CD163, CD169, CD206 and CCR5 compared to the hPSCs prior to modulation of KLF1.
  • the altered phenotype may be one in which the level of at least 2 of or at least 3 or all of the CD163, CD169, CD206 and CCR5 is increased in a sample of the hPSCs following activation with KLF1 (i.e. following increasing the activity of KLF1).
  • Any known protein detection method may be used to detect the level of protein of a marker of interest (e.g. CD163, CD169, CD206 and CCR5) in a sample.
  • a marker of interest e.g. CD163, CD169, CD206 and CCR5
  • protein detection methods comprise contacting an agent that selectively binds to a protein, for example an anti-CD163 an anti-CD169, an anti-CD206 or an anti- CCR5, with a sample to determine the level of the specific protein in the sample.
  • the agent or antibody is labeled, for example with a detectable label.
  • Suitable antibodies may be polyclonal or monoclonal.
  • An antibody fragment such as a Fab or F(ab')2 may be used.
  • labeling refers to direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance.
  • the level of a specific protein marker in a sample may be determined by techniques known in the art, such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (BIA), radioimmunoassay (RIA), Western blot analysis, Flow cytometry and Lateral Flow Devices (LFDs) utilizing a membrane bound antibody specific to the protein biomarker.
  • ELISAs enzyme linked immunosorbent assays
  • BIOA enzyme immunoassay
  • RIA radioimmunoassay
  • Western blot analysis Flow cytometry and Lateral Flow Devices
  • LFDs Lateral Flow Devices
  • Methods of the invention may further comprise comparing the level or activity of the at least one marker in the test sample (i.e. a sample of hPSC derived macrophages following modulation of the activity of KLF1) with the level or activity of the at least one biomarker in a control sample (i.e. a sample of the hPSC derived macrophages prior to the modulation of the level of KLF1).
  • a control sample i.e. a sample of the hPSC derived macrophages prior to the modulation of the level of KLF1
  • methods of the invention include contacting a control sample with a compound or agent capable of detecting a specific biomarker mRNA (e.g. CD163, CD169, CD206 and/or CCR5), and comparing the level of the biomarker mRNA in the control sample with the level of biomarker mRNA in the test sample.
  • a compound or agent capable of detecting a specific biomarker mRNA e.g. CD163, CD169, CD206 and/or CCR5
  • the methods of the invention include contacting the control sample with a compound or agent capable of detecting a specific biomarker protein (e.g. CD163, CD169, CD206 and CCR5), and comparing the level of the biomarker protein in the control sample with the presence of the biomarker protein in the test sample.
  • a specific biomarker protein e.g. CD163, CD169, CD206 and CCR5
  • reference level refers to a hPSC derived macrophage sample having a normal level of biomarker (e.g. CD163, CD169, CD206 and CCR5) expression which would equate to the typical level of the biomarker hPSC derived macrophage population of the same type prior to modulation of KLF1.
  • biomarker e.g. CD163, CD169, CD206 and CCR5
  • the reference level may be comprised of a biomarker expression level from a reference database, which may be used to generate a pre-determined cut off value, i.e. a score that is statistically predictive of a significant alteration of the maker of interest.
  • control sample or reference sample is obtained using the same method as the method used to obtain a test sample.
  • control sample or reference sample is normalized as discussed below.
  • predictions may be based on the normalized expression level of the specific biomarker.
  • Expression levels are normalized by correcting the absolute expression level of the biomarker in a sample by comparing its expression to the expression of a reference nucleic acid that is not a marker, e.g., an mRNA or protein that is constitutively expressed. This normalization allows the comparison of the expression level in one sample to another sample, or between samples from different sources. This normalized expression can then optionally be compared to a reference standard or control.
  • the level of the biomarker or biomarkers of interest in the test sample is increased by at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1 , at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.1 , at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4.0, at least 4.1 , at least 4.2, at least 4.3, at least 4.4, at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5.0 fold compared to the control sample or predetermined reference sample.
  • the level of the at least one biomarker in the test sample is increased by at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 5 fold, at least 7.5 fold, at least 10 fold, at least 15 fold etc compared to the control sample or predetermined reference level.
  • By“significantly increased” it is meant a statistically significant increase calculated used a mean +/- standard error mean with an increase being considered statistically significant where the p-value is less than 0.05.
  • the altered phenotype may be one in which the level of at least one of CD163, CD169, CD206 and CCR5 the level of mRNA is increased at least two fold or at least 3 fold or at least 4 fold or at least 5 fold.
  • the altered phenotype may be one in which percentage of hPSCs in a sample which express at least one of CD163, CD169, CD206 and CCR5 is significantly increased and/or is increased by at least 5% or at least 6% or at least 6% or at least 7% or at least 10%.
  • the altered phenotype may be one in which the mean fluorescence intensity of at least one of CD163, CD169, CD206 and CCR5 in a sample is significantly increased and/or is increased by at least 500 or at least 600 or at least 750 or at least 1000.
  • the altered phenotype may be one which comprises enhanced phagocytosis.
  • Various methods for determining phagocytosis are known in the art.
  • One preferable method is the phagocytosis assay as detailed in the Examples. Using this assay, the number of iPSC-DMs that ingest beads (the phagocytic cell fraction) and the average number of beads that each iPSC-DM has ingested (phagocytic index) can be measured.
  • an altered which has enhanced phagocytosis may be one in which the phagocytic fraction after a set time period such as 175 minutes is significantly increased (e.g. by at least 0.05 or at least 0.1 or at least 0.2) and/or the phagocytic index after a set time period such as 175 minutes is significantly increased (e.g. by at least 5 or at least 10 or at least 20).
  • the method of the invention may increase the level of one or more further markers of interest.
  • the method of the invention may increase the level of one or more markers selected from the group consisting of: CD11A, CD1 1 B, CD64, TNFa and PECAM1.
  • the increase in the levels of any of the further markers may be measured in accordance with the methods provided for CD163, CD169, CD206 and CCR5.
  • the increase in the levels of any of the further markers may be measured by mRNA expression levels (such as by rtPCR).
  • the hPSC derived macrophages used in the method of the invention may be iPSC derived macrophages.
  • the method of the invention may comprise the step of providing the human pluripotent stem cell derived macrophages with IL-10.
  • the inventors have surprisingly found that modulation of KLF1 activity in combination with the provision of IL-10 increases the alteration of phenotype in the hPSC derived macrophages beyond that obtained by the modulation of KLF1 activity or provision of IL-10 on their own.
  • the modulation of KLF1 activity in combination with the provision of IL-10 increases the phagocytic activity of the hPSC derived macrophages.
  • IL-10 provided to the macrophages may be allogenic or autologous.
  • allogenic IL-10 may be provided to the macrophages by contacting the macrophages with IL-10.
  • the macrophages may be contacted with IL-10 by being cultured in the presence of IL-10.
  • autologous IL-10 may be provided to the macrophages by increasing the production of IL-10 by the macrophages, for example by increasing the expression of IL-10.
  • Methods for increasing the expression of IL-10 will be known to those skilled in the art.
  • IL-10 may be provided to the macrophages simultaneously with modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population.
  • IL-10 may be provided to the macrophages sequentially to modulating the activity of KLF1.
  • IL-10 may be provided sequentially, it may be provided before or after KLF1 activity has been modulated.
  • IL-10 may be provided after KLF1 activity has been modulated.
  • IL-10 may be provided to the macrophages at a concentration of at least 1 ng/ml, at least 2 ng/ml, at least 3 ng/ml, at least 4 ng/ml, at least 5ng/ml, at least 6 ng/ml, at least 7ng/ml, at least 8ng/ml, at least 9 ng/ml, at least 10ng/ml, or more. More suitably, IL-10 may be provided at a concentration of at least 5 ng/ml.
  • IL-10 is provided to the macrophages at a concentration of 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5ng/ml, 6 ng/ml, 7ng/ml, 8ng/ml, 9 ng/ml, 10ng/ml, or more. More suitably, IL-10 may be provided at a concentration of 5ng/ml.
  • IL-10 is provided to the macrophages at a concentration of between 1 and 10 ng/ml, 2 to 9 ng/ml, 3 to 8 ng/ml, or 4 to 7 ng/ml.
  • the methods of invention may activate or increase expression of KLF 1.
  • Any method of gene activation of KLF1 may be used included CRISPR activation methods.
  • the activation of KLF1 may be via a transgene encoding KLF1.
  • transgene it is meant a gene encoding KLF1 which has either been transferred by e.g. genetic engineering techniques to change the phenotype of an organism.
  • the method of the invention may comprise incorporating a transgene into a population of hPSC derived macrophages (iPSC-DM).
  • the transgene may be integrated into the safe harbour AAVS1 locus of the cells.
  • KLF1 may be under the control of a constitutive promoter or expression of KLF1 may be controlled such as through the use of an inducible transgene.
  • the Examples utilise a tamoxifen inducible transgene, the skilled person is readily aware of various ways to provide induced expression of KLF1.
  • the transgene may be an inducible fusion transgene whereby KLF1 is fused to, for example, an ERT2 domain and the activity can be induced by addition of tamoxifen or similar agent.
  • the method may comprise CRISPR activation (CRISPRa).
  • CRISPRa CRISPR activation
  • Various ways of using CRISPRa to increase the activity levels of KLF1 are known in the art. For example, Fidanza, A., Lopez-Yrigoyen, M., Romano, N., Taylor, H. & Forrester, L.M. (2017) An all-in- one UniSam vector system for efficient gene activation. Scientific Reports 7: 6394 discloses one way of using CRISPRa to activate the expression of KLF1.
  • the present invention further provides use of a human pluripotent stem cell derived macrophage to increase enucleation of CD34 + (e.g. umbilical cord derived CD34 + ) erythroid cells in vitro or ex vivo.
  • CD34 + e.g. umbilical cord derived CD34 +
  • the present inventors have surprising found that the maturation and enucleation of CD34 + H PC-derived erythroid cells are enhanced by co-culture with KLF-1 activated macrophages - see Figure 3 and the section entitled“activation of KLF1 in iPSC- DMs enhanced maturation of erythroid cells in the Examples. It can be seen that co-culture with KLF1 activated iPSC-DMs surprisingly increased the average baseline level of enucleation from 22% to 55% at day 14 and from 30% to 73% at day 21.
  • the present invention further provides an in vitro or ex vivo method for producing enucleated erythroid cells wherein the method comprises: culturing CD34 + (e.g. umbilical cord derived CD34 + ) hematopoietic progenitor cells with at least one of the following: i) a population of human pluripotent stem cell derived macrophages in which the activity (e.g., level) of KLF1 is increased; or ii) the culture media of a population of human pluripotent stem cell derived macrophages in which the activity (e.g., level) of KLF1 is increased; or iii) IL33 and optionally, one or more of selected from the group consisting of ANGPTL7,SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
  • CD34 + e.g. umbilical cord derived CD34 +
  • hematopoietic progenitor cells with at least one of the following: i
  • CD34 + e.g. umbilical cord derived CD34 +
  • hematopoietic progenitor cells with a population of human pluripotent stem cell derived macrophages in which the level of KLF1 is increased significantly enhances the maturation and enucleation of the erythroid cells - see Figure 3.
  • An“enucleated erythroid cell” as used herein refers to an erythroid cell which is CD235a + , CD7T, and does not stain with the DNA dye Hoescht.
  • the CD34+ haematopoietic progenitor cells may be umbilical cord derived CD34+ haematopoietic progenitor cells
  • the population of human pluripotent stem cell derived macrophages may be human pluripotent stem cell derived macrophages produced a method of the invention or the population may comprise a human pluripotent stem cell derived macrophages of the invention.
  • the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone prior to step a.
  • the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone for at least 11 days or at least 12 days or at least 13 days or at least 14 days.
  • the culturing step a may be carried out for at least 11 days or at least 12 days or at least 13 days or at least 14 days.
  • the method may result in a statistically significant increase in the number of enucleated erythroid cells when compared to a control of CD34 + (e.g. umbilical cord derived CD34 + ) hematopoietic progenitor cells cultures under identical conditions in the absence of: i) a population of human pluripotent stem cell derived macrophages in which the activity (e.g., level) of KLF1 is increased; and ii) the culture media of a population of human pluripotent stem cell derived macrophages in which the activity (e.g.
  • CD34 + e.g. umbilical cord derived CD34 +
  • the average baseline level of enucleation may an increase of at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%, or more as compared to the control.
  • the present invention further provides an in vitro or ex vivo model for studying erythropoiesis, said model comprising a combination of a human pluripotent stem cell derived erythroblastic island macrophage and umbilical cord blood derived CD34+ haematopoietic progenitor cells.
  • the human pluripotent stem cell derived erythroblastic island macrophage may be an induced pluripotent stem cell derived erythroblastic island macrophage.
  • the present invention further provides IL-33 for use in the treatment of dyserythropoietic anaemia in a subject and/or a method of treating a subject having dyserythropoietic anaemia, said method comprising administering a therapeutically effective amount of IL33.
  • the method may further comprise administering a therapeutically effective amount of one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
  • diserythropoietic anaemia it is meant anaemia, characterized by ineffective erythropoiesis, and resulting from a decrease in the number of red blood cells (RBCs) in the body and a less than normal quantity of haemoglobin in the blood.
  • the anaemia may be the result of mutation in KLF1.
  • IL33 belongs to IL1 superfamily that can apparently act as a cytokine or as a nuclear factor within the cell [29] It has been implicated in inflammation, wound healing and various disease processes. Interestingly IL33 is expressed in erythroid progenitor cells and released during haemolysis. Without wishing to be bound by theory, it is postulated that this then stimulates the maturation of new RBCs [30] Thus, administration of IL33 may aid maturation of RBCs in a subject suffering from dyserythropoietic anaemia as a result of a mutation in KLF1.
  • IL33 may be used in combination with one or more further cytokines.
  • IL33 may be used in combination with one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL to treat dyserythropoietic anaemia.
  • IL33 may be used in combination with ANGPTL7 and SERPINB2.
  • IL33 may be used in combination with ANGPTL7, SERPINB2 and NRG1.
  • IL33 may be used in combination with ANGPTL7, SERPINB2 and IGFBP6.
  • IL33 may be used in combination with ANGPTL7, SERPINB2 and CCL13.
  • IL33 may be used in combination with ANGPTL7, SERPINB2 and TNFS10.
  • IL33 and/or SERPINB2 may be used at a concentration of at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/ml, at least 100 ng/ml, at least 110 ng/ml, at least 120 ng/ml or more. More suitably, IL33 and/or SERPINB2 may be used at a concentration of at least 75 ng/ml.
  • IL33 and/or SERPINB2 may be used at a concentration of 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 110 ng/ml, 120 ng/ml, or more. More suitably, IL33 and/or SERPINB2 may be used at a concentration of 75 ng/ml.
  • IL33 and/or SERPINB2 may be used at a concentration of between 10 ng/ml and 120 ng/ml, 25 ng/ml and 100 ng/ml, or 70 and 80 ng/ml.
  • ANGPTL7 may be used at a concentration of at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/ml, at least 100 ng/ml, or more. More suitably, ANGPTL7 may be used at a concentration of at least 60 ng/ml.
  • ANGPTL7 may be used at a concentration of 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, or more. More suitably, ANGPTL7 may be used at a concentration of 60 ng/ml.
  • ANGPTL7 may be used at a concentration of between 10 ng/ml and 100 ng/ml, 30ng/ml and 80ng/ml, or 55 and 65 ng/ml.
  • NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/ml, at least 100 ng/ml, or more. More suitably, NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of at least 50 ng/ml.
  • NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, or more. More suitably, IL33 and/or SERPINB2 may be used at a concentration of 50 ng/ml.
  • NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of between 10 ng/ml and 100 ng/ml, 25 ng/ml and 75 ng/ml, or 45 and 55 ng/ml.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted disorder (i.e. dyserythropoietic anaemia) or symptom.
  • IPSC-DM express low levels of KLF1
  • MAF is also reported to be marker for yolk sac macrophages
  • KLF1 was expressed at lower levels in iPSC-DM compared to monocyte-derived macrophages (MDMs) ( Figure 1A).
  • iPSC-DMs were generated in a stepwise protocol by first generating embryoid bodies (EBs) in the presence VEGF, BMP4 and SCF then transferred to gelatin-coated plates and cultured in presence of IL-3 and MCSF. From day 16, myeloid progenitor cells were harvested from the supernatant and cultured in MCSF for 9-12 days [17] Tamoxifen was added to iPSC-DMs for the last 4 days to activate KLF1.
  • IPSC-DMs derived from the iKLF1.2 iPSC line were comparable in size and morphology to those derived from the parental iPSC line, SFCi55 and the addition of tamoxifen had no obvious effect on these parameters (Figure 1 C).
  • iPSC-DMs generated from the iKLF1.2 iPSC line demonstrated a high expression of KLF1 mRNA expression compared macrophages derived from monocytes or control iPSC-DMs ( Figure 1 D).
  • the inventors demonstrated that the KLF1-ERT2 fusion protein is expressed in the cytoplasm of iKLF1.2 iPSC-DMs and translocates to the nucleus upon tamoxifen addition thus demonstrating that the fusion protein activation strategy can function and that the KLF1-ERT2 transgene is not silenced in differentiated iPSC-DMS (Figure 5A).
  • macrophages from additional three independently derived iPSC lines iKLF 1.6, iKLF1.7 and iKLF1.12 carrying the CAG-KLF1-ER 2 transgene were tested.
  • Activation of KLF1 in iPSC-DMs generated from all three cell lines resulted in an up- regulation of El-related genes: CD163, CD169, CD11A and CD1 1 B comparable to the result observed in iPSC-DMs generated from the iKLF1.2 line ( Figure 8A) and enhanced phagocytic activity (Figure 8B, C).
  • KLF1 activation on the mRNA expression of previously reported KLF1 target genes including VCAM1 , DNASE2A; cell adhesion molecules involved in macrophage-erythroblast interaction (ITGAV, EMP/MAEA, PECAM1 , CD163 and CD169); El macrophage markers (CD64, CD68, CD1 1a, CD1 1 b, CD1 1c) and extrinsic regulators of erythropoiesis (PALD, IFN-b and TNF-a) [10, 15, 21-26]
  • Activation of KLF1 by the addition of tamoxifen resulted in the increased expression of a subset of these transcripts including CD163, CD169, CD11A, CD11 B, CD64, TNFa and PECAM 1 in macrophages derived from iKLF1.2 iPSCs ( Figure 2A).
  • KLF1 activation increased the proportion of iPSC-DMs expressing El-associated markers (CD206, CD163, CD169 and CCR5) ( Figure 2B) and on the level of expression of these markers on the cell surface as measured by the mean fluorescent intensity (MFI) ( Figure 2C).
  • IPSC-DMs expressed the marker 25F9 irrespective of tamoxifen treatment but the level of expression per cell was significantly higher after KLF1 activation ( Figure 2B,C).
  • CD34+ cord blood derived HPCs were cultured in SCF, EPO, IL3 and hydrocortisone for 7 days then at day 8 the cells were co-cultured with iKLF1.2 iPSC-DM in the presence or absence of tamoxifen.
  • the proportion of fully mature, enucleated cells was determined at day 14 and 21 by assessing the number of CD235a-expressing erythroid cells that were negative for CD71 and the DNA dye, Hoechst [18] ( Figure 6).
  • the percentage of mature enucleated erythroid cells (CD235a+, CD71-, Hoechst-) was higher in cells that were co-cultured with macrophages and this was further increased when co-cultured with KLF1.2 derived macrophages that had been treated with tamoxifen ( Figure 3A-B).
  • KLF1 -activated iPSC-DMs increased the proportion of enucleated cells from 12 to 30% and from 28 to 53% at day 14 and 21 , respectively.
  • KLF1 up-regulates cell communication and protein binding associated genes in iPSC-DMs
  • Receptors for ANGPTL7 (NEURL1 and NEURL1), IL33 ( IL1R1 ) and SERPINB2 (PLAUR) were all expressed in differentiating (day 14) UCB-CD34 + cells and their expression level increased significantly when all three cytokines were added to the protocol ( Figure 4C).
  • ANGPTL7, IL33 and SERPINB2 was assessed the combined effect of ANGPTL7, IL33 and SERPINB2 on the differentiation and maturation of UCB-CD34+ cells, in absence of macrophages.
  • KLF1 and IL10 enhances macrophage phagocytosis
  • the inventors have surprisingly found that phagocytic activity of macrophages can be enhanced by the KLF1 and IL10 provided together. Since the combined treatment of macrophages with KLF1 and IL10 resulted in a greater increase in phagocytic activity than IL10 or KLF1 on their own, this suggests that phagocytosis induced by KLF1 and IL10 are mediated by different mechanisms.
  • DISCUSSION The production of RBCs in vitro could solve many of the problems associated with blood transfusion such as limitations in supply, transfusion transmitted infection, and immune compatibility. Indeed, culture conditions have been developed for the production of RBC in vitro from human CD34+ HPCs, PSCs and immortalised erythroid progenitor cell lines, known as BELA [4-9] However, regardless of the starting populations, culture conditions are relatively inefficient and a low proportion of the resultant cell populations undergo the enucleation process that marks the final steps of erythroid maturation and this has severely hampered clinical translation. Enucleation occurs in vivo within the El niche that consists of a central macrophage surrounded by up to 30 developing erythroblasts. Understanding the cellular and molecular interactions that take place within this niche will help in the development of protocols to generate mature RBCs.
  • IPSC-derived macrophages have been reported to have a tissue resident-like phenotype [16, 17] and in-keeping with this the inventors demonstrate that MAF was expressed at a higher level in iPSC-DMs compared to monocyte derived macrophages (MDMs).
  • MDMs monocyte derived macrophages
  • KLF1 was expressed at low level both iPSC-DMs and MDMs and, the inventors hypothesised that this transcription factor would programme macrophages into an El-like phenotype.
  • the inventors used an inducible KLF1-ERT2 transgenes, targeted to the AAVS1 locus to allow conditional activation in differentiated IPSC-DMs.
  • the inventors have previously shown that the production and function of macrophages from iPSCs is unaffected by AAVS1 targeting per se and is resistant to epigenetic silencing [27], providing an ideal platform for testing the specific effect of transcription factor activation.
  • Activation of KLF1 in iPSC-DM increased the expression of some El-associated genes and cell surface markers.
  • KLF1 target genes are activated in this system which can be explained by the fact that transcriptional control by KLF1 is context dependent, involving a number of protein partners that will be differ between cell types.
  • KLF1- activated iPSC-DMs have an enhanced rate of phagocytosis which is in keeping with fact that El macrophages are reported to be more phagocytic. This function has likely evolved to clear free nuclei.
  • KLF1 -activated El-like macrophages increased the production of fully mature and enucleated erythroid cells from UCB-CD34+ HPCs, consistent with the report of an extrinsic role for KLF1 in the murine system [20, 21]
  • the inventors demonstrate that cell contact is not absolutely required for the increase in enucleation observed, indicating that the action of KLF1 target genes is in part mediated by secreted factors.
  • the inventors identified a number of secreted factors by RNA sequencing and have shown that a combination of three of these secreted factors (ANGPTL7, IL33 and SERPINB2) has a significant impact on the maturation of UBC-CD34+ HPCs.
  • IL33 appeared to have the most significant effect because the inventors observed a significant reduction in the enucleation rate at all stages when this cytokine was removed from the protocol.
  • IL33- belongs to IL1 superfamily that can apparently act as a cytokine or as a nuclear factor within the cell [29] It has been implicated in inflammation, wound healing and various disease processes. Interestingly IL33 is expressed in erythroid progenitor cells and released during haemolysis can imagine this then stimulates the maturation of new RBCs [30] Thus it is possible that the inventors have uncovered a novel feedback mechanism that could be used in therapy to treat anaemia.
  • ANGPTL7 has been shown to regulate the expansion and repopulation of human HSC and HPCs
  • SerpinB2 a serine protease inhibitor of the serpin superfamily is coagulation factor known to be present in macrophages. TGFb-responsive lineage fate determinant of human bone marrow stromal cells [32]
  • the human iPSCs lines SFCi55 and SFCi55-KLF1.2, SFCi55-KLF1.6, SFCi55-KLF1.7 and SFCi55-KLF1.12 were generated in house and were confirmed to be pluripotent and have normal karyotype [19] iPSCs were maintained in StemPro hESC SFM media (Gibco) with 20ng/ml bFGF (R&D). Wells were coated with CELLstart at least 1 hour before plating and cells were passaged using the StemPro EZPassage tool (ThermoFisher Scientific). Media change was performed every day and cells passaged at a ratio of 1 :4 every 3-4 days, when cells were 70% confluent.
  • the inventors adapted published differentiation protocol that resulted in optimal macrophage production [17, 33]
  • Maintenance media on one confluent well of a 6-well plate was replaced with 1.5 ml of Day 0 mix which consisted of StemPro hESC SFM (Gibco) supplemented with BMP4 (50ng/ml), VEGF (50ng/ml) and SCF (20ng/ml).
  • Cells were passaged using the EZPassage tool into two wells with 2.25ml of Day 0 mix cultured in Ultralow Attachment 6 well plates for four days to induce embryoid body (EB) formation.
  • EB embryoid body
  • EBs were transferred to gelatin-coated tissue culture grade 6 well plates in 3ml Day 4 mix (X-VIV015 media supplemented with M-CSF(100ng/ml), IL3 (25ng/ml), Penicillin-Streptomycin (1 %), Glutamax (2mM) and b-mercaptoethanol (0.055) and media was changed every 3-4 days.
  • non-adherent monocyte-like precursors were harvested from the supernatant and plated into untreated bacteriological plates or six-well plates in Maturation mix (X- VIV015 media supplemented with M-CSF (100ng/ml), Glutamax (2mM) and Penicillin- Streptomycin (1 %) for 9-11 days.
  • IL-10 5ng/ml of IL-10 (Preprotech Cat# 200-10) were added to adherent iKLF1.2 iPSC-DMs (IL10 activation only).
  • 100nM of tamoxifen was added to adherent macrophages for 4 days, every 2 days (KLF1 activation only).
  • 100nM of tamoxifen were added to iKLF1.2 iPSC-DMs for 48h; then one more dose of 100nM of tamoxifen + 5ng/ml of IL-10 were added for 48h more.
  • Umbilical cord blood (UCB) derived CD34+ cell expansion and differentiation was carried out using a step-wise protocol [9] Briefly cells were cultured (104 cell/ml) in ISHIT base media (Iscove’s Basal Media (Biochrom AG), Human AB+ serum (5%), Heparin (3U/ml) and Insulin (10pg/ml)) supplemented with SCF (60ng/ml), IL3 (5ng/ml), EPO (3U/ml), Hydrocortisone (1 mM) and holo-T ransferrin (200 pg/ml) for 8 days.
  • Cell density was adjusted to 105 cells /ml in ISHIT media supplemented with SCF (10ng/ml), EPO (3U/ml), Hydrocortisone 1 pM and holo-Transferrin (300pg/ml), cultured for a further 3 days then cultured at a density of 106 cells /ml in ISHIT medium supplemented with EPO (3U/ml) and holo-transferrin (300 pg/ml) until day 21. Media was changed every 3-4 days throughout the protocol.
  • ANGPTL7 (Preprotech Cat# 130-22), IL33 (Preprotech Cat# 200-33) and SERPINB2 also known as PAI 2 (Preprotech Cat# 140-06) was 60, 75 and 75 ng/ml respectively.
  • the final concentrations of NRG1/ Heregul ⁇ -1 (Preprotech Cat# 100-03), IGFBP6 (Preprotech Cat# 350-07B), NOV (Preprotech Cat# 120-26), CCL13 (Preprotech Cat# 300-24), TRAIL/TNSF10 Preprotech Cat# 310-04) was the same for all of them: 50 ng/ml.
  • Cytospins of macrophages were prepared by re-suspending 5x104 in 200 pi PBS. Cytospins of erythroid cells were prepared by re-suspending 1x105 cells in 200mI PBS. Cells were cyto- centrifuged into polylysine slides at 800RPM for 8 minutes in a Thermo Shandon Cytospin 4 and allowed to air-dry overnight. Cells were stained according to manufacturer’s instructions (Thermo-fisher #9990702, https://assets.thermofisher.com/TFSAssets/LSG/ manuals/238570%20Kwik-DiffStaining%20IFU.pdf).
  • Single cell suspensions were obtained by StemPro Accutase Cell Dissociation Reagent (Gibco) and re-suspended in PBS with 1 %BSA and 5 mM EDTA. Cells were blocked with MACS FcR Blocking Reagent (#130-059-901) for 40 min on ice according to manufacturer instructions. 1x105 cell were washed and stained with appropriate antibodies (Supplementary Table S1) for 20 minutes at room temperature.
  • iPSC-DM 8 x 104 were plated in tissue-culture grade 96-well plates (CellCarrier, PerkinElmer) at least 2 days before assessing their phagocytic activity as previously described [17] Briefly cells were stained with the nuclear stain, Hoechst33342 and CellMaskTMdeep red plasma membrane stain then pHrodoTM Green Zymosan A BioParticles were applied immediately prior to live imaging using the Operetta High-Content Imaging System (Perkin Elmer). The number of iPSC-DMs that had ingested beads (phagocytic cell fraction) and the average number of beads that each cell had ingested (phagocytic index) was quantified using Columbus Image data storage and analysis system.
  • RNA extraction was carried out using the RNAeasy Mini Kit (Qiagen); cDNA was generated from 500ng of total RNA using the High Capacity cDNA synthesis Kit (Applied Biosystem). cDNA (2ng) was amplified per reaction and each reaction was performed in technical triplicates using the LightCycler 384 (Roche) with SYBR Green Master Mix II (Roche). GADPH, b-Actin and B2M were used as reference genes and the geometrical mean was used to normalize the data. Primer sequences and efficiencies are reported (Supplementary Table S2).
  • RNA extraction was carried out using the RNAeasy Mini Kit (Qiagen) and RNA quantity and quality was assessed using the Agilent Technologies TapeStation and its software. Edinburgh Genomics generated 8 TruSeq stranded mRNA-seq libraries (lllumina) from total RNA samples. Sequence data was generated in HiSeq 400075PE (lllumina) to yield at least 290M + 290M reads (1 lane). The reference used for mapping was the Homo sapiens genome from Ensembl, assembly GRCh38, annotation version 84. A principal components analysis was undertaken on normalised and filtered expression data to explore observed patterns with respect to experimental factors.
  • Klf1 affects DNase ll-alpha expression in the central macrophage of a fetal liver erythroblastic island: a non-cell-autonomous role in definitive erythropoiesis. Mol Cell Biol, 2011. 31(19): p. 4144-54.
  • ANGPTL7 regulates the expansion and repopulation of human hematopoietic stem and progenitor cells. Haematologica, 2015. 100(5): p. 585-94.
  • SERPINB2 is a novel TGFbeta-responsive lineage fate determinant of human bone marrow stromal cells. Sci Rep, 2017. 7(1): p. 10797.

Abstract

The present invention relates to: an in vitro or ex vivo method of producing a population of human pluripotent stem cell derived macrophages or enucleated erythroid cells; human pluripotent stem cells derived macrophage comprising a KLF1 transgene and uses of human pluripotent stem cell derived macrophages or enucleated erythroid cells as well as being directed to IL-33 for use in the treatment of dyserythropoietic anaemia in a subject.

Description

Macrophage Use
[0001] This invention relates to methods of producing human pluripotent stem cell derived macrophages with an altered phenotype in response to KLF1 activation which have utility in increasing the efficiency of enucleation of erythroid cells, uses of such macrophages and the provision of an in vitro erythroblastic-island niche to study erythropoiesis.
BACKGROUND
[0002] Blood transfusion remains the most prominent means of treating emergency situations and chronic haematological disorders. Over 100 million red blood cell (RBC) concentrates per annum are collected from donors and distributed throughout the world [1] However, blood transfusion services have continuing problems with their donor supply and cell quality and there are significant risks associated with infection transmission and immune incompatibility [2, 3] To circumvent these issues, attempts have been made to produce RBCs in vitro from different starting cell populations including CD34+ haematopoietic progenitor cells (HPCs), pluripotent stem cells (PSCs) and more recently immortalised erythroid progenitor cell lines [4-9] RBC production from all of these sources is relatively inefficient and a low proportion of the resultant cell populations undergo the enucleation process that marks the final steps of erythroid maturation.
[0003] Enucleation involves multiple molecular and cellular processing including histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions and vesicle trafficking and these all operate in a well-orchestrated signalling network within the erythroblastic island (El) niche [10, 11] Els consist of a central macrophage that has been proposed to act as a ‘nurse cell’ surrounded by 5-30 developing erythroblasts. The macrophage-erythroblast interaction provides both positive and negative regulators of cell differentiation and development at early and late stages of erythroid maturation [12] Recreating the El niche in vitro would provide a model to study these molecular interactions in more detail and aid in the understanding of the later stages of erythropoiesis. Human monocyte-derived macrophages can promote primary erythroblast proliferation and survival but studies have reported different effects on maturation and enucleation [13] [14] This discrepancy likely reflects the source and heterogeneous phenotype of the macrophage cell populations that were used. Furthermore, monocyte-derived macrophage might not accurately reflect the El niche because they have a distinct developmental origin and phenotype to tissue resident macrophages [15-18]
[0004] Accordingly, there is a need for an in vitro El niche to study erythropoiesis and improved methods of producing enucleated mature erythroid cells. [0005] The inventors previously demonstrated that activation of the transcription factor, KLF1 enhanced the maturation of iPSC-derived erythroid cells but this effect was only observed at a time point when the differentiating culture consisted of a heterogeneous mixture of haematopoietic cells [19] An extrinsic role of KLF1 within the murine erythroid island (El) niche had been reported [20, 21] However, it was not known that increased activity of the transcription factor KLF1 could alter macrophage phenotype, let alone that an in vitro El-like niche could be produced.
BRIEF SUMMARY OF THE DISCLOSURE
[0006] The present invention is predicated on the surprising findings that KLF1 activation in differentiating iPSCs could be mediated, in part, by its action in non-erythroid support cells and the further surprising finding that a transcription factor KLF1 could alter the phenotype of human pluripotent stem cell derived macrophages and in a way to generate a more El- like phenotype. The inventors have surprisingly shown that KLF1 activation programmes induced pluripotent stem cell-derived macrophages (iPSC-DMs) into cells that have an El- like phenotype in terms of cell surface marker expression, an increase in phagocytic activity and were able to support the maturation and enucleation of differentiating erythroid cells.
[0007] Accordingly, the present invention provides an in vitro or ex vivo method of producing a population of human pluripotent stem cell derived macrophages with an altered phenotype, the method comprising modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population.
[0008] Suitably, the method may comprise increasing the activity of KLF1.
[0009] Suitably, the altered phenotype may comprise enhanced phagocytic activity and/or the altered phenotype may comprise an erythroblastic island macrophage phenotype.
[0010] Suitably, the method may increase the level of a combination of two or more of, three or more of or all of: CD163, CD169, CD206 and CCR5 in the population. Suitably, the method may increase the level of such markers on the cell surface.
[0011] Suitably, the method may increase the level of one or more markers selected from the group consisting of: CD1 1A, CD1 1 B, CD64, TNFa and PECAM 1. Suitably, the method may increase the RNA expression level of such markers.
[0012] Suitably, the human pluripotent stem cell derived macrophages may be induced pluripotent stem cell (iPSC) derived macrophages.
[0013] Suitably, the method may comprise the step of incorporating a transgene comprising a gene encoding KLF1 into the cells; optionally wherein the transgene is integrated into the safe harbour AAVS1 locus of the cell. Suitably, the transgene may be an inducible transgene and the method may further comprise the step of inducing the activity of KLF 1. For example, suitably the transgene may be an inducible fusion transgene whereby KLF1 is fused to, for example, an ERT2 domain and the activity can be induced by addition of tamoxifen or similar agent. Suitably, in addition or in the alternative, the method may comprise CRISPR activation (CRISPRa).
[0014] Suitably, the method comprises the step of providing the human pluripotent stem cell derived macrophages with IL-10.
[0015] The present invention further provides a human pluripotent stem cell derived macrophage comprising a KLF1 transgene.
[0016] In another aspect, the present invention provides the use of a human pluripotent stem cell derived erythroblastic island macrophage to increase enucleation of CD34+ HPC- derived erythroid cells in vitro or ex vi vo; optionally wherein the CD34+ H PC-derived erythroid cells are derived from the umbilical cord blood. Suitably, the human pluripotent stem cell derived erythroblastic island macrophage may be produced by a method of the invention.
[0017] In a further aspect, the present invention provides an in vitro or ex vivo method for producing enucleated erythroid cells, said method comprising:
a. culturing derived CD34+ haematopoietic progenitor cells with at least one of the following:
i. I L33 and, optionally one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL;
ii. population of human pluripotent stem cell derived macrophages in which the activity of KLF1 is increased; or
iii.the culture media of a population of human pluripotent stem cell derived macrophage in which the activity of KLF1 is increased.
[0018] Suitably, the CD34+ haematopoietic progenitor cells may be umbilical cord derived CD34+ haematopoietic progenitor cells. Suitably, the CD34+ haematopoietic progenitor cells may be derived from iPSCs, as shown by Lopez-Yrigoyen, et al. in Nature Communications 2018 Feb 20;10(1):881.
[0019] Suitably, the population of human pluripotent stem cell derived macrophages may be human pluripotent stem cell derived macrophage produced a method of the invention or the population may comprise a human pluripotent stem cell derived macrophage of the invention. [0020] Suitably, the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone prior to step a. Suitably, the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone for at least 11 days.
[0021] Suitably, the culturing step a, may be carried out for at least 1 1 days.
[0022] The present invention further provides an in vitro or ex vivo model for studying erythropoiesis, said model comprising a combination of a human pluripotent stem cell derived erythroblastic island macrophage and umbilical cord blood derived CD34+ haematopoietic progenitor cells.
[0023] Suitably, the human pluripotent stem cell derived erythroblastic island macrophage may be an induced pluripotent stem cell derived erythroblastic island macrophage.
[0024] In another aspect, the present invention provides IL-33 for use in the treatment of dyserythropoietic anaemia in a subject.
[0025] Suitably, IL-33 may be used in combination with one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
[0026] In a further aspect, the present invention provides a method of treating a subject having dyserythropoietic anaemia, said method comprising administering a therapeutically effective amount of IL33.
[0027] Suitably, the method of treating a subject having dyserythropoietic anaemia may further comprising administering a therapeutically effective amount of one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
[0029] Figure 1 shows that AAVS1 -targeted KLF1 transgene was expressed in iPSC- DM. (A) Expression of ΈG macrophage related transcription factors (c-MAF and KLF1) by qRT-PCR analyses (n=5, Mann Whitney Test); (B) Differentiation protocol used to generate macrophage from an iPSC cell line (iKLF1.2) carrying the AAVS1 -targeted KLF1- ERT2 transgene; (C) Kwik-Diff stained cytospin preparations of iKLF1.2-derived macrophages stained with line (Scale bar, 20mhi); and (D) qRT-PCR analysis of KLF1 in iPSC-DM derived from control (SFCi55) and iKLF1.2 iPSC lines (n=4 Kruskal- Wallis Test with Dunn’s correction for Post-test) [*p<0.05, **p<0.01 , ***p<0.001 ,
****p<0.0001]. [0030] Figure 2 shows that activation of KLF1 in iPSC-DM up-regulated El related markers and enhanced phagocytic activity. (A) qRT-PCR analyses of El macrophage related genes in iPSC-DM derived from iKLF1.2 cells in the presence and absence of tamoxifen (Tam)(n=4, non-parametric Wilcoxon Test); (B) Flow cytometry analyses of El- related cell surface marker expression in control and iKLF 1 -derived- iPSC-DMs in the presence and absence of tamoxifen (n=4, non-parametric Kruskal Wallis Test and Dunn’s Post-test); (C) Mean fluorescence intensity (MFI) of cell surface marker expression in parental and iKLF1.2-derived iPSC-DMs in presence and absence of tamoxifen (n=4, non- parametric Kruskal Wallis Test and Dunn’s Post-test); (D) Images captured at 175 minutes after addition of Zymosan-green beads to control and iKLF1.2-iPSC-DMs cells in the presence and absence of tamoxifen. (40X objective); (E) Phagocytic fraction analyses as measured by the proportion of phagocytic cells from 0-175 hours in control and iKLF 1 iPSC- DMs (+/- tamoxifen); and (F) Phagocytic index as calculated by level of fluorescence per cell green (n=5, two-way ANOVA and Bonferoni post-test) [*p<0.05, **p<0.01 , ***p<0.001 ,
****p<0.0001]
[0031] Figure 3 shows that maturation and enucleation of UCB CD34+ derived erythroid cells were enhanced when co-cultured with KLF1 -activated macrophages. (A/B) Flow cytometry analyses of live CD235a+-gated cells (day 14) of UCB CD34+ -derived erythroid cells cultured alone or in co-culture with iPSC-DM in the presence and absence of tamoxifen (Tam) at day 14 (A) and day 21 (B) stained with anti-CD71 antibody and Hoechst dye (see Figure 6 for gating strategy and FMO controls); (C) Quantification and statistical analysis of replicate co-culture experiments (n=4; 2-way ANOVA with Tukey’s post test); (D) Cytospin of co-culture of KLF1 expressing iPSC-DM and UCB CD34+ cells showing close association and a fully mature erythroid cell with biconcave shape (arrow) (scale bar, 20mhi) and (E) Cytospins of UCB CD34+ cells (from left to right) cultured alone, alone plus tamoxifen, with iPSC-DM, with iPSC-DM and tamoxifen (KLF1 activation); arrows point to enucleated cells (E) Cytospins of UCB CD34+ erythroid cells cultured alone or in co-culture with iPSC-DM in the presence and absence of tamoxifen (Tam) at day 14 (upper panels) and day 21 (lower panels) (day 21); arrows point to enucleated cells (scale bar, 20mhi) [*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001].
[0032] Figure 4 shows that KLF1 activation enhanced the effects of iPSC-DMs in a paracrine manner. A. Flow cytometry analyses of live CD235a+ cells with CD71 and Hoechst staining of UBC-CD34+ cells that were differentiated alone or in the presence of iKLF1-derived macrophages that were separated in a transwell assay -/+ tamoxifen (n=4; 2 way ANOVA with Tukey’s post-test). B. qRT-PCR validation of genes encoding for three secreted factors ANGPTL7, IL33 and SERPINB2 in iKLF1.2-derived iPSC-DMs in the presence and absence of tamoxifen (n=5 Wilcoxon matched paired signed ranked test). C. qRT-PCR analyses of cytokine-(ANGPTL7, IL33 and SERPINB2) related receptors or known targets in UCB CD34+ cells at day 14 of erythroid differentiation with and without the addition of all three cytokines, (n=4, One way ANOVA with Sidak's Multiple Comparisons). D. Flow cytometry analyses of live CD235a+ cells with CD71 and Hoechst staining of UBC-CD34+ cells that were differentiated in control conditions (-3 secreted factors) and in the presence of ANGPTL7, IL33 and SERPINB2 (+3 secreted factors), at days 1 1 , 14 and 21 (n=5, 2 Way ANOVA with Dunnette’s post-test). E. As above with conditions where each cytokine was excluded then analysed at days 1 1 , 14 and 21 (n=5, 2 Way ANOVA with Dunnette’s post-test). F. Flow cytometry analyses of live CD235a+ cells with CD71 and Hoechst staining of UBC-CD34+ cells that were differentiated in the absence and presence of IL33 alone at day 1 1 and 14 (n=4, 2 Way ANOVA).[*p<0.05,
**p<0.01 , ***p<0.001 , ****p<0.0001
[0033] Figure 5 shows (A) Immunohistochemistry of iPSC-DM from control (SFCi55) or iKLF1.2 iPSCs in the presence and absence of tamoxifen using an anti-HA antibody that detects the HA-KLF1-ER 2 fusion protein. The insert showed higher magnification demonstrating fusion protein subcellular localisation in cytoplasm in absence of tamoxifen and in the nucleus after tamoxifen addition; and (B) Expression of some known KLF1 target genes in control iPSC in the presence and absence of tamoxifen demonstrating that they are not regulated by tamoxifen along and that the genes upregulated in iKLF1.2-iPSCS (Figure 2A) are indeed due to KLF1 activation.
[0034] Figure 6 shows the Gating strategy for analysis of erythroid maturation. Single, live cells that were CD235a+ were gated, then analysed for the expression of CD71 and Hoechst DNA stain (fluorescence minus one (FMO) controls are shown). The positive control is peripheral blood showing fully mature, CD7T, enucleated red blood cells.
[0035] Figure 7 shows that maturation and enucleation of UCB derived CD34+ erythroid cells was enhanced when KLF 1 -activated macrophages were cultured in a transwell culture where contact was inhibited. (A/B) Representative Flow cytometry analyses of live CD235a+- gated cells (day 14) of UCB CD34+ erythroid cells cultured alone or in co-culture with iPSC- DM in the presence and absence of tamoxifen (Tam) at day 14 (A) and day 21 (B) stained with anti-CD71 antibody and Hoechst dye (see Figure 6 for gating strategy and FMO controls and Figure 4A for quantification); (C) Cytospins of UCB CD34+ cells (from left to right) cultured alone, alone plus tamoxifen, with iPSC-DM, with iPSC-DM and tamoxifen (KLF1 activation); arrows point to enucleated cells (E) Cytospins of UCB CD34+ erythroid cells cultured alone or in co-culture with iPSC-DM in the presence and absence of tamoxifen (Tam) at day 14 (upper panels) and day 21 (lower panels) (day 21); arrows point to enucleated cells (scale bar, 20pm).
[0036] Figure 8 shows that KLF1 activation in iPSC-DM up-regulates El related markers and enhances phagocytosis in a number of independently derived KLF1-ERT2-expressing iPSC lines. Quantitative RT-PCR analyses of El-macrophage related genes in macrophages derived from 3 independently generated iPSC-lines (iKLF 1.6, iKLF1.7 and iKLF 1.12) carrying the CAG-KLF1-ER 2 transgene in the AAVS1 locus in the presence and absence of tamoxifen (Tam)(n=4 biologically independent samples, non-parametric Wilcoxon Test)(A). Phagocytic fraction analyses (measured by the proportion of macrophages containing fluorescent beads) from 0 to 210 minutes in control iPSC-DMs and iKLF1-DMs (iKLF 1.6, iKLFI .7 and iKLF 1.12) in the presence or absence of tamoxifen, (n=5 biologically independent samples, two-way ANOVA and Bonferoni post-test) (B). Phagocytic index (calculated by level of green fluorescence per cell) in control and iKLF 1 iPSC-DM (iKLF 1.6, iKLF1.7 and iKLF1.12), in the presence or absence of tamoxifen (n=5 biologically independent samples, two-way ANOVA and Bonferoni post-test). [*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001] (C).
[0037] Figure 9 shows that KLF1 activation and IL10 activation are additive. Images were captured at 240 minutes after addition of Zymosan-green beads to iKLF1.2-iPSC-DMs cells in the presence and absence of tamoxifen and/or IL10 (40X) (A). Phagocytic fraction analyses as measured by the proportion of phagocytic cells from 0-240 minutes in iKLF1.2 iPSC-DMs in the presence and absence of tamoxifen and/or IL10 (n=5, two-way ANOVA and Bonferoni post-test) [*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001] (B).
[0038] Figure 10 shows the proportion of enucleated cells (CD71-/Hoechst-) UCB cells at 11 , 14 and 21 days in erythroid differentiation conditions in basal media or upon the addition of IL33, SerpinBI and Angptl7 (3 cytokines) or IL33, SERPINB1 , ANGPTL7, NRG1 , NOV, IGFBP6, CCL3 and TRAIL (8 cytokines) (n=4, 2 Way ANOVA with Dunnette’s post-test)) [*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001]
[0039] Figure 11 shows the proportion of enucleated cells (CD71-/Hoechst-) UCB cells at 11 , 14 and 21 days in erythroid differentiation conditions in basal media or upon the addition of IL33, SerpinBI and Angptl7 or IL33, SERPINB1 , ANGPTL7, NRG1 , NOV, IGFBP6, CCL3 and TRAIL (8 secreted factors) and in conditions where individual factors are removed. (n=4, 2 Way ANOVA with Dunnette’s post-test) [*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001]
[0040]
DETAILED DESCRIPTION The present invention provides an in vitro or ex vivo method of producing a population of human pluripotent stem cell derived macrophages with an altered phenotype, the method comprising modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population.
[0041] “Human pluripotent stem cell derived macrophages” as used herein include macrophages produced from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The maturation of macrophages from such cells can be detected by the presence of mature macrophage specific markers. For example, a human pluripotent stem cell derived macrophages will be positive for the human macrophage specific 25F9 (human macrophage specific) and, optionally, CD11 b.
[0042] Various methods of producing macrophages from pluripotent stem cells such as ESCs and iPSCs are known in the art, see Yeung et ai. 2012 (“Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function”. Sc. Rep. 2015 Mar 10;5:8909. doi: 10. 1038/srep08908), Zhuang et ai. 2012 (“Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance.” J Immunol Methods. 2012 Nov 30;385(1 -2): 1 -14. doi: 10.1016/j.jim.2012.06.008. Epub 2012 Jun 18.), Sneju et ai. (“Application of iPS cell-derived macrophages to cancer therapy” Oncoimmunology. 2014; 3: e27927), Hale et ai. (“Induced Pluripotent Stem Cell Derived Macrophages as a Cellular System to Study Salmonella and Other Pathogens” PLOS, http://dx.doi.org/10.1371/journal.pone.0124307), Zhang et ai. (“Functional Analysis and Transcriptomic Profiling of iPSC-derived Macrophages and Their Application in Modeling Mendelian Disease” Circ Res. 2015 Jun 19;117(1):17-28) Mucci et ai. (“Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency” Stem Cell Reports^ 2016 Aug 9;7(2):292-305.) and van Wigenburt et al. , (“Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly- Defined and Fully-Defined Conditions” PLOS ONE 8(8): e71098. doi: 10.1371/journal. pone.0071098). Suitably, any method for generating human iPSC-DMs may be used. For example, one method which may be used in accordance with the invention for generating human iPSC-DMs is provided in Haideri, S.S., et al., Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury. Regenerative Medicine, 2017. 2 (article 14).
[0043] Suitably, the method of generating human iPSCs may be that detailed in the Examples section of the patent.
[0044] ESC derived macrophages (ESDMs) may be generated by culturing the ESCs in the presence of colony stimulating factor-1 (CSF-1) (also known as M-CSF) and IL-3 to form embryoid bodies (EB). Whilst EBs adhere to tissue culture plastic, macrophage progenitor cells are non-adherent and thus are released into the medium. The macrophage progenitor cells may then be harvested at various time points, for example after 10 or 20 days and plated onto non-treated Petri dishes and cultured in the presence of CSF-1 alone. This process can give rise to monocyte-like cells that adhere to the plastic forming a monolayer and mature into ESDM. The maturation of the ESC into ESDM can be monitored by detecting the presence of mature macrophage specific markers F4/80 (mouse macrophage specific) or 25F9 (human macrophage specific) and CD1 1 b. Advantageously, the method described yields a substantially homogenous population of ESDMs. Suitably, ESDMs for use in the invention may be human ESCs and hence the marker 25F9, optionally in combination with CD1 1 b can be used to determine maturation into ESDMs.
[0045] Alternatively, macrophages may be derived from iPSC. Suitably, the method for differentiation of iPSCs to macrophages may involve supplementing culture medium with a cytokine Mix 1 (comprising bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF) and stem cell factor (SCF)). Cells may be cut, dislodged, divided and re cultured in fresh media supplemented with cytokine mix 1. Cells may be cultured in suspension for 3 days with a cytokine top up on Day 2, to form EBs. The EBs may then be transferred to media supplemented with cytokine Mix 2 (comprising M-CSF, IL3, Glutamax, Penicillin/Streptomycin and b-mercaptoethanol). EBs can be maintained in this medium for the remaining duration of the protocol, with spent medium being replaced with fresh medium every 3-4 days. After about 2 weeks, the EBs produced macrophage progenitors in the culture supernatant that were harvested and transferred to medium supplemented with cytokine Mix 3 (M-CSF, Glutamax, Penicillin/Streptomycin) and allowed to mature into iPSC- derived macrophages (iPSC-DM). Macrophage progenitors may continue to be harvested twice a week for approximately 2 months.
[0046] Preferably, the iPSC-derived macrophages (iPSC-DM) are from human iPSCs.
[0047] If desired, the resulting ESDMs or iPSC-DM may be subsequently polarised in vitro to adopt either a M 1 -like phenotype by treatment with LPS and IFNy. Alternatively, ESDMs or iPSC-DM may be polarised to yield Alternatively Activated Macrophage with IL-4. Alternatively, ESDMs may be polarized to yield AAMs with IL-4, IL-13 and CSF-1. M1 -like polarised macrophages are also known as classically activated macrophages (CAM). AAM derived from ESDMs may be characterised by their high expression of Chil3 (Ym1), Retnla (Fizz), Mrd (Mannose Receptor 1), and Arg1 (Arginase). In contrast, M1 -like polarised macrophages (such as ESDM, BMDM or iPSC-DM) may be characterised by a significant increase in NO production and increased gene expression of iNos and Cd86. [0048] Suitably, the human pluripotent stem cell derived macrophages may not be polarised, such macrophages may be referred to as“naive”.
[0049] Various ways of generating human pluripotent stem cell derived macrophages are known in the art and the methods of the invention contemplate the use of any such macrophages.
[0050] By“phenotype” it is meant an observable set of characteristics resulting from the interaction of the genotype of the cell with the environment. In the context of the method of the invention, modulating the activity of KLF1 will alter the phenotype of the population of human pluripotent stem cell derived macrophages. The invention has surprising found that modulation of expression of the transcription factor KLF1 will alter the phenotype of human pluripotent stem cell derived macrophages.
[0051] Advantageously, activation (i.e. increasing the level of activity of KLF1) can alter the phenotype of a human pluripotent stem cell derived macrophage to that of a human pluripotent stem cell derived erythroblastic island macrophage.
[0052] A“pluripotent stem cell derived erythroblastic island (El) macrophage” may be defined as a pluripotent stem cell derived macrophage in which one or more El markers are significantly increased. For example, a population of “pluripotent stem cell derived erythroblastic island (El) macrophages” may have increased levels of at least one of the following markers following activation of KLF1 : CD 163, CD 169, CD206 and CCR5.
[0053] Any known method may be used to increase the activity of KLF1. Suitably, one way is to increase the level of KLF1 present in the macrophages.
[0054] By“increased levels” it is meant that at least one of the following is increased: the percentage of cells within the population expressing the marker of interest; the level of the marker on the surface of the cells within the population is significantly increased; or the mRNA expression of the marker is increased.
[0055] Any known method may be used to determine the percentage of cells within a sample of the population that express a desired marker such as flow cytometry analysis.
[0056] The presence, level or absence of a marker polypeptide or nucleic acid molecule (e.g. mRNA) in a population of macrophages can be determined by contacting the sample population with a compound or an agent capable of specifically detecting (e.g. specifically binding) the specific marker polypeptide or nucleic acid molecule.
[0057] Routine methods may be used to obtain sample from a cell population. For example, by immersing the cell population in a buffer for extracting protein or mRNA. [0058] The level of any specific marker in a cell population can be can be measured in a number of ways, including: measuring the mRNA that encodes the protein marker; measuring the amount of protein marker; or measuring the activity of the protein biomarker.
[0059] Any known mRNA detection method may be used to detect the level of mRNA of a marker of interest (e.g. CD163, CD169, CD206 or CCR5) in a sample.
[0060] For example, the level of a specific mRNA in a sample can be determined both by in situ and by in vitro formats. mRNA may be detected using Northern blot analysis, polymerase chain reaction, probe arrays or RNA sequencing. In one embodiment, a sample may be contacted with a nucleic acid molecule (i.e. a probe, such as a labeled probe) that can specifically hybridize to the specific mRNA of the marker of interest (e.g. e.g. CD163, CD169, CD206 or CCR5). The probe may be, for example, a complement to a full-length nucleic acid molecule, or a portion thereof, such as a nucleic acid molecule of at least 10, 15, 30, 50, 100, 250 or 350 nucleotides in length and which specifically hybridizes under stringent conditions to specific mRNA of interest.
[0061] The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies. Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide binding complex, as taught in Berger and Immel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below. Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences. In a preferred aspect, the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g. 50°C and 0.2xSSC). In a more preferred aspect, the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringency conditions (e.g. 65°C and O.lxSSC).
[0062] Alternatively, the level of a specific mRNA in a sample may be evaluated with nucleic acid amplification, for example by RT-PCR, ligase chain reaction, self-sustained sequence replication, transcriptional amplification or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art including RNA sequencing.
[0063] Suitably, the level of at least one of CD163, CD169, CD206 and CCR5 may be measured by RT-PCR analysis. Suitably, following an increase in KLF1 , the hPSCs have an altered phenotype having a significant increase in at least CD163, CD169, CD206 and CCR5 compared to the hPSCs prior to modulation of KLF1.
[0064] Suitably, the altered phenotype may be one in which the level of at least 2 of or at least 3 or all of the CD163, CD169, CD206 and CCR5 is increased in a sample of the hPSCs following activation with KLF1 (i.e. following increasing the activity of KLF1).
[0065] Any known protein detection method may be used to detect the level of protein of a marker of interest (e.g. CD163, CD169, CD206 and CCR5) in a sample.
[0066] Generally, protein detection methods comprise contacting an agent that selectively binds to a protein, for example an anti-CD163 an anti-CD169, an anti-CD206 or an anti- CCR5, with a sample to determine the level of the specific protein in the sample. Preferably, the agent or antibody is labeled, for example with a detectable label. Suitable antibodies may be polyclonal or monoclonal. An antibody fragment such as a Fab or F(ab')2 may be used.
[0067] As used herein the term "labeled", refers to direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance.
[0068] The level of a specific protein marker in a sample may be determined by techniques known in the art, such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (BIA), radioimmunoassay (RIA), Western blot analysis, Flow cytometry and Lateral Flow Devices (LFDs) utilizing a membrane bound antibody specific to the protein biomarker. Alternatively, the level of a specific biomarker protein in a sample can be detected and quantified using mass spectrometry. Such methods are routine in the art.
[0069] Methods of the invention may further comprise comparing the level or activity of the at least one marker in the test sample (i.e. a sample of hPSC derived macrophages following modulation of the activity of KLF1) with the level or activity of the at least one biomarker in a control sample (i.e. a sample of the hPSC derived macrophages prior to the modulation of the level of KLF1).
[0070] In one embodiment, methods of the invention include contacting a control sample with a compound or agent capable of detecting a specific biomarker mRNA (e.g. CD163, CD169, CD206 and/or CCR5), and comparing the level of the biomarker mRNA in the control sample with the level of biomarker mRNA in the test sample.
[0071] In another embodiment, the methods of the invention include contacting the control sample with a compound or agent capable of detecting a specific biomarker protein (e.g. CD163, CD169, CD206 and CCR5), and comparing the level of the biomarker protein in the control sample with the presence of the biomarker protein in the test sample.
[0072] As used herein "reference level" or“control”, refers to a hPSC derived macrophage sample having a normal level of biomarker (e.g. CD163, CD169, CD206 and CCR5) expression which would equate to the typical level of the biomarker hPSC derived macrophage population of the same type prior to modulation of KLF1.
[0073] Alternatively, the reference level may be comprised of a biomarker expression level from a reference database, which may be used to generate a pre-determined cut off value, i.e. a score that is statistically predictive of a significant alteration of the maker of interest.
[0074] Suitably, the control sample or reference sample is obtained using the same method as the method used to obtain a test sample. Alternatively, or in addition, the control sample or reference sample is normalized as discussed below.
[0075] Alternatively, predictions may be based on the normalized expression level of the specific biomarker. Expression levels are normalized by correcting the absolute expression level of the biomarker in a sample by comparing its expression to the expression of a reference nucleic acid that is not a marker, e.g., an mRNA or protein that is constitutively expressed. This normalization allows the comparison of the expression level in one sample to another sample, or between samples from different sources. This normalized expression can then optionally be compared to a reference standard or control.
[0076] In one embodiment, the level of the biomarker or biomarkers of interest in the test sample is increased by at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1 , at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.1 , at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4.0, at least 4.1 , at least 4.2, at least 4.3, at least 4.4, at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5.0 fold compared to the control sample or predetermined reference sample. In one embodiment, 1 the level of the at least one biomarker in the test sample is increased by at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 5 fold, at least 7.5 fold, at least 10 fold, at least 15 fold etc compared to the control sample or predetermined reference level. [0077] By“significantly increased” it is meant a statistically significant increase calculated used a mean +/- standard error mean with an increase being considered statistically significant where the p-value is less than 0.05.
[0078] Suitably, the altered phenotype may be one in which the level of at least one of CD163, CD169, CD206 and CCR5 the level of mRNA is increased at least two fold or at least 3 fold or at least 4 fold or at least 5 fold.
[0079] Suitably, the altered phenotype may be one in which percentage of hPSCs in a sample which express at least one of CD163, CD169, CD206 and CCR5 is significantly increased and/or is increased by at least 5% or at least 6% or at least 6% or at least 7% or at least 10%.
[0080] Suitably, the altered phenotype may be one in which the mean fluorescence intensity of at least one of CD163, CD169, CD206 and CCR5 in a sample is significantly increased and/or is increased by at least 500 or at least 600 or at least 750 or at least 1000.
[0081] Suitably, the altered phenotype may be one which comprises enhanced phagocytosis. Various methods for determining phagocytosis are known in the art. One preferable method is the phagocytosis assay as detailed in the Examples. Using this assay, the number of iPSC-DMs that ingest beads (the phagocytic cell fraction) and the average number of beads that each iPSC-DM has ingested (phagocytic index) can be measured.
[0082] Suitably, an altered which has enhanced phagocytosis may be one in which the phagocytic fraction after a set time period such as 175 minutes is significantly increased (e.g. by at least 0.05 or at least 0.1 or at least 0.2) and/or the phagocytic index after a set time period such as 175 minutes is significantly increased (e.g. by at least 5 or at least 10 or at least 20).
[0083] Suitably, the method of the invention may increase the level of one or more further markers of interest. For example, the method of the invention may increase the level of one or more markers selected from the group consisting of: CD11A, CD1 1 B, CD64, TNFa and PECAM1.
[0084] Suitably, the increase in the levels of any of the further markers may be measured in accordance with the methods provided for CD163, CD169, CD206 and CCR5. Suitably, the increase in the levels of any of the further markers may be measured by mRNA expression levels (such as by rtPCR).
[0085] Suitably, the hPSC derived macrophages used in the method of the invention may be iPSC derived macrophages. [0086] Suitably, the method of the invention may comprise the step of providing the human pluripotent stem cell derived macrophages with IL-10. The inventors have surprisingly found that modulation of KLF1 activity in combination with the provision of IL-10 increases the alteration of phenotype in the hPSC derived macrophages beyond that obtained by the modulation of KLF1 activity or provision of IL-10 on their own. Suitably, the modulation of KLF1 activity in combination with the provision of IL-10 increases the phagocytic activity of the hPSC derived macrophages.
[0087] It will be appreciated that IL-10 provided to the macrophages may be allogenic or autologous.
[0088] Suitably, allogenic IL-10 may be provided to the macrophages by contacting the macrophages with IL-10. The macrophages may be contacted with IL-10 by being cultured in the presence of IL-10.
[0089] Suitably, autologous IL-10 may be provided to the macrophages by increasing the production of IL-10 by the macrophages, for example by increasing the expression of IL-10. Methods for increasing the expression of IL-10 will be known to those skilled in the art.
[0090] Suitably, IL-10 may be provided to the macrophages simultaneously with modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population. Alternatively or additionally, IL-10 may be provided to the macrophages sequentially to modulating the activity of KLF1. When IL-10 is provided sequentially, it may be provided before or after KLF1 activity has been modulated. Suitably, IL-10 may be provided after KLF1 activity has been modulated.
[0091] Suitably, IL-10 may be provided to the macrophages at a concentration of at least 1 ng/ml, at least 2 ng/ml, at least 3 ng/ml, at least 4 ng/ml, at least 5ng/ml, at least 6 ng/ml, at least 7ng/ml, at least 8ng/ml, at least 9 ng/ml, at least 10ng/ml, or more. More suitably, IL-10 may be provided at a concentration of at least 5 ng/ml.
[0092] Suitably IL-10 is provided to the macrophages at a concentration of 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5ng/ml, 6 ng/ml, 7ng/ml, 8ng/ml, 9 ng/ml, 10ng/ml, or more. More suitably, IL-10 may be provided at a concentration of 5ng/ml.
[0093] Suitably IL-10 is provided to the macrophages at a concentration of between 1 and 10 ng/ml, 2 to 9 ng/ml, 3 to 8 ng/ml, or 4 to 7 ng/ml.
[0094] Throughout the description and claims of this specification, the words“comprise” and“contain” and variations of them mean“including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0095] Various methods for modulating the expression of gene are known in the art. Suitably, the methods of invention may activate or increase expression of KLF 1. Any method of gene activation of KLF1 may be used included CRISPR activation methods.
[0096] Suitably, the activation of KLF1 may be via a transgene encoding KLF1. By “transgene” it is meant a gene encoding KLF1 which has either been transferred by e.g. genetic engineering techniques to change the phenotype of an organism.
[0097] Suitably, the method of the invention may comprise incorporating a transgene into a population of hPSC derived macrophages (iPSC-DM). Suitably, the transgene may be integrated into the safe harbour AAVS1 locus of the cells. KLF1 may be under the control of a constitutive promoter or expression of KLF1 may be controlled such as through the use of an inducible transgene. Whilst the Examples utilise a tamoxifen inducible transgene, the skilled person is readily aware of various ways to provide induced expression of KLF1. For example, suitably the transgene may be an inducible fusion transgene whereby KLF1 is fused to, for example, an ERT2 domain and the activity can be induced by addition of tamoxifen or similar agent.
[0098] Suitably, in addition or in the alternative, the method may comprise CRISPR activation (CRISPRa). Various ways of using CRISPRa to increase the activity levels of KLF1 are known in the art. For example, Fidanza, A., Lopez-Yrigoyen, M., Romano, N., Taylor, H. & Forrester, L.M. (2017) An all-in- one UniSam vector system for efficient gene activation. Scientific Reports 7: 6394 discloses one way of using CRISPRa to activate the expression of KLF1.
[0099] The present invention further provides use of a human pluripotent stem cell derived macrophage to increase enucleation of CD34+ (e.g. umbilical cord derived CD34+) erythroid cells in vitro or ex vivo. The present inventors have surprising found that the maturation and enucleation of CD34+H PC-derived erythroid cells are enhanced by co-culture with KLF-1 activated macrophages - see Figure 3 and the section entitled“activation of KLF1 in iPSC- DMs enhanced maturation of erythroid cells in the Examples. It can be seen that co-culture with KLF1 activated iPSC-DMs surprisingly increased the average baseline level of enucleation from 22% to 55% at day 14 and from 30% to 73% at day 21.
[00100] The present invention further provides an in vitro or ex vivo method for producing enucleated erythroid cells wherein the method comprises: culturing CD34+ (e.g. umbilical cord derived CD34+) hematopoietic progenitor cells with at least one of the following: i) a population of human pluripotent stem cell derived macrophages in which the activity (e.g., level) of KLF1 is increased; or ii) the culture media of a population of human pluripotent stem cell derived macrophages in which the activity (e.g., level) of KLF1 is increased; or iii) IL33 and optionally, one or more of selected from the group consisting of ANGPTL7,SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
[00101] It has been surprisingly shown that co-culture of CD34+ (e.g. umbilical cord derived CD34+) hematopoietic progenitor cells with a population of human pluripotent stem cell derived macrophages in which the level of KLF1 is increased significantly enhances the maturation and enucleation of the erythroid cells - see Figure 3.
[00102] Furthermore, it has been surprisingly shown that activation of KLF1 in iPSC-DMs enhances terminal maturation and enucleation in part by a paracrine mechanism - see the Examples and Figure 4a.
[00103] An“enucleated erythroid cell” as used herein refers to an erythroid cell which is CD235a+, CD7T, and does not stain with the DNA dye Hoescht.
[00104] Suitably, the CD34+ haematopoietic progenitor cells may be umbilical cord derived CD34+ haematopoietic progenitor cells
[00105] Suitably, the population of human pluripotent stem cell derived macrophages may be human pluripotent stem cell derived macrophages produced a method of the invention or the population may comprise a human pluripotent stem cell derived macrophages of the invention.
[00106] Suitably, the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone prior to step a. Suitably, the CD34+ haematopoietic progenitor cells may be cultured with SCF, EPO, IL3 and hydrocortisone for at least 11 days or at least 12 days or at least 13 days or at least 14 days.
[00107] Suitably, the culturing step a, may be carried out for at least 11 days or at least 12 days or at least 13 days or at least 14 days.
[00108] Suitably, the method may result in a statistically significant increase in the number of enucleated erythroid cells when compared to a control of CD34+ (e.g. umbilical cord derived CD34+) hematopoietic progenitor cells cultures under identical conditions in the absence of: i) a population of human pluripotent stem cell derived macrophages in which the activity (e.g., level) of KLF1 is increased; and ii) the culture media of a population of human pluripotent stem cell derived macrophages in which the activity (e.g. level) of KLF1 is increased; and iii) IL33 and optionally, one or more of selected from the group consisting of ANGPTL7,SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL. [00109] Suitably, the average baseline level of enucleation may an increase of at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%, or more as compared to the control.
[00110] The present invention further provides an in vitro or ex vivo model for studying erythropoiesis, said model comprising a combination of a human pluripotent stem cell derived erythroblastic island macrophage and umbilical cord blood derived CD34+ haematopoietic progenitor cells.
[00111] Suitably, the human pluripotent stem cell derived erythroblastic island macrophage may be an induced pluripotent stem cell derived erythroblastic island macrophage.
[00112] The present invention further provides IL-33 for use in the treatment of dyserythropoietic anaemia in a subject and/or a method of treating a subject having dyserythropoietic anaemia, said method comprising administering a therapeutically effective amount of IL33. Suitably, the method may further comprise administering a therapeutically effective amount of one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
[00113] By “dyserythropoietic anaemia” it is meant anaemia, characterized by ineffective erythropoiesis, and resulting from a decrease in the number of red blood cells (RBCs) in the body and a less than normal quantity of haemoglobin in the blood. Suitably, the anaemia may be the result of mutation in KLF1.
[00114] There is various literature sources known in the art which linked mutations in KLF1 with certain types of anaemia. For example, see “Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anaemia type IV in a patient of Taiwanese origin: review of all reported cases and development of a clinical diagnostic paradigm” (Jafray JA et al, Blood Cells Mol Dis. 2013 Aug; 51 (2):71 -5, doi: 10.1016/j.bcmd.2013.02.006. Epub 2013 Mar 20.); and“A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia” (Arnaud L et al. Am J Hum Genet. 2010 Nov 12;87(5):721-7. doi: 10.1016/j.ajhg.2010.10.010. Epub 2010 Nov 4).
[00115] The present invention has surprisingly shown a significant reduction in the enucleation rate at all stages in the absence of IL33. IL33 belongs to IL1 superfamily that can apparently act as a cytokine or as a nuclear factor within the cell [29] It has been implicated in inflammation, wound healing and various disease processes. Interestingly IL33 is expressed in erythroid progenitor cells and released during haemolysis. Without wishing to be bound by theory, it is postulated that this then stimulates the maturation of new RBCs [30] Thus, administration of IL33 may aid maturation of RBCs in a subject suffering from dyserythropoietic anaemia as a result of a mutation in KLF1.
[00116] Suitably, IL33 may be used in combination with one or more further cytokines. Suitably, IL33 may be used in combination with one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL to treat dyserythropoietic anaemia. Suitably, IL33 may be used in combination with ANGPTL7 and SERPINB2. Suitably, IL33 may be used in combination with ANGPTL7, SERPINB2 and NRG1. Suitably IL33 may be used in combination with ANGPTL7, SERPINB2 and IGFBP6. Suitably IL33 may be used in combination with ANGPTL7, SERPINB2 and CCL13. Suitably IL33 may be used in combination with ANGPTL7, SERPINB2 and TNFS10.
[00117] Suitably, IL33 and/or SERPINB2 may be used at a concentration of at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/ml, at least 100 ng/ml, at least 110 ng/ml, at least 120 ng/ml or more. More suitably, IL33 and/or SERPINB2 may be used at a concentration of at least 75 ng/ml.
[00118] Suitably, IL33 and/or SERPINB2 may be used at a concentration of 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 110 ng/ml, 120 ng/ml, or more. More suitably, IL33 and/or SERPINB2 may be used at a concentration of 75 ng/ml.
[00119] Suitably, IL33 and/or SERPINB2 may be used at a concentration of between 10 ng/ml and 120 ng/ml, 25 ng/ml and 100 ng/ml, or 70 and 80 ng/ml.
[00120] Suitably, ANGPTL7 may be used at a concentration of at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/ml, at least 100 ng/ml, or more. More suitably, ANGPTL7 may be used at a concentration of at least 60 ng/ml.
[00121] Suitably, ANGPTL7 may be used at a concentration of 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, or more. More suitably, ANGPTL7 may be used at a concentration of 60 ng/ml.
[00122] Suitably, ANGPTL7 may be used at a concentration of between 10 ng/ml and 100 ng/ml, 30ng/ml and 80ng/ml, or 55 and 65 ng/ml.
[00123] Suitably, NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml, at least 80 ng/ml, at least 90 ng/ml, at least 100 ng/ml, or more. More suitably, NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of at least 50 ng/ml. [00124] Suitably, NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, or more. More suitably, IL33 and/or SERPINB2 may be used at a concentration of 50 ng/ml.
[00125] Suitably, NRG1 , IGFBP6, CCL3 and/or TRAIL may be used at a concentration of between 10 ng/ml and 100 ng/ml, 25 ng/ml and 75 ng/ml, or 45 and 55 ng/ml.
[00126] As used herein, the terms“treat”,“treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a disorder or symptom. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted disorder (i.e. dyserythropoietic anaemia) or symptom.
[00127] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[00128] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
[00129] EXAMPLES
[00130] RESULTS
[00131] IPSC-DM express low levels of KLF1
The inventors assessed the expression of two transcription factors (MAF and KLF1) that play a role in macrophages associated with the erythroid island niche [21] (Figure 1A). The expression of C-MAF was expressed at a significantly higher level in iPSC-DMs compared in monocyte-derived macrophages. As MAF is also reported to be marker for yolk sac macrophages, this result supports the idea that the phenotype of iPSC-DMs is comparable to tissue resident macrophages [16, 17] KLF1 was expressed at lower levels in iPSC-DM compared to monocyte-derived macrophages (MDMs) (Figure 1A). The inventors hypothesised that enhancing the expression of KLF1 would direct iPSC-DMs into an El-like phenotype.
[00132] Generation of macrophages from the iKLF1.2 iPSC line
The inventors established an iPSC line carrying an inducible KLF1-ERT2 transgene targeted to the safe harbour AAVS1 locus, herein referred to as iKLF1.2 (Figure 1 B) [19] iPSC-DMs were generated in a stepwise protocol by first generating embryoid bodies (EBs) in the presence VEGF, BMP4 and SCF then transferred to gelatin-coated plates and cultured in presence of IL-3 and MCSF. From day 16, myeloid progenitor cells were harvested from the supernatant and cultured in MCSF for 9-12 days [17] Tamoxifen was added to iPSC-DMs for the last 4 days to activate KLF1.
[00133] IPSC-DMs derived from the iKLF1.2 iPSC line were comparable in size and morphology to those derived from the parental iPSC line, SFCi55 and the addition of tamoxifen had no obvious effect on these parameters (Figure 1 C). As expected, iPSC-DMs generated from the iKLF1.2 iPSC line demonstrated a high expression of KLF1 mRNA expression compared macrophages derived from monocytes or control iPSC-DMs (Figure 1 D). Using an anti-HA antibody the inventors demonstrated that the KLF1-ERT2 fusion protein is expressed in the cytoplasm of iKLF1.2 iPSC-DMs and translocates to the nucleus upon tamoxifen addition thus demonstrating that the fusion protein activation strategy can function and that the KLF1-ERT2 transgene is not silenced in differentiated iPSC-DMS (Figure 5A).
[00134] Effect of KLF1 is reproducible in iPSC-DMs derived from independent iPSC lines
To demonstrate the reproducibility of the method of the invention to alter the functional phenotype of macrophages, macrophages from additional three independently derived iPSC lines (iKLF 1.6, iKLF1.7 and iKLF1.12) carrying the CAG-KLF1-ER 2 transgene were tested. Activation of KLF1 in iPSC-DMs generated from all three cell lines resulted in an up- regulation of El-related genes: CD163, CD169, CD11A and CD1 1 B comparable to the result observed in iPSC-DMs generated from the iKLF1.2 line (Figure 8A) and enhanced phagocytic activity (Figure 8B, C).
[00135] Activation of KLF1 in iPSC-DMs up-regulates El related genes and cell surface markers, and enhances phagocytosis
The inventors assessed the effect of KLF1 activation on the mRNA expression of previously reported KLF1 target genes including VCAM1 , DNASE2A; cell adhesion molecules involved in macrophage-erythroblast interaction (ITGAV, EMP/MAEA, PECAM1 , CD163 and CD169); El macrophage markers (CD64, CD68, CD1 1a, CD1 1 b, CD1 1c) and extrinsic regulators of erythropoiesis (PALD, IFN-b and TNF-a) [10, 15, 21-26] Activation of KLF1 by the addition of tamoxifen resulted in the increased expression of a subset of these transcripts including CD163, CD169, CD11A, CD11 B, CD64, TNFa and PECAM 1 in macrophages derived from iKLF1.2 iPSCs (Figure 2A). These genes were not upregulated upon tamoxifen addition of iPSC-DMs derived from the control SFCi55 iPSCs confirming that this phenotype change was associated with KLF1 activation rather than an non-specific effect of tamoxifen (Figure 5B).
[00136] KLF1 activation increased the proportion of iPSC-DMs expressing El-associated markers (CD206, CD163, CD169 and CCR5) (Figure 2B) and on the level of expression of these markers on the cell surface as measured by the mean fluorescent intensity (MFI) (Figure 2C). IPSC-DMs expressed the marker 25F9 irrespective of tamoxifen treatment but the level of expression per cell was significantly higher after KLF1 activation (Figure 2B,C).
[00137] To assess whether activation of KLF1 altered iPSC-DM function, the inventors used a quantitative assay of phagocytic activity [27] As the pHrodoTM Green Zymosan A BioParticles fluoresce green when ingested into cells, the number of iPSC-DMs that take up the beads (phagocytic fraction) and the average number of beads per cell (phagocytic index) can be assessed by live cell imaging (Figure 2D). There was a gradual increase in the number of iPSC-DMs containing fluorescent beads over the time frame of the experiments in all iPSC-DM cell populations as previously reported [27] However, the increase in both phagocytic cell fraction and the phagocytic index in iKLF1-derived-iPSC-DMs that were treated with tamoxifen was significantly higher compared to control iPSC-DMs and to iKLF 1 - iPSC-DMs in the absence of tamoxifen (Figure 2E, F).
[00138] Activation of KLF1 in iPSC-DM enhanced terminal maturation of erythroid cells
Umbilical cord blood (UCB) derived CD34+ haematopoietic progenitor cells (HPC) enucleate in vitro but the efficiency is relatively low (23-40%) [28] The inventors hypothesised that the enucleation efficiency could be enhanced by co-culture with ΈI-like’ iPSC-DMs. CD34+ cord blood derived HPCs were cultured in SCF, EPO, IL3 and hydrocortisone for 7 days then at day 8 the cells were co-cultured with iKLF1.2 iPSC-DM in the presence or absence of tamoxifen. The proportion of fully mature, enucleated cells was determined at day 14 and 21 by assessing the number of CD235a-expressing erythroid cells that were negative for CD71 and the DNA dye, Hoechst [18] (Figure 6). The percentage of mature enucleated erythroid cells (CD235a+, CD71-, Hoechst-) was higher in cells that were co-cultured with macrophages and this was further increased when co-cultured with KLF1.2 derived macrophages that had been treated with tamoxifen (Figure 3A-B). Quantification of replicate experiments demonstrated that co-culture with iPSC-DMs in which KLF1 had been activated increased the average baseline level of enucleation from 22% to 55% at day 14 and from 30% to 73% at day 21 (Figure 3C). Morphological analysis by cytospin preparation of co cultured cells highlighted the close association between macrophages and differentiating erythroid cells (Figure 3D) and confirmed the increase in enucleation in a qualitative manner (Figure 3E).
[00139] Activation of KLF1 in iPSC-DM enhances terminal maturation and enucleation in part by a paracrine mechanism
To assess whether cell contact was required for the enhanced maturation and enucleation in iPSC-DM co-cultures, the inventors used a transwell assay where media and secreted factors could be exchanged but direct cell contact was inhibited. The transwell culture setup reduced the average baseline level of enucleation but the inventors noted a significant increase in the proportion of enucleated cells when KLF1 -activated iPSC-DMs were present on the other side of the transwell (Figure 4A, Figure 7). KLF1 -activated iPSC-DMs increased the proportion of enucleated cells from 12 to 30% and from 28 to 53% at day 14 and 21 , respectively. Although it was not possible to compare the absolute numbers of enucleated cells in contact and transwell cultures directly, the data suggests that both cell-cell contact and secreted factors are involved in mediating the effects of KLF1 activation. When contact was prevented the average proportion of CD71-, enucleated CD235a+ cells increased from 28 to 53% (Figure 4A) but in contact cultures that proportion was further increased to 73% (Figure 3C).
[00140] KLF1 up-regulates cell communication and protein binding associated genes in iPSC-DMs
To identify KLF1 target genes in iPSC-DMs that encode potential mediators of the observed effect on erythroid maturation, we carried out RNA sequencing of iKLF1.2 iPSC-DMs in the presence and absence of tamoxifen (Lopez-Yrigoyen, 2019). We selected genes encoding secreted factors (ANGPTL7, IL33 and SERPINB2) that were commercially available and that were validated to be upregulated by KLF1 activation (Figure 4B). To first assess whether differentiating UCB-CD34+ cells have the potential to be responsive to these selected cytokines we analysed the expression of their putative receptors (Figure 4C). Receptors for ANGPTL7 (NEURL1 and NEURL1), IL33 ( IL1R1 ) and SERPINB2 (PLAUR) were all expressed in differentiating (day 14) UCB-CD34+ cells and their expression level increased significantly when all three cytokines were added to the protocol (Figure 4C). In addition, upregulation of GATA3, a known downstream target of IL33 signaling, suggests that this pathway is functionally active. We next assessed the combined effect of ANGPTL7, IL33 and SERPINB2 on the differentiation and maturation of UCB-CD34+ cells, in absence of macrophages. When all three cytokines were included in the differentiation protocol, the proportion of CD7T enucleated cells was significantly increased compared to control cultures and this was apparent at all stages (day 11 , 14 and 21) of the differentiation protocol (Figure 4D). To assess the contribution of individual cytokines, we used an elimination strategy and assessed the proportion of CD7T enucleated cells at day 11 , 14 and 21 (Figure 4E). At day 11 , the proportion of CD7T enucleated cells was significantly decreased when IL33 was removed compared to cultures with all three cytokines (Figure 4E). We noted that the decrease was specifically due to the loss of CD71 expression at a stage prior to the enucleation process suggesting that this cytokine enhanced the timing of maturation. At day 14 and 21 , the highest proportion of CD7T enucleated cells was observed in cultures when all three cytokines were included and a significant reduction was observed when each one of the three cytokines was removed. Statistical analyses indicate that IL33 is the most important player because the effect of its removal at both days 14 and 21 was more significant (p=0.0009, p<0.0001) than the removal of either ANGPTL7 (p=0.0139, p=0.0194) or SERPINB2 (p=0.0035, p=0.0003) ( Figure 4E). Based on these findings we tested the effect of IL33 alone but surprisingly the proportion of CD7Tenucleated cells was comparable to control cultures with no secreted factors added (Figure 4F).
Furthermore, the inventors have shown that addition of five other factors, also upregulated factors (NRG1 , NOV, IGFBP6, CCL3 and TRAIL) could further increase the effects of IL33/ANGPTL7/SERPINB2 (Figure 10). Inclusion of all 8 factors resulted in the highest number of mature RBCs at the endpoint of the protocol (Day 21) but it was noted that inclusion of NRG1 , NOV, IGFBP6, CCL3 and TRAIL were detrimental to the enhancing effects of IL33, ANGPTL7 and SERPINB1 at earlier stages in the protocol. Without wishing to be bound to this hypothesis, the inventors propose that this might be due to an inhibitory effect of one or more of these cytokines at the early stages but clearly the system is highly complex and fine tuning the timing and concentration of cytokines will be required to unpick the complexity.
To define which one of these factors are critical we assess the RBC output under conditions where each one of the factors was removed. Removal of NRG1 , IGFBP6, CCL3 and TRAIL but not NOV reduced the enhancing effect indicating that these are important (Figure 11).
[00141] KLF1 and IL10 enhances macrophage phagocytosis
The inventors have surprisingly found that phagocytic activity of macrophages can be enhanced by the KLF1 and IL10 provided together. Since the combined treatment of macrophages with KLF1 and IL10 resulted in a greater increase in phagocytic activity than IL10 or KLF1 on their own, this suggests that phagocytosis induced by KLF1 and IL10 are mediated by different mechanisms.
[00142] DISCUSSION [00143] The production of RBCs in vitro could solve many of the problems associated with blood transfusion such as limitations in supply, transfusion transmitted infection, and immune compatibility. Indeed, culture conditions have been developed for the production of RBC in vitro from human CD34+ HPCs, PSCs and immortalised erythroid progenitor cell lines, known as BELA [4-9] However, regardless of the starting populations, culture conditions are relatively inefficient and a low proportion of the resultant cell populations undergo the enucleation process that marks the final steps of erythroid maturation and this has severely hampered clinical translation. Enucleation occurs in vivo within the El niche that consists of a central macrophage surrounded by up to 30 developing erythroblasts. Understanding the cellular and molecular interactions that take place within this niche will help in the development of protocols to generate mature RBCs.
[00144] Generation of El-like macrophages by genetic programming.
IPSC-derived macrophages have been reported to have a tissue resident-like phenotype [16, 17] and in-keeping with this the inventors demonstrate that MAF was expressed at a higher level in iPSC-DMs compared to monocyte derived macrophages (MDMs). The inventors noted that KLF1 was expressed at low level both iPSC-DMs and MDMs and, the inventors hypothesised that this transcription factor would programme macrophages into an El-like phenotype. The inventors used an inducible KLF1-ERT2 transgenes, targeted to the AAVS1 locus to allow conditional activation in differentiated IPSC-DMs. The inventors have previously shown that the production and function of macrophages from iPSCs is unaffected by AAVS1 targeting per se and is resistant to epigenetic silencing [27], providing an ideal platform for testing the specific effect of transcription factor activation. Activation of KLF1 in iPSC-DM increased the expression of some El-associated genes and cell surface markers. Interestingly not all KLF1 target genes are activated in this system which can be explained by the fact that transcriptional control by KLF1 is context dependent, involving a number of protein partners that will be differ between cell types. The inventors demonstrate that KLF1- activated iPSC-DMs have an enhanced rate of phagocytosis which is in keeping with fact that El macrophages are reported to be more phagocytic. This function has likely evolved to clear free nuclei.
[00145] In co-culture experiments, KLF1 -activated El-like macrophages increased the production of fully mature and enucleated erythroid cells from UCB-CD34+ HPCs, consistent with the report of an extrinsic role for KLF1 in the murine system [20, 21]
[00146] The inventors demonstrate that cell contact is not absolutely required for the increase in enucleation observed, indicating that the action of KLF1 target genes is in part mediated by secreted factors. The inventors identified a number of secreted factors by RNA sequencing and have shown that a combination of three of these secreted factors (ANGPTL7, IL33 and SERPINB2) has a significant impact on the maturation of UBC-CD34+ HPCs. IL33 appeared to have the most significant effect because the inventors observed a significant reduction in the enucleation rate at all stages when this cytokine was removed from the protocol. IL33- belongs to IL1 superfamily that can apparently act as a cytokine or as a nuclear factor within the cell [29] It has been implicated in inflammation, wound healing and various disease processes. Interestingly IL33 is expressed in erythroid progenitor cells and released during haemolysis can imagine this then stimulates the maturation of new RBCs [30] Thus it is possible that the inventors have uncovered a novel feedback mechanism that could be used in therapy to treat anaemia.
[00147] There was also some reduction on the enucleation at the later stage when ANGPTL7 and SERPINB2 were removed indicating that they may plan a synergistic role at this stage. ANGPTL7 has been shown to regulate the expansion and repopulation of human HSC and HPCs [31] SerpinB2 a serine protease inhibitor of the serpin superfamily is coagulation factor known to be present in macrophages. TGFb-responsive lineage fate determinant of human bone marrow stromal cells [32]
[00148] This is the first time that macrophages have been manipulated through the enforced expression of a transcription factor and show significant changes in phenotype. The inventors have used this system to study molecular processes involved in the final step of human erythroid maturation that are inaccessible to study but the strategy can be applied to other biological systems as a tool to study the role of macrophage biology or to lock plastic macrophages into a specific therapeutic phenotype.
[00149] MATERIALS AND METHODS
[00150] Maintenance of human iPSCs
The human iPSCs lines SFCi55 and SFCi55-KLF1.2, SFCi55-KLF1.6, SFCi55-KLF1.7 and SFCi55-KLF1.12 were generated in house and were confirmed to be pluripotent and have normal karyotype [19] iPSCs were maintained in StemPro hESC SFM media (Gibco) with 20ng/ml bFGF (R&D). Wells were coated with CELLstart at least 1 hour before plating and cells were passaged using the StemPro EZPassage tool (ThermoFisher Scientific). Media change was performed every day and cells passaged at a ratio of 1 :4 every 3-4 days, when cells were 70% confluent.
[00151] Generation of human iPSC derived macrophages (IPSC-DM)
The inventors adapted published differentiation protocol that resulted in optimal macrophage production [17, 33] Maintenance media on one confluent well of a 6-well plate was replaced with 1.5 ml of Day 0 mix which consisted of StemPro hESC SFM (Gibco) supplemented with BMP4 (50ng/ml), VEGF (50ng/ml) and SCF (20ng/ml). Cells were passaged using the EZPassage tool into two wells with 2.25ml of Day 0 mix cultured in Ultralow Attachment 6 well plates for four days to induce embryoid body (EB) formation. 10-15 EBs were transferred to gelatin-coated tissue culture grade 6 well plates in 3ml Day 4 mix (X-VIV015 media supplemented with M-CSF(100ng/ml), IL3 (25ng/ml), Penicillin-Streptomycin (1 %), Glutamax (2mM) and b-mercaptoethanol (0.055) and media was changed every 3-4 days. After 3 weeks, non-adherent monocyte-like precursors were harvested from the supernatant and plated into untreated bacteriological plates or six-well plates in Maturation mix (X- VIV015 media supplemented with M-CSF (100ng/ml), Glutamax (2mM) and Penicillin- Streptomycin (1 %) for 9-11 days. Harvesting was repeated every 3-4 days for up to 3 months. To activate KLF1 in iKLF1.2 iPSC-DMs, 100nM of tamoxifen was added to the maturing macrophage population for the last 4 days of the differentiation process (every 2 days)
iPSC-DM activation with IL-10
5ng/ml of IL-10 (Preprotech Cat# 200-10) were added to adherent iKLF1.2 iPSC-DMs (IL10 activation only). To activate KLF1 in iKLF1.2 iPSC-DMs, 100nM of tamoxifen was added to adherent macrophages for 4 days, every 2 days (KLF1 activation only). For dual activation (KLF1 and IL10), 100nM of tamoxifen were added to iKLF1.2 iPSC-DMs for 48h; then one more dose of 100nM of tamoxifen + 5ng/ml of IL-10 were added for 48h more.
[00152] Culture of umbilical cord blood-derived CD34+ cells.
Umbilical cord blood (UCB) derived CD34+ cell expansion and differentiation was carried out using a step-wise protocol [9] Briefly cells were cultured (104 cell/ml) in ISHIT base media (Iscove’s Basal Media (Biochrom AG), Human AB+ serum (5%), Heparin (3U/ml) and Insulin (10pg/ml)) supplemented with SCF (60ng/ml), IL3 (5ng/ml), EPO (3U/ml), Hydrocortisone (1 mM) and holo-T ransferrin (200 pg/ml) for 8 days. Cell density was adjusted to 105 cells /ml in ISHIT media supplemented with SCF (10ng/ml), EPO (3U/ml), Hydrocortisone 1 pM and holo-Transferrin (300pg/ml), cultured for a further 3 days then cultured at a density of 106 cells /ml in ISHIT medium supplemented with EPO (3U/ml) and holo-transferrin (300 pg/ml) until day 21. Media was changed every 3-4 days throughout the protocol.
[00153] The same media was used in co-culture experiments that were set up from day 8 at a ratio of 3: 1 (iPSC-DM: CD34+-derived cells).
[00154] For experiments where secreted factors were tested, the factors were added from day 8 and topped up every two days. The final concentration of ANGPTL7 (Preprotech Cat# 130-22), IL33 (Preprotech Cat# 200-33) and SERPINB2 also known as PAI 2 (Preprotech Cat# 140-06) was 60, 75 and 75 ng/ml respectively. The final concentrations of NRG1/ Heregul^-1 (Preprotech Cat# 100-03), IGFBP6 (Preprotech Cat# 350-07B), NOV (Preprotech Cat# 120-26), CCL13 (Preprotech Cat# 300-24), TRAIL/TNSF10 Preprotech Cat# 310-04) was the same for all of them: 50 ng/ml.
[00155] Cytospins and Rapid-Chrome Kwik-Diff Staining.
Cytospins of macrophages were prepared by re-suspending 5x104 in 200 pi PBS. Cytospins of erythroid cells were prepared by re-suspending 1x105 cells in 200mI PBS. Cells were cyto- centrifuged into polylysine slides at 800RPM for 8 minutes in a Thermo Shandon Cytospin 4 and allowed to air-dry overnight. Cells were stained according to manufacturer’s instructions (Thermo-fisher #9990702, https://assets.thermofisher.com/TFSAssets/LSG/ manuals/238570%20Kwik-DiffStaining%20IFU.pdf).
[00156] Immunocytochemistry.
For KLF1 localization, 6x104 macrophages were cultured in a gelatin coated Nunc® Lab- Tek® Chamber Slide System (Sigma). Cells were fixed in 4% PFA in PBS at room temperature for 10 minutes, permeabilized in PBS-T (PBS+Triton-X100 (0.4%) for 20 minutes and blocked in PBS-T with 1 % BSA and 3% goat serum for 2 hours and incubated in anti-HA 1 :500 (Clontech #631207) for 1.5 hours. Cells were washed with PBS-T thrice for 15min, incubated in Alexa488 anti-rabbit 1 : 1000 (Thermofisher scientific #A- 11008) for 1.5h in the dark, washed with PBS-T thrice for 15 min then counter-stained with DAPI 1 : 1000 (Sigma) for 5 min.
[00157] Flow-cytometry
Single cell suspensions were obtained by StemPro Accutase Cell Dissociation Reagent (Gibco) and re-suspended in PBS with 1 %BSA and 5 mM EDTA. Cells were blocked with MACS FcR Blocking Reagent (#130-059-901) for 40 min on ice according to manufacturer instructions. 1x105 cell were washed and stained with appropriate antibodies (Supplementary Table S1) for 20 minutes at room temperature. To assess enucleation, single cell suspensions were stained with Hoechst33342 1 :20 (Thermofisher #R37605) for 20 min, washed with PBS 1 %BSA and 5mM EDTA then stained with CD71-APC 1 :200 (Thermofisher, 17-0719-42), CD235a-FITC 1 : 1000 (EBioscience #1 1-9987) and LIVE/DEAD™ Fixable Near-IR Dead Cell Stain 1 : 100 (Thermofisher #L101 19) for 20min at room temperature. Cells were washed with PBS with 1 %BSA and 5mM EDTA and kept on ice prior to analysis using LSR Fortessa Analyser (BD) and FlowJo Software. Dead cells were gated out using DAPI.
[00158] Phagocytosis assay
iPSC-DM (8 x 104) were plated in tissue-culture grade 96-well plates (CellCarrier, PerkinElmer) at least 2 days before assessing their phagocytic activity as previously described [17] Briefly cells were stained with the nuclear stain, Hoechst33342 and CellMaskTMdeep red plasma membrane stain then pHrodoTM Green Zymosan A BioParticles were applied immediately prior to live imaging using the Operetta High-Content Imaging System (Perkin Elmer). The number of iPSC-DMs that had ingested beads (phagocytic cell fraction) and the average number of beads that each cell had ingested (phagocytic index) was quantified using Columbus Image data storage and analysis system.
[00159] Gene expression analyses
Total RNA extraction was carried out using the RNAeasy Mini Kit (Qiagen); cDNA was generated from 500ng of total RNA using the High Capacity cDNA synthesis Kit (Applied Biosystem). cDNA (2ng) was amplified per reaction and each reaction was performed in technical triplicates using the LightCycler 384 (Roche) with SYBR Green Master Mix II (Roche). GADPH, b-Actin and B2M were used as reference genes and the geometrical mean was used to normalize the data. Primer sequences and efficiencies are reported (Supplementary Table S2).
[00160] RNA-sequencing
RNA extraction was carried out using the RNAeasy Mini Kit (Qiagen) and RNA quantity and quality was assessed using the Agilent Technologies TapeStation and its software. Edinburgh Genomics generated 8 TruSeq stranded mRNA-seq libraries (lllumina) from total RNA samples. Sequence data was generated in HiSeq 400075PE (lllumina) to yield at least 290M + 290M reads (1 lane). The reference used for mapping was the Homo sapiens genome from Ensembl, assembly GRCh38, annotation version 84. A principal components analysis was undertaken on normalised and filtered expression data to explore observed patterns with respect to experimental factors. The cumulative proportion of variance associated with each factor was used to study the level of structure in the data, while associations between continuous value-ranges in principal components and categorical experimental factors were assessed with an ANOVA test. Points were assigned as outliers in each component if they occurred outside the interquartile range + 1.5. No outliers were found. Differential analysis was carried out with EdgeR (version 3.16.5)36. Gene ontology analyses were carried out with the online tools in panther Gene ontology classification system [32,33]
[00161] Statistical analyses
Statistical analyses were performed using Graph Pad software version 6.0c. P-values less than 0.05 were considered statistically significant (*p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001) .
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Claims

1. An in vitro or ex vivo method of producing a population of human pluripotent stem cell derived macrophages with an altered phenotype, the method comprising modulating the activity of KLF1 in a population of human pluripotent stem cell derived macrophages and thereby altering the phenotype of the population.
2. The method of claim 1 , wherein the method comprises increasing the activity of KLF1.
3. The method of claim 1 or claim 2, wherein the altered phenotype comprises enhanced phagocytic activity.
4. The method of claim 2 or claim 3, wherein the altered phenotype comprises an erythroblastic island macrophage phenotype.
5. The method of claim 4, wherein the method increases the level of CD163, CD169, CD206 and CCR5 in the population.
6. The method of any one of claims 2 to 5, wherein the method increases the level of one or more markers selected from the group consisting of: CD11A, CD11 B, CD64, TNFa and PECAM1.
7. The method according to any one of the preceding claims, wherein the human pluripotent stem cells are induced pluripotent stem cell (iPSC) derived macrophages.
8. The method according to any one of the preceding claims, wherein the method comprises the step of incorporating a transgene comprising a gene encoding KLF1 into the cells; optionally wherein the transgene is integrated into the safe harbour AAVS1 locus of the cell.
9. The method of claim 8, wherein the transgene is an inducible transgene and the method further comprises the step of inducing activity of KLF1.
10. The method according to any one of the preceding claims, wherein the method further comprises the step of providing the human pluripotent stem cell derived
macrophages with IL-10.
11. A human pluripotent stem cell derived macrophage comprising a KLF1 transgene.
12. Use of a human pluripotent stem cell derived erythroblastic island macrophage to increase enucleation of CD34+ HPC-derived erythroid cells in vitro or ex vivo; optionally wherein the CD34+ cells are umbilical cord blood derived of CD34+ erythroid cells, or are derived from iPS cells.
13. An in vitro or ex vivo method for producing enucleated erythroid cells, said method comprising:
a. culturing umbilical cord blood derived CD34+ haematopoietic progenitor cells with at least one of the following:
i. IL33 and optionally, one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL;
ii. A population of human pluripotent stem cell derived macrophages in which the activity of KLF1 is increased; or iii. the culture media of a population of human pluripotent stem cell derived macrophage in which the activity of KLF1 is increased.
14. The method according to claim 13, wherein the population of human pluripotent stem cell derived macrophages are human pluripotent stem cell derived macrophage produced by any one of claims 1 to 10 or wherein the population comprises a human pluripotent stem cell derived macrophage according to claim 11.
15. The method according to claim 13 or claim 14, wherein the umbilical cord blood derived CD34+ haematopoietic progenitor cells are cultured with SCF, EPO, IL3 and hydrocortisone prior to step a.
16. The method of claim 15, wherein the umbilical cord blood derived CD34+ haematopoietic progenitor cells are cultured with SCF, EPO, IL3 and hydrocortisone for at least 11 days.
17. The method of any one of claims 13 to 16, wherein the culturing step a, is carried out for at least 11 days
18. An in vitro or ex vivo model for studying erythropoiesis, said model comprising a combination of a human pluripotent stem cell derived erythroblastic island macrophage and umbilical cord blood derived CD34+ haematopoietic progenitor cells.
19. The in vitro or ex vivo model of claim 18, wherein the human pluripotent stem cell derived erythroblastic island macrophage is an induced pluripotent stem cell derived erythroblastic island macrophage.
20. IL-33 for use in the treatment of dyserythropoietic anaemia in a subject.
21. IL-33 for use according to claim 20, wherein IL-33 is in combination with one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , NOV, IGFBP6, CCL3 and TRAIL.
22. A method of treating a subject having dyserythropoietic anaemia, said method comprising administering a therapeutically effective amount of IL33.
23. The method of claim 22, said method further comprising the step of administering a therapeutically effective amount of one or more of selected from the group consisting of ANGPTL7, SERPINB2, NRG1 , IGFBP6, CCL3 and TRAIL.
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