[go: up one dir, main page]

WO2018037091A1 - Procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques - Google Patents

Procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques Download PDF

Info

Publication number
WO2018037091A1
WO2018037091A1 PCT/EP2017/071372 EP2017071372W WO2018037091A1 WO 2018037091 A1 WO2018037091 A1 WO 2018037091A1 EP 2017071372 W EP2017071372 W EP 2017071372W WO 2018037091 A1 WO2018037091 A1 WO 2018037091A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
hematopoietic
list
hoxb4
Prior art date
Application number
PCT/EP2017/071372
Other languages
English (en)
Inventor
Michael Dennis MILSOM
Paul KASCHUTNIG
Original Assignee
Hi-Stem Ggmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hi-Stem Ggmbh filed Critical Hi-Stem Ggmbh
Publication of WO2018037091A1 publication Critical patent/WO2018037091A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • 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
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors

Definitions

  • This invention relates to a novel approach for the identification, characterization and isolation of hematopoietic stem and progenitor cells (HSPCs), and to the use of particular cell-surface markers in such methods.
  • the invention furthermore relates to novel populations of HSPCs, novel antibodies and kits.
  • Implant stem cells are long-lived tissue-specific cells that have the ability to give rise to various specialized cell types, while retaining the ability to self-renew, a prerequisite for sustained tissue maintenance. These multipotent stem cells have been identified in various self-renewing organs and tissues including brain, muscles, skin, teeth, liver, the intestinal epithelium and the blood system (Cotsarelis et al. , 1990; Potten et al., 1997; Till and Mc, 1961 ).
  • the cells are thought to reside in a specific microenvironment of their surrounding tissues called the stem cell niche, where they remain quiescent (non-dividing) for long periods of time until they are activated by a need for cells to maintain existing tissues or upon injury repair (Arai et al., 2004; Wilson et al., 2008).
  • HSCs hematopoietic stem cells
  • hematologic diseases including distinct forms of leukaemia, lymphoma and myeloma since the late 1960s (Bortin, 1970; Gatti et al., 1968).
  • epidermal skin stem cells are clinically used to grow sheets of new skin for severe burn patients.
  • tissue-specific stem cell therapy approaches face the same commonly shared problem, namely the very low number of available cells due to the lack of efficient stem cell expansion protocols and human leukocyte antigen (HLA)-matched donor material.
  • Experimental methodologies aiming for an efficient and robust in vitro generation of multipotent tissue-specific stem cells from different cellular source material possess great potential to circumvent the low number of clinically available cells in the near future.
  • HSCs would allow direct therapeutic application via BM transplantation and in addition represent a source of mature hematopoietic cells for red blood cell and platelet transfusions as well as a model for studying hematologic malignancies (Easterbrook et al., 2016; Singbrant et al., 2015). Although the complex intrinsic and extrinsic characteristics of HSCs have been studied extensively, up to now it is not possible to expand isolated HSCs under defined culture conditions for clinical usage.
  • PSCs pluripotent stem cells
  • pluripotent cells like embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into the spotlight.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • iPSCs can be generated by reprogramming of somatic cells through expression of a set of pluripotency-associated transcription factors (Pou5f1 , Klf4, Sox2 and Myc), which makes them a potential autologous source for any cell type (Takahashi et al., 2007).
  • Hoxa and Hoxb gene clusters have shown to be highly expressed in definitive, but not yolk sac cells (Lawrence et al., 1996; McGrath and Palis, 1997; Sauvageau et al., 1994). Based on the described expression profiles Kyba et al. demonstrated that ectopic expression of Hoxb4 endowed both yolk sac and ESC-derived hematopoietic cells with multi-lineage reconstitution potential (Kyba et al., 2002) (Table 1) ( Figure 6).
  • mice and human cells confirmed the conversion of fibroblast cells into myeloid-restricted progenitors (Pulecio et al., 2014; Szabo et al., 2010).
  • Other groups used starting cells with a more similar epigenetic profile to functional HSCs, including human microvascular endothelial cells (TFs: FOSB, GFI1 , RUNX1 & SPI1) as well as murine primary lymphoid and myeloid progenitors (TFs: Runxl , Hlf, Lmo2, Prdm5, Pbx1 & Zfp37) (Table 1) ( Figure 6).
  • i-HSCs definitive progenitors termed induced-HSCs
  • hPSC-derived hematopoietic progenitors have emerged, the generation of HSCs from teratomas in vivo.
  • Direct injection of pluripotent cells (hiPSCs) into NOD-scid IL2rynull mice produced human CD45+ cells capable of mobilization and engraftment via teratoma formation (Amabile et al., 2013).
  • Suzuki et al. co-injected murine or human iPSCs with OP9 stromal cells and observed migration of iPSC-derived HSCs from teratomas to the mouse BM (Suzuki et al., 2013).
  • HSPCs hematopoietic stem and progenitor cells
  • HSPCs hematopoietic stem and progenitor cells
  • HSPCs hematopoietic stem and progenitor cells
  • the present invention relates to a method for the identification of hematopoietic stem and progenitor cells (HSPCs) in a cell population comprising the steps of:
  • identifying cells characterized by the presence of one or more proteins on the cell surface selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1
  • identifying cells characterized by the additional presence of one or more proteins on the cell surface selected from the list of: CD34, CD41 , CD93 (AA4.1), EMCN, ENG, ESAM, ICAM2, JAM1/F1 1 R, THSD1 , and VECAD; in particular CD93 (AA4.1).
  • the present invention relates to a method for the isolation of hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:
  • HSPCs hematopoietic stem and progenitor cells
  • step (b) selecting hematopoietic stem and progenitor cells (HSPCs) that have been identified in step (a).
  • HSPCs hematopoietic stem and progenitor cells
  • the present invention relates to a method for isolating hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:
  • step (b) adding cytokines BMP-4 (Bone morphogenetic protein 4), Activin-A, FGF2 (Fibroblast growth factor 2) and VEGF (Vascular endothelial growth factor) to the culture according to step (a);
  • BMP-4 Bone morphogenetic protein 4
  • Activin-A Activin-A
  • FGF2 Fibroblast growth factor 2
  • VEGF Vascular endothelial growth factor
  • steps (c) continuing the culture of the ESC-based cells under hypoxic conditions (5% O2) for additional 3 to 4 days, in particular 3.5 days; in particular wherein the duration of steps (a) to (c) together is from 5 to 7 days, in particular from 5.5 to 6.5 days, more particularly 6 days; and
  • the present invention relates to an isolated population of cells, consisting to at least 50%, particularly to at least 60%, particularly to at least 70% of hematopoietic stem and progenitor cells (HSPCs), each characterized by the simultaneous presence of at least four cell surface proteins selected from the list of: ADAM15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAI1 , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and
  • HSPCs hema
  • the present invention relates to an antibody directed against a cell surface protein selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFIT
  • the present invention relates to a kit comprising at least two antibodies, wherein at least one antibody is directed against a cell surface protein selected from the list of: ADAM15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA
  • Figure 1 shows roadmaps of blood stem cell differentiation:
  • the classical model envisions that oligopotent progenitors such as CMPs are essential intermediate stages from which My/Er/Mk differentiation originates.
  • the redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent.
  • the grayed planes represent theoretical tiers of differentiation (Figure was taken from Notta et al., 2016).
  • Figure 2 shows timing of hematopoietic maturation across species: The relative timing of hematopoiesis at specific anatomic sites in the human (blue), mouse (red), and zebrafish (green) are shown. Although the pace of hematopoietic maturation varies in each organism, hematopoiesis matures in highly conserved patterns through analogous organs prenatally and postnatally, with primitive hematopoiesis occurring in extraembryonic mesoderm-derived cells. (Figure was taken from Rowe et al., 2016).
  • Figure 3 shows a model of blood cell formation from the hemangioblast: The specific phenotype of the cell populations as well as the key regulators, transcription factors and signalling pathways, involved in hematopoietic development are indicated. (Figure was taken from Lancrin et al., 2010).
  • Figure 4 shows a model for Jagged 1/Notch1 and Wnt/p-catenin function during HSC specification in the AGM:
  • A Mouse embryo at E10.5.
  • the dashed line represents a transversal section of the dorsal aorta.
  • B Aortic endothelium and
  • C emerging clusters.
  • D Specific endothelial cells (c-kit-) activate ⁇ -catenin. This activation is required for the specification of HSCs.
  • E In the cell clusters, cells activate Notchl through Jaggedl , which activates Gata2 and its repressor Hes1 , which ensures the right levels of Gata2 required for functional HSC generation.
  • Figure was taken from Bigas et al., 2013).
  • Figure 5 shows critical transcription factors of hematopoietic development: The stages at which hematopoietic development is blocked in the absence of a given transcription factor, as determined through conventional gene knockouts, are indicated by red bars. The factors depicted in black have been associated with oncogenesis. Those factors in light font have not yet been found translocated or mutated in human/mouse hematologic malignancies.
  • LT-HSC long- term hematopoietic stem cell
  • ST-HSC short-term hematopoietic stem cell
  • CMP common myeloid progenitor
  • CLP common lymphoid progenitor
  • MEP megakaryocyte/erythroid progenitor
  • GMP granulocyte/macrophage progenitor
  • RBCs red blood cells.
  • Figure 6 shows routes for HSC engineering: Directed differentiation of ES and iPS cells relies on morphogens and growth factors to recapitulate hematopoietic development in vitro. Direct conversion utilizes TFs to force somatic cells to switch cell fate without transitioning through normal developmental intermediates. TF combinations employed to convert heterologous cell types to hematopoietic cells are listed. Combinations in lowercase indicate conversions in mouse cells and those in uppercase represent conversions in human cells. Conversions from PSCs comprise a distinct approach.
  • the pink arrows show direct conversions between cell types
  • the blue arrows show direct hematopoietic induction from PSCs using TF combinations
  • the green arrows represent a hybrid strategy of directed differentiation and direct conversion, termed "respecification.” Extensive molecular analysis must be combined with functional interrogation to assess the relatedness of the engineered cell types to their native counterparts.
  • ES/iPS embryonic stem/induced PSCs
  • MPP multipotent progenitor
  • MLP multilymphoid progenitor
  • NK natural killer
  • CMP common myeloid progenitor
  • ES-HPC embryonic stem-derived hematopoietic progenitor cells
  • YS- HPC yolk sac-derived hematopoietic progenitor cells.
  • FIG. 7 shows mechanistic insights into HOXB4-mediated induction of hematopoietic differentiation: HOXB4 can act in both a cell autonomous and non-cell autonomous manner to enhance hematopoietic differentiation of ESCs.
  • Gene expression profiling of HOXB4 target genes in differentiating ESCs by several groups revealed the upregulation of genes associated with several processes (marked 1-4) that are involved in the production and expansion of hematopoietic cells. These include genes associated with HSC expansion (1), HSC programming (2) and those associated with a number of signalling pathways involved in the interaction of HSC with their niche (3).
  • HOXB4 The induction of HOXB4 at an early time point during ESC differentiation can enhance the production of paraxial mesoderm that gives rise to the endogenous ESC-derived hematopoietic niche (4). This possibly explains the paracrine effect of HOXB4 via an increase in the production of Frzb and other hematopoietic growth factors (5). Arrows in red indicate the processes that are enhanced by enforced expression of HOXB4. ( Figure was taken from Forrester and Jackson, 2012).
  • Figure 8 shows the targeting strategy for introducing the YFP reporter at the Hoxb4 locus:
  • A The YFP variant sequence, Venus, and an frt-flanked blasticidin selection cassette were inserted at the start codon of Hoxb4 in a BAC clone.
  • a 5.2-kb Hind 111 fragment was subcloned from the BAC and used for gene targeting in ES cells.
  • Schematic diagrams illustrate the Hoxb4 locus and the correctly targeted alleles before and after blasticidin selection cassette removal, with the use of FLPe- mediated recombination.
  • B Southern blot of BamHI-digested DNA probed with 5' probe (P5).
  • Figure 9 shows the generation and validation of reporter ES cell lines from Hoxb4-YFP mice:
  • A Cross-correlation analysis between Hoxb4-YFP levels and HSC rate.
  • LSK Hoxb4-YFPhigh cell compartment demonstrates significant HSC enrichment (> 80%) in comparison to LSK Hoxb4-YFPmed/low cells ( ⁇ 20%).
  • Virtually all gated HSCs from BM and FL (13.5) express high levels of Hoxb4.
  • B Schematic representation ES cell derivation. Timed matings were set up between Hoxb4-YFPTG/+ mice.
  • Endoderm gut-like or respiratory epithelium (black arrow); Mesoderm: cartilage (white arrow-head) or muscle (black arrow-head); Ectoderm: squamous epithelium with keratin deposition (white arrow).
  • Figure 10 shows the in vitro differentiation of Hoxb4-YFP reporter ESCs into hematopoietic cells:
  • A Schematic workflow of the cytokine-induced embryoid body (EB) differentiation protocol. ESCs were cultured in EB differentiation medium under hypoxic conditions (5% O2) for 6 days with addition of BMP4, Activin-A, FGF2 and VEGF at a final concentration of 5 ng/ml on day 2.5.
  • Figure 11 shows the gene expression analysis and functional characterization of in vitro specified HOXB4+ cells:
  • C Colony-forming unit
  • E Representative microscopy bright field (BF) images of hematopoietic colonies derived on OP9 stromal cells by plating of sorted HOXB4+ and HOXB4- cells originating from D6 EBs (100 X amplification). Error bars depict mean ⁇ SD. * P ⁇ 0.05; **P ⁇ 00. 1 ; ***P ⁇ 0.001 ; * *** P ⁇ 0.0001 ; by unpaired (two-tailed) t-test..
  • Figure 12 shows the immunophenotypic kinetics of in vitro differentiated of HOXB4+ cells:
  • B FACS-based immunophenotypic cross-correlation analysis of HOXB4+ cells with endothelial and hematopoietic marker proteins expressed during day 4.5 to day 6 of in vitro EB differentiation.
  • FIG. 13 shows that Hoxb4-YFP ES cells transiently express HOXB4 during hematopoietic in vitro differentiation:
  • A Schematic illustration depicting workflow of EB reaggregation analysis. Differentiated EBs were dissociated and individual cells sorted based on expression of developmental stage-specific markers. Sorted cells were reaggregated and continued differentiation process monitored via FACS for 24- 72 hours.
  • C Schematic timeline of FLK1 , KIT, CD41 and HOXB4 expression kinetics during in vitro differentiation of Hoxb4-YFP ESCs including immunophenotypic classification of Upstream, HOXB4+ and Downstream cell populations.
  • Figure 14 shows the identification and molecular characterization of in vitro differentiated HSC-like HOXB4+ AA4.1 + cells:
  • A Representative FACS plot panel depicting percentages of hematopoietic marker (VECAD, TIE2, KIT, CD41 , CD34 and AA4.1) expressing cells in correlation to HOXB4 protein expression levels (HOXB4hi, HOXB4low and HOXB4-).
  • FIG. 1 Circle diagram depicting FACS gating refinement for in vitro differentiated HSPC-like cells (HOXB4hi AA4.1 +) including marker expression and cell population sizes.
  • C Schematic workflow of global gene expression profiling for sorted HOXB4- AA4.1 + and HOXB4+ AA4.1 + cell populations.
  • D GSEA comparison of HOXB4- AA4.1 + and HOXB4+ AA4.1+ cells based on Hallmark, Cell signalling and HSPC-specific genesets. Statistical significance was assessed using 1000 permutations. ES, enrichment score; NES, normalized enrichment score; FDR, false-discovery rate..
  • Figure 15 shows that Hoxb4-YFP ES cells undergo characteristic transcriptomic shifts during hematopoietic in vitro differentiation:
  • A Schematic illustration of sorted cell populations used for global gene expression profiling (lllumina Mouse WG-6 v2.0), including ESCs, Upstream, Hoxb4hi, HOXB4+ AA4.1 + and Downstream cells.
  • B Hierarchical clustering of individually sorted cell populations. Euclidean distance measure and single-linkage clustering were applied using R/Bioconductor through the graphical user interface Chipster (v3.8).
  • Figure 16 shows the molecular profile of in vitro differentiated HOXB4+ AA4.1 + cells strongly resembles profile of AGM- and FL-HSCs:
  • FLK1 expressing endothelial Upstream cells showed gene signature enrichment in early hematopoiesis-inducing signalling cascades (TGF-beta, Wnt, Hhex targets) as well as in epithelial to mesenchymal transition, ECM organization, angiogenesis and cell adhesion.
  • GSEA was assessed using 1.000 permutations. Circle area represents NES score. Colour intensity represents FDR.
  • B Individual GSEA enrichment plots comparing HOXB4+ AA4.1 + cells to early Upstream and late-stage Downstream cells. Indicated gene sets are categorized into inflammatory signalling, HSPC development, lymphoid development, HSC-Niche- and general stem cells signatures.
  • Figure 17 shows the identification of CSM encoding genes coexpressed on HOXB4+ AA4.1+ cells:
  • Upregulated genes (log2 fold change > 1) of both individual comparisons were analysed via Venn analysis (Venny v2.1.0) in order to identify common genes exclusively expressed on HOXB4+ AA4.1 + cells.
  • Differential gene expression was analysed via two-group test (Chipster v3.8). Test type: empirical Bayes; corrected by Benjamini Hochberg (BH) method; P-value cut-off, 0.05.
  • BH Benjamini Hochberg
  • Figure 18 shows MRM mass spectrometry based proteomic verification of cell surface proteins co-expressed on HOXB4+ AA4.1 + cells:
  • B Table depicting relevant information of undetected proteins as well as proteins without clear regulation across the distinct cell populations.).
  • Figure 19 shows the whole Proteome analysis of differentiated cell populations representing consecutive stages of in vitro HSPC specification:
  • HRM Hyper Reaction Monitoring
  • B Two dimensional unsupervised hierarchical clustering of cell populations based on the individual protein expression levels.
  • C Comparison of differentially regulated (log2 fold change > 1 ) hematopoietic proteins across the individually sorted cell populations.
  • D Gene list enrichment analysis (Enrichr ® ) tools for signalling analysis (WikiPathway) and transcription factor enrichment analysis (ChEA) have been applied to lists of differentially regulated proteins across the compared cell populations. Top 10 candidates are depicted in horizontal column graphs. Additional enriched signalling pathways and hematopoietic transcription factors are summarized in green and red boxes, respectively. Ranking according to combined Enrichr ® score, representing a combination of calculated p- value and z-score (Chen et al., 2013).
  • Figure 20 shows the functional knockout of EVI2A and LYVE1 proteins result in severe hematopoietic differentiation defects:
  • Heatmap Gene-E based on normalized microarray expression values of individual developmental stages (McKinney-Freeman et al., 2012).
  • FIG. 1 Schematic workflow of CRISPR/Cas9 knockout screen. Individual cell surface proteins were knocked out at the ES cell stage and selected clones subsequently differentiated to assess hematopoietic potential.
  • C Representative FACS contour plot panel depicting hematopoietic differentiation potential of individual representative knockout clones based on CD41 and KIT expression levels. Parental WT clone(s), Runxl positive control(s) and functionally compromised KO-clones are illustrated in green, blue and red, respectively.
  • Figure 21 shows the immunophenotypic analysis of EVI2A and LYVE1 KO ES clones reveal differentiation defects during endothelial to hematopoietic transition:
  • A Mean percentage as determined by FACS analysis and representative FACS contour plots of CD41 + KIT+ expressing cells after hematopoietic differentiation (D6) of EVI2A and LYVE1 KO ES clones in comparison to WT parental clones and Runxl positive control cells. Numbers (n) as indicated.
  • (C) Percentage of cells expressing early HSC marker protein AA4.1 after hematopoietic differentiation of EVI2A and LYVE1 KO ES clones in comparison to WT parental clones and Runxl positive control. Error bars depict mean ⁇ SD, n 3. * P ⁇ 0.05; ** *P ⁇ 0.001 ; * *** P ⁇ 0.0001 ; by unpaired (two-tailed) t-test.
  • Figure 22 shows the developmental arrest in EVI2A and LYVE1 KO ES clones during EHT transition.
  • Figure 23 shows the crRNA oligo design and PX459 plasmid Vector map.
  • A Designed gRNA sequences (blue) were synthesized with sticky end sequences (red) complementary to Bbsl (Fermentas) digested PX459 plasmid.
  • B Vector map of the mammalian expression plasmid PX459 containing the expression cassettes for human-optimized SpCas9 (s. pyogenes), and the single guide RNA (sgRNA) scaffold (Addgene Plasmid #62988). Guide sequence(s) were cloned into the plasmid using Bbsl sites and positive cell clones subsequently selected via puromycin.
  • Figure 24 shows human microarray data based in in vitro differentiation of hiPSCs: mesoderm cells: FLK1/KDR+; hemogenic endothelial progenitors (HEP): VEcad+ CD34+ CD45-; hematopoietic progenitors (HP): VEcad- CD34+ CD45+; FL- HSCs: CD34+.
  • HEP hemogenic endothelial progenitors
  • HP hematopoietic progenitors
  • FIG. 25 shows that Ifitm 1 gene expression is enriched in HSCs: Long-term HSC (LT-HSC), multipotent progenitor 1 (MPP1), MPP2, MPP3/4, pre-granulocyte- macrophage progenitor (PreGM), common lymphoid progenitor (CLP), pre- megakaryocyte-erythroid progenitor (PreMegE), Granulocyte-macrophage progenitor (GMP), megakaryocyte progenitor (MkP), Pre-colony forming Unit-erythroid (Pre- CFU-E), colony forming Unit-erythroid (CFU-E) populations were sorted form wildtype mice and subjected to qPCR profiling of the Ifitml gene.
  • LT-HSC Long-term HSC
  • MPP1 multipotent progenitor 1
  • PreGM pre-granulocyte- macrophage progenitor
  • CLP common lymphoid progenitor
  • PreMegE
  • Figure 26 shows lfitm3-eGFP expression in hematopoietic cells: IFITM3_eGFP expression in Long-term HSC (LT-HSC), multipotent progenitor 1 (MPP1), MPP2, MPP3/4, Lineage-Sca-1-cKit+ (LS-K), Lineage- cKit+, Lineage- (lin-) and total bone marrow (tBM) compartments; left panel: exemplary histograms, right panel: quantifications.
  • LT-HSC Long-term HSC
  • MPP1 multipotent progenitor 1
  • MPP2 MPP2/MP3/4
  • LS-K Lineage-Sca-1-cKit+
  • tBM total bone marrow
  • Figure 27 shows that homeostatic interferon signalling activity is a powerful indicator of sternness:
  • A Frequency of phenotypic LSKCD150+CD48-CD34- LT- HSC in IFITM3- and IFITM3+ total bone marrow.
  • B Transplantation of 120,000 IFITM3- and IFITM3+ cKit+ progenitors into lethally irradiated mice. Blood chimerism was analyzed at indicated time points. 12 weeks post transplantation, secondary transplantations were performed. Significance was determined using the Two-tailed unpaired student T-test * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 , **** p ⁇ 0.0001 , NS: Nonsignificant.
  • Figure 28 shows the results from expression analyses of two of the key markers for HSPCs (Evi2A and Lyvel ) in sorted cell populations from human embryos.
  • the different sorted populations were iPSCs (induced pluripotent stem cells), EC (endothelial cells), HE cells (hemogenic endothelium cells), HC (definitive hematopoietic stem cell/progenitor), and HCcom (committed (i.e. differentiated) definitive hematopoietic cell).
  • A Expression analyses of the transcription factor RUNX1 ;
  • B expression analyses of VE-CADHERIN;
  • C expression analyses of HSPC marker Evi2A;
  • (D) expression analyses of HSPC marker Lyvel Each dot represents a sample from an individual embryo.
  • the present invention relates to a method for the identification of hematopoietic stem and progenitor cells (HSPCs) in a cell population comprising the steps of:
  • identifying cells characterized by the presence of one or more proteins on the cell surface selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1
  • hematopoietic stem and progenitor cells abbreviated HSPCs, collectively refers to hematopoietic stem cells (HSCs) and progenitors thereof, which are the first stage of differentiation of HSCs.
  • LT- HSCs long-term HSCs
  • ST-HSCs short-term reconstituting HSCs
  • the blood cell differentiation hierarchy comprises pools of progenitor or transit amplifying cells, which divide rapidly and generate a larger number of differentiated progeny.
  • CMPs common myeloid progenitors
  • CLPs common lymphoid progenitors
  • MEPs megakaryocyte/erythrocyte progenitors
  • GMPs granulocyte/macrophage progenitors
  • said one or more proteins on the cell surface are selected from the list of: EVI2A, LYVE1 , PTPRE and TIE1. In other particular embodiments, said one or more proteins on the cell surface are selected from the list of: IFITM1 and IFITM3. In other embodiments, said one or more proteins on the cell surface are selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 and both of IFITM1 and IFITM3 (i.e. EVI2A, LYVE1 , PTPRE, and TIE1 , and/or IFITM1 and IFITM3).
  • step (a) comprises the identification of cells characterized by the presence of two or more proteins on the cell surface selected from said list, particularly three or more, particularly four or more, particularly five or more.
  • step (a) comprises the identification of cells characterized by the presence of two or more proteins on the cell surface selected from said list, particularly three or more, particularly four or more, particularly 5 or more.
  • said population of cells is a cell population selected from the list of:
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the present invention relates to a method for the isolation of hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:
  • HSPCs hematopoietic stem and progenitor cells
  • step (b) selecting hematopoietic stem and progenitor cells (HSPCs) that have been identified in step (a).
  • HSPCs hematopoietic stem and progenitor cells
  • said step of selecting is performed by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the present invention relates to a method for isolating hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:
  • step (b) adding cytokines BMP-4 (Bone morphogenetic protein 4), Activin-A, FGF2 (Fibroblast growth factor 2) and VEGF (Vascular endothelial growth factor) to the culture according to step (a);
  • BMP-4 Bone morphogenetic protein 4
  • Activin-A Activin-A
  • FGF2 Fibroblast growth factor 2
  • VEGF Vascular endothelial growth factor
  • steps (c) continuing the culture of the ESC-based cells under hypoxic conditions (5% O2) for additional 3 to 4 days, in particular 3.5 days; in particular wherein the duration of steps (a) to (c) together is from 5 to 7 days, in particular from 5.5 to 6.5 days, more particularly 6 days; and
  • ESCs induced pluripotent stem cells
  • iPSCs induced pluripotent stem cells
  • the group of Gordon Keller developed an in vitro model based on the differentiation of pluripotent embryonic stem cells (ESCs). Using this system, the group was able to identify a precursor, termed the blast colony forming cell (BL-CFC), which generates both hematopoietic and endothelial cells, and as such represents the in vitro counterpart to the hemangioblast.
  • BL-CFC blast colony forming cell
  • the BL-CFC expresses the vascular endothelial growth factor receptor 2 (FLK1) and the mesodermal marker Brachyury but no markers of blood or endothelial cells (with the exception of FLK1) (Fehling et al., 2003).
  • FLK1 vascular endothelial growth factor receptor 2
  • mesodermal marker Brachyury but no markers of blood or endothelial cells (with the exception of FLK1) (Fehling et al., 2003).
  • FLK1 vascular endothelial growth factor receptor 2
  • VECAD vascular endothelial growth factor receptor 2
  • CD31 the cells at that stage are capable of forming endothelial networks in 3D culture (Lancrin et al., 2009).
  • TFs transcription factors
  • hematopoiesis belong to different classes of DNA-binding proteins, with some of them located at the site of common chromosomal translocations that drive leukemogenesis (Orkin, 2000).
  • T-cell acute lymphocytic leukemia protein 1 (Scl/Tal1) as critical regulator during the initial formation of blood cells ( Figure 5). Scl- /- embryos die before E9.5 and demonstrate a complete absence of primitive erythrocytes and myeloid progenitors. Accordingly Scl-/- ES cells fail to form any hematopoietic cells upon differentiation (Porcher et al., 1996; Shivdasani et al., 1995).
  • Runt-related transcription factor 1 (Runxl ) ( Figure 5).
  • Runxl Runt-related transcription factor 1
  • Figure 5 Another crucial transcription factor specifically required for the development of definitive hematopoietic progenitors.
  • Runxl is Runt-related transcription factor 1 (Runxl ) ( Figure 5).
  • Runxl knockout results in a differentiation block and the subsequent accumulation of hemogenic endothelial cells, an effect that can be reversed by reactivation of this transcription factor (Lancrin et al., 2009).
  • Runxl is dispensable for the formation of hemogenic endothelium, but required for the subsequent production of definitive progenitors, which is consistent with studies demonstrating that Runxl is required in TIE2+ and VECAD+ cells for hematopoietic development (Chen et al., 2009; Li et al., 2006; Liakarskaia et al., 2009).
  • Gfi1 and Gfil b have been identified as direct targets of RUNX1 and critical regulators of EHT (Figure 5).
  • the two genes are highly expressed in developing hematopoietic progenitors (TIE2hi KIT+ FLK1+ CD41+) and are able to trigger the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic for the EHT during early hematopoietic development (Lancrin et al., 2012).
  • Etv2 a member of the ETS transcription factor family has been described to be at or near the top of a hierarchy of factors involved in early specification of endothelial as well as subsequent hematopoietic lineages from early mesoderm (Lee et al., 2008). Etv2 contributes to the efficient expression of Flk1 , Scl, Cd31 and Tie2 during mouse embryogenesis. Among these, Tie2 plays a key role in definitive hematopoiesis and is preferentially expressed in HSCs (Takakura et al., 1998; Terskikh et al., 2003). Hence, Etv2 represents an important regulator of HSC development and maintenance by regulating Tie2 expression (Lee et al., 2011 ).
  • step (d) two or more of said selection steps, or three or more of said selection steps, are performed.
  • said selection steps are performed by fluorescence-activated cell sorting (FACS).
  • one or more antibodies directed against said one or more cell surface proteins are used.
  • the present invention relates to an isolated population of cells, consisting to at least 50%, particularly to at least 60%, particularly to at least 70% of hematopoietic stem and progenitor cells (HSPCs), each characterized by the simultaneous presence of at least four cell surface proteins selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and TY
  • HSPCs hema
  • the present invention relates to an antibody directed against a cell surface protein selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1, IFITM1, IFITM3, IGNGR1, LAPTM5, LRMP, LTC4S, LYVE1, MRC1, ORAM, PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1, CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1, PDZK1IP1, PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1, IFITM1, IFITM3, IGNGR1,
  • the present invention relates to a kit comprising at least two antibodies, wherein at least one antibody is directed against a cell surface protein selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1, IFITM1, IFITM3, IGNGR1, LAPTM5, LRMP, LTC4S, LYVE1, MRC1, ORAM, PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, TIE1, CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1, PDZK1IP1, PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular selected from the list of: ADAM 15, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH,
  • the kit further comprises at least one cytokine selected from the list of: BMP-4 (Bone morphogenetic protein 4), Activin-A, FGF2 (Fibroblast growth factor 2) and VEGF (Vascular endothelial growth factor).
  • BMP-4 Bone morphogenetic protein 4
  • Activin-A Activin-A
  • FGF2 Fibroblast growth factor 2
  • VEGF Vascular endothelial growth factor
  • the TF HOXB4 is of specific interest for the field of in vitro HSC specification since its ectopic expression confers long-term repopulating activity on pluripotent ES cells and early yolk sac progenitors suggesting that HOXB4 is involved in programming definitive hematopoietic cells (Kyba et al., 2002; Wang et al., 2005). If HOXB4 does prove to offer a route to the production of hPSC-derived HSCs is not yet clear since it has been demonstrated to induce leukemia in large animal models (Zhang et al., 2008). Hence, the assessment of the cellular and molecular mechanism of action of HOXB4 is of utmost importance in order to generate more efficient and safer protocols for clinical translation.
  • HOXB4 target genes associated with numerous cellular processes have been identified indicating multiple modes of action for this transcription factor.
  • HOXB4 target genes were described by Schiedlmeier et al. via usage of a tamoxifen-inducible form of HOXB4 and subsequent comparative gene expression analysis of LSK HPCs that had been expanded in the presence or absence of tamoxifen (Schiedlmeier et al., 2007). More than 700 differentially expressed genes were identified including genes associated with cell proliferation, cell cycle, apoptosis, and those critical for the self-renewal, survival and maintenance of HSCs (e.g. Cnkni b, Mad, Foxo3a, Ptgs2, and Zfx).
  • Oshima et al. used both ChlP-on-chip and microarray analysis to identify HOXB4 target genes in ESC-derived KIT+ CD41 + cells that had been transduced with HOXB4 and further cultured on OP9 stromal cells for 7 days (Oshima et al., 201 1).
  • HOXB4 can act in a cell autonomous manner by modulating the expression of genes involved in cell proliferation and survival and thus contribute to the expansion of the HSPC compartment (Figure 7).
  • the ability of HOXB4 to directly and simultaneously regulate key hematopoietic transcription factors suggests that it could also act as a HSC reprogramming factor, switching pre- HSCs (primitive HPCs) into HSCs with an adult, definitive phenotype.
  • the expression of genes encoding components of cellular signalling pathways are regulated by HOXB4, which could result in the altered response to specific factors involved in hematopoietic differentiation ( Figure 6) (Forrester and Jackson, 2012).
  • Notch signalling has been shown to be essential for definitive but not primitive hematopoiesis with the hematopoietic transcription factors RUNX1 and GATA2 being downstream of Notch activation (Bigas et al., 2010; Robert-Moreno et al., 2005).
  • HECs CD45- VECAD+
  • direct precursors of HSC in the AGM were also Hoxb4-YFP+, which supports the widely accepted hypothesis of a common origin of endothelial cells and developing HSCs.
  • the transgenic Hoxb4-YFP mouse model developed by Hills et al., enabled for the first time the analysis of Hoxb4 expression in HSCs of embryonic and adult tissues as well as direct functional testing of Hoxb4-expressing cells throughout the consecutive steps of hematopoietic development and represents the basis for the reporter ES cell lines used in this study. Our group was able to confirm the strong correlation of Hoxb4-YFP expression levels and cellular HSC characteristics in these mice.
  • the LSK Hoxb4-YFPhigh cell fraction is highly enriched for HSCs (> 80% LSK CD150+ CD48-), while the LSK Hoxb4-YFPmid/low cell fraction contains a high rate of MPPs, (> 80% LSK CD150- or CD48+).
  • virtually all HSCs from BM and FL (E 13.5) expressed high levels of Hoxb4 (Figure 9A).
  • Example 1 In vitro differentiation of Hoxb4-YFP reporter ESCs into hematopoietic cells
  • Hoxb4-YFP ESCs were cultured under hypoxic conditions (5% 0 2 ) for 6 days only interrupted by the addition of four hematopoietic fate inducing cytokines, namely BMP-4 (Bone morphogenetic protein 4), Activin-A, FGF2 (Fibroblast growth factor 2) and VEGF (Vascular endothelial growth factor) on day 2.5 of EB differentiation ( Figure 10A).
  • BMP-4 Bone morphogenetic protein 4
  • Activin-A Activin-A
  • FGF2 Fibroblast growth factor 2
  • VEGF Vascular endothelial growth factor
  • Hematopoietic differentiation efficiency was assessed by FACS analysis for the hematopoietic marker protein CD41 (ITGA2B), representing the most reliable marker of early steps of primitive and definitive hematopoiesis during ontogeny and ESC specification, as well as for the stem cell factor (SCF) receptor CD1 17 (KIT), predominantly expressed on the most primitive hematopoietic progenitors (Li et al., 2005; Mitjavila-Garcia et al., 2002; Ogawa et al., 1991).
  • IGA2B hematopoietic marker protein CD41
  • SCF stem cell factor
  • this immunophenotypically defined double positive cell compartment contains all functional ES cell-derived HSPCs (Irion et al., 20 0; McKinney-Freeman et al., 2009).
  • KIT+ cells ⁇ 15%
  • CD41 + KIT+ cells Figure 10B
  • Hoxb4-YFP reporter ESCs were sorted on day 6 of EB differentiation based on YFP expression levels (Figure 10D).
  • Gene expression analysis via qRT- PCR demonstrated significantly higher expression levels (up to 10 fold) of genes associated with the consecutive steps of early HSC development within the sorted HOXB4+ cell compartment as compared to HOXB4- cells ( Figure 1 1A).
  • the set of genes tested includes endothelial, hemangioblast and hemogenic endothelial marker genes (Flk1 , Pecaml , Scl, Vecad, Tie2), genes involved in the endothelial to hematopoietic (EHT) transition process (Runxl , Sox17, Gfi1 , Mpl) as well as genes expressed on definitive hematopoietic cells including HSPCs (Gatal , Gata2, Pu.1 , Nfe2, Lmo2) (Clarke et al., 2013; Lancrin et al., 2012; Lizama et al., 2015; Petit- Cocault et al., 2007).
  • Sorted HOXB4+ and HOXB4- cells were plated out in cytokine- supplemented methylcellulose medium and clonally formed hematopoietic colonies were assessed after incubation at 37°C for 7-14 days.
  • the plated cells were capable of comprehensive myeloid-committed differentiation, including the formation of granulocytic, monoytic, erythroid and megakaryocyte colonies. Threshold for counted colonies was defined by the size of at least 50 cells.
  • HOXB4+ cells demonstrated significantly higher colony formation potential than HOXB4- cells, peaking at a colony-forming ratio (CFR) of 1 :1000 on day of EB differentiation ( Figure 1 1 B).
  • FACS sorting for additional hematopoietic cell surface markers (VECAD, CD41 , TIE2, KIT) within the HOXB4+ cell compartment resulted in further enrichment for hematopoietic progenitors and an additive increase of formed hematopoietic colonies up to a CFR of 1 :500 ( Figure 1 1C).
  • HOXB4 sorted cells from D6 EBs were plated on OP9 stromal cells supporting hematopoietic differentiation and colony formation (Weisel et al., 2006). Consistent with the results from the methylcellulose-based assays, HOXB4+ cells were able to develop significantly more and larger hematopoietic colonies (CFR: 1 :1000) in comparison to HOXB4- cells (CFR: 1 :5000) ( Figure 1 1 D & 11 E).
  • Example 3 Immunophenotypic kinetics of in vitro differentiated HOXB4+ cells
  • TIE2 hemogenic endothelial markers
  • HSPC hematopoietic stem and progenitor cell
  • HPCs definitive hematopoietic progenitor cells
  • Endothelial cells (FLK1 +) express Podxl, Etv2, Flt1 and Eng, which is in line with studies demonstrating that during murine embryogenesis and ES cell specification, definitive hematopoietic potential is restricted to a subset of vascular endothelial cells expressing Podxl, Etv2 or Eng in addition to Flk1 (Borges et al., 2012; Wareing et al., 2012; Zhang et al., 2014).
  • HOXB4+ cells expressed genes previously described to be involved in early HSC emergence and migration including Vecad, Gfi1 , Sox17, Robo4, Gata2, Epcr and AA4.1 among others (Clarke et al., 2013; Shibata et al., 2009; Thambyrajah et al., 2016; Yamane et al., 2009; Yokota et al., 2009).
  • cells of the HOXB4- CD41 + KIT+ cell compartment showed high expression of genes representative for lineage-committed hematopoietic cells (Epor, Pu.1 , Gatal) ( Figure 13E).
  • Example 4 Immunophenotypic analysis identifies differentiated cells expressing early HSC marker AA4.1 within HoxB4hi cell fraction
  • Gene expression profiling (Affymetrix Mouse 430.2) was conducted in order to evaluate if selection for Hoxb4-YFP expression within the AA4.1 + cell compartment adds to the isolation of the most primitive hematopoietic stem- and progenitor cells (Figure 6C).
  • AA4.1+ HOXB4- cells demonstrated gene signature enrichment for signalling pathways described to regulate developmental steps preceding HSPC emergence, such as Wnt, Hedgehog and TGF- ⁇ signalling (Bigas et al., 2013; Kim and Letterio, 2003). This was further supported by the enrichment of early endothelial and EHT related biological processes as for instance angiogenesis, cell adhesion, ECM organization and epithelial to mesenchymal transition, in these cells (Figure 14D).
  • transcriptomic analysis via GSEA indicated that the earliest in vitro differentiated cells holding HSPC characteristics express the transcription factor HOXB4 as well as the cell surface protein AA4.1 , and further confirm that our Hoxb4- YFP reporter ESCs represent a powerful platform that facilitates the analysis of developing HSPCs during in vitro specification.
  • Example 5 Global Gene expression profiling confirms early HSC-like molecular profile of HOXB4+ aa4.1+ double positive cells
  • Upstream cells expressed CD40 and Icam2 two marker encoding genes whose sequential expression defines progressive steps of blood formation, specifically the transition from hemangioblast cells (CD40+ Icam2+) to definitive hemogenic endothelial cells (CD40- Icam2+). This process is reflected during in vitro differentiation by the transition of Upstream cells (CD40+ Icam2+) to HOXB4 expressing cell populations (CD40- Icam2+) ( Figure 15C) (Pearson et al., 2010).
  • HOXB4+ AA4.1 + double positive cells demonstrated a HEC/HSPC-like transcriptomic profile, by high expression of HEC-specific factors (Gfi1 , Gfil b, Hhex, Mpl, Sox17, Vecad, Lmo2 and Runx ) as well as transcripts typically expressed in HSCs, with some of them encoding for potent HSC purification markers (Hoxb5, Fgd5, Esam, CD9, Gata2, Tie2, CD34, Thsdl and Emcn) (Chen et al., 2016; Gazit et al., 2014; Matsubara et al., 2005; Takayanagi et al., 2006). Highest regulated transcripts (Top 40) are illustrated in Table 2.
  • HOXB4+ AA4.1 + double positive cells demonstrated expression of genes involved in T cell receptor (TCR) and B cell receptor (BCR) signalling (Vav1 , Vav3, Jun, Lat, Ptpn6, II4 and CD79b) as well as in inflammatory signalling (Statl , Stat5a, Myd88, Nfkbl , Irf2, Irf3 and Ifnar2) (Figure 14D & 15G-15H).
  • Endothelial-like FLK1 + Upstream cells were enriched for early endothelial and hematopoiesis-inducing signalling cascades as well as ECM-, cell adhesion- and angiogenesis- specific gene sets preceding HSPC emergence (blue circles).
  • Table 2 Top 40 differentially regulated genes between HOXB4+ AA4.1 + and respective Up-/Downstream cell populations.
  • Example 6 Proteomic analysis of in vitro specified HSC-like HOXB4+ AA4.1 + cells
  • Microarray data was analysed for cell surface proteins expressed exclusively by the differentiated HSC-like HOXB4+ AA4.1 + cell population, hypothesizing a potential role for some of these proteins and the respective downstream signalling process, at the onset of in vitro and in vivo HSC emergence.
  • the marker encoding genes were ranked according to the degree of differential expression and novelty (Table 3).
  • the thereby selected microarray-identified genes were validated by qRT-PCR confirming exclusively high expression levels within the HOXB4+ AA4.1+ cells ( Figure 17D).
  • MRM Multiple Reaction Monitoring
  • enrichment analysis confirmed the already observed gene signature data (HSC differentiation-, BCR/TCR- and inflammatory signatures), previously obtained from the microarray analyses (lllumina & Affymetrix), also at protein level (Figure 19D).
  • Transcription factor enrichment analysis (Enrichr ® ) of the proteome dataset identified the hematopoietic master regulatory proteins MECOM (EVI1 ), SPI1 , GFI1 B, TAL1 , MEIS1 , RUNX1 , GATA1 , GATA2 and HOXB4 among others as the defining transcription factors within the double positive cells (HOXB4+ AA4.1 +) (Figure 19D).
  • Proteomic analysis confirmed the previously identified microarray-based HSPC-specific molecular profile of HOXB4+ AA4.1 + cells on protein level by demonstrating high expression of transcription factors, cell surface proteins and signalling processes exclusively ascribed to HSCs. Furthermore, we were able to verify the expression of 30 newly identified marker proteins coexpressed on HOXB4+ AA4.1 + cells via targeted mass spectrometry (MRM). These novel cell surface receptors, not yet described in the context of HSC biology, could enhance the isolation of definitive hematopoietic cells in various ES/iPS cell lines with some markers putatively representing key players of cell signalling processes required for definitive HSC development during in vitro ES specification as well as embryonic hematopoietic development.
  • MRM mass spectrometry
  • Example 7 Crispr/Cas9 knockout screen reveals functional requirement of newly identified cell surface receptors during hematopoietic in vitro specification of HSPCS
  • Top candidates were then cross- correlated with the published whole transcriptome dataset of McKinney-Freeman et al., encompassing expression data of the individual stages of hematopoietic development during murine embryogenesis (McKinney-Freeman et al., 2012). Specific focus was placed on marker proteins highly expressed in both microarray datasets, in particular on candidates with predominant expression within early HSCs isolated from the AGM or FL (E12.5) of developing mouse embryos ( Figure 20A).
  • CRISPR/Cas9-based screening revealed that the individual KO of four screened marker proteins (EVI2A, LYVE1 , PTPRE and TIE1) resulted in significant hematopoietic differentiation defects assessed by CD41 and KIT expression levels (Figure 12C).
  • EVI2A screened marker proteins
  • LYVE1 LYVE1
  • PTPRE screened marker proteins
  • TIE1 hematopoietic differentiation defects assessed by CD41 and KIT expression levels
  • Figure 12C On day 6 of EB differentiation, average hematopoietic CD41 + KIT+ double positive cell population size observed in WT parental clones and empty vector controls (data not shown) accounted for 35% ⁇ 5% of total cells.
  • EVI2A- and LYVE1-KO phenotype revealed severe differentiation defects already at the stage of EHT transition.
  • EVI2A-KO as well as LYVE1-KO ES cells are still able to generate VECAD+ cells but exhibit severe defects during further differentiation into CD41 + VECAD+ HECs (EVI2A: 3% ⁇ 1 %; LYVE1 : 5% ⁇ 3%; WT: 20% ⁇ 5%) and subsequent CD41 + VECAD- hematopoietic cells (EVI2A: 3% ⁇ 1 %; LYVE1 : 3% ⁇ 1 %; WT: 25% ⁇ 5%) compared to the WT parental ES cells.
  • Figure 24 shows the results from Human Array Data which demonstrate that the novel targets are expressed in either human iPS cells that have been induced to differentiate into blood, or in human fetal liver (both enriched for putative HSC compartment).
  • Figures 25 and 26 show the results from IFITM1 and IFITM3 expression analyses. It could be shown that these novel markers are differentially expressed in mouse adult HSCs and that using a transgenic mouse system to isolate IFITM3 expressing cells, HSCs with the highest transplantation efficiency can be purified.
  • Example 10 Expression Analyses of HSPC Markers Evi2A and Lyvel in Human Embryonic Cell Populations
  • Figure 28 shows the results from expression analyses of two of the key markers for HSPCs (Evi2A and Lyvel) in sorted cell populations from human embryos.
  • the different sorted populations were iPSCs (induced pluripotent stem cells), EC (endothelial cells), HE cells (hemogenic endothelium cells), HC (definitive hematopoietic stem cell/progenitor), and HCcom (committed (i.e. differentiated) definitive hematopoietic cell).
  • the expression analyses were performed in parallel to the expression analyses of the transcription factor RUNX1 , which is expressed to the highest degree in definitive hematopoietic stem and progenitor cells and their hemogenic endothelial precursors, and which is then downregulated in more differentiated definitive hematopoietic cells (see Figure 28A), and of VE-CADHERIN, which is expressed at highest levels in endothelial cells and also in the hemogenic endothelium, which is the direct precursor to definitive hematopoietic stem cells (see Figure 28B). It could be shown that Evi2A is expressed as the start of hematopoietic specification in the HE population, and is maintained in hematopoietic stem cells and their differentiated progeny (see Figure 28C). Furthermore, it could be shown that Lyvel is expressed as endothelial cells commit to hematopoietic differentiation (see Figure 28D).
  • DMEM Dulbecco's Modified Eagle Medium
  • ESGRO LIF mouse leukemia inhibitory factor
  • Hoxb4-YFP reporter ES cell lines were generated as previously described (Tesar, 2005). Hoxb4-YFP transgenic mice were obtained on a C57BL/6J background (Hills et al., 2011) and crossed with congenic CD45.1 positive B6.SJL- Ptprca Pepcb/BoyJ mice (The Jackson Laboratory, Bar Harbor, Maine, USA). Mice were housed in individually ventilated cages in the DKFZ animal facility and all experimental procedures were performed in accordance to the institutional and governmental animal welfare guidelines.
  • mice Female mice (6-10 weeks) were induced to super ovulate via intraperitoneal (i.p.) injection of 7 international units (lUs) equine chorionic gonadotropin (eCG; Intergonan) followed by a second injection of 7 lUs human chorionic gonadotropin (hCG) 48 h later.
  • Injected females were placed with heterozygous Hoxb4-YFP males and mating was confirmed by the presence of a vaginal plug.
  • 2.5 days after injection females were sacrificed via cervical dislocation and embryos were isolated from the oviducts and transferred to M2 medium. Zonae pellucidae were removed through brief exposure to Tyrode's saline acidified to pH 2.5.
  • the Hoxb4-YFP reporter ESC lines were maintained under 2i/LIF culture conditions on MEF feeder cells, which provide an additional growth substrate for the ES cells and secrete factors necessary for ESC pluripotency.
  • MEFs were initially seeded in MEF medium at a density of 2-3 x 10 4 cells/cm 2 . Medium was replaced by ESC medium prior to seeding ES cells onto feeder layers. ESC medium was subsequently replaced on a daily basis, while the feeder layer was renewed weekly. ES cells were passaged every 48h to avoid confluency and acidification of the media. Cells were incubated at 37°C and 5% C0 2 .
  • Confluent Hoxb4-YFP ESCs were washed twice with PBS and subsequently harvested (Trypsin-EDTA, Gibco). Feeder cells were separated from cell suspension by plating into TPP tissue culture flasks (Corning, Ney York, USA) for 30-40 minutes. Supernatant was transferred to fresh tube and ESCs were counted. Subsequently cells were seeded into embryoid body (EB) differentiation medium containing Ultra Low Attachment cell culture flasks (Corning) as follows.
  • EB embryoid body
  • EBs dissociation was carried out by addition of 250 ⁇ (T25) or 750 ⁇ (T75) dissociation enzyme mix (1.2) and subsequent incubation at 37°C for 20 minutes in the water bath. The residual cell aggregates were then fully dissociated by trituration within 8 ml enzyme-free dissociation buffer (Life Technologies). Cells were collected via centrifugation at 300 g for 5 minutes, resuspended and then assessed for hematopoietic activity via flow cytometry-based analysis or by performing functional colony-forming unit assays (CFUs).
  • CFUs functional colony-forming unit assays
  • cytokine-enriched semisolid media (Methocult). 300 ⁇ IMDM medium containing 3x105 cells (1x105 cells /ml) was added on top of 2.7 ml pre-aliquoted MethoCult (Stem Cell Technologies). The cell mixture was vortexed thoroughly and then incubated for 5-10 min at room temperature (RT) in order to avoid transfer of formed air bubbles. 1 ml of the cell suspension was inoculated into a 35 mm Petri dish (Corning) and then incubated in a humidified incubator at 37°C and 5% CO 2 . Hematopoietic colonies were counted and characterized after 7 to 10 days of incubation.
  • RT room temperature
  • the ViiA 7 Software 1.1 was used for data acquisition and analysis was based on the 2-AACt method. Expression data of individual target genes was normalized against the housekeepers Oaz1 and Sdha.
  • FACS samples were analysed on a LSRII or LSR-Fortessa flow cytometer (BD Biosciences). FACS-Sort experiments were performed via Aria I, II or III flow cytometers (BD Biosciences). Gating of marker-expressing cells based on unstained cell controls. Dead cells were excluded by using 7-Aminoactinomycin (7AAD; Invitrogen).
  • 7AAD 7-Aminoactinomycin
  • Table 4 List of primers used for quantitative real-time PCR
  • EBs were dissociated (as described in 2.3 above) on day 5 of differentiation and subsequently sorted according to the following immunophenotypic marker proteins:
  • RNA Integrity Number represents the quality of the analyzed RNA samples and ranges from 1-10, with 1 standing for the most degraded profile and 10 for very high integrity. Samples demonstrating a RIN value higher than 7 were considered for cDNA synthesis, biotin labeling and on-chip probe hybridization. Two distinct chip systems were used: lllumina MouseWG-6 v2 BeadChip ®
  • lllumina chips were laser-scanned via the lllumina iScan system®, Affymetrix chips with the GeneChip® Scanner 3000.
  • GSEA Gene set enrichment analysis
  • Phenotype labels Respective .cls file
  • Chip platform Respective .chip file
  • Bubble GUM GSEA Unlimited Map
  • NES maximal Normalized Enrichment Score
  • FDR False Discovery Rate
  • the web-based ENRICHR® database provides a comprehensive set of functional tools to identify biological interactions behind large gene lists extracted from gene expression profiling (Chen et al., 2013; Kuleshov et al., 2016).
  • the tool was used for pathway (KEGG, WikiPathways), ChRIP-based transcription factor enrichment (ChEA 2015, TRANSFAC) and gene ontology (GO) analyses of extracted microarray data.
  • FACS sorted cells were washed with PBS and lysed with RIPA buffer (50 mM Tris, 150 mM NaCI, 1 % NP-40, 0.5% Sodium-Deoxycholate, 0.1 % SDS, complete Protease Inhibitor Cocktail, Roche) before subjecting to sonication (15% amplitude) and one freeze-thaw cycle. Lysates were cleared by centrifugation and the protein concentration of individual samples was determined using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific). Proteins were reduced with 5 mM dithiothreitol at 60°C for 30 min followed by alkylation with 15 mM iodoacetamide for 30 min at 37°C.
  • RIPA buffer 50 mM Tris, 150 mM NaCI, 1 % NP-40, 0.5% Sodium-Deoxycholate, 0.1 % SDS, complete Protease Inhibitor Cocktail, Roche
  • the samples were resolubilized in 0.1 % RapiGest solution (Waters) in tryptic digestion buffer (50 mM Tris-HCI, 1 mM CaCI2) and digested with Trypsin (1 :50, w/w) for 15 h at 37°C. Following acidification (0.5% TFA), samples were incubated for 30 min at 37°C and separated from detergent byproducts by centrifugation at 20000 x g for 10 min.
  • tryptic digestion buffer 50 mM Tris-HCI, 1 mM CaCI2
  • Peptides were desalted using a Peptide Desalting Lab-in-a-Plate Flow-Thru-plate (C18, Glygen), dried and resuspended in 3% acetonitrile, 0.1 % formic acid, 0.01 % TFA in water containing the heavy peptide pool (see below). [00165] Based on SRM Atlas data, up to four proteotypic peptides per target protein were selected. Peptides were restricted to a mass range of 600-2000 Da and methionine and cysteine containing peptides were excluded if possible.
  • heavy peptide standards lysine (13C615N2) or arginine (13C615N4) at the C terminus
  • light Intavis
  • SRM analysis was performed on a QTRAP 6500 mass spectrometer (AB SCIEX) operated with Analyst software (v1.6.2) and coupled to a nanoAcquity UPLC (Waters). Reversed-phase chromatography was performed on an Acquity UPLC M-Class CSH C18 column (300 ⁇ x 15 cm, 130 A) (Waters). Samples were separated over 120 min at a flow rate of 6 ⁇ /min using a 4 to 30% (1-1 10 min), 30- 85% (1 0-1 15 min acetonitrile gradient in 0.1% formic acid, 0.01 % TFA.
  • MS/MS spectra were acquired in the ion trap mode (enhanced product ion) with dynamic fill time, Q1 resolution low, scan speed of 10000 Da/s and m/z range of 100-2000. Two transitions for each peptide were selected based on maximum signal intensities. For the final SRM quantification experiment, two reproducibly detectable peptides per protein with at least 2 charges were targeted with two SRM transition signals per heavy or light peptide. This resulted in a total of 314 transitions for 78 peptides deriving from 43 proteins.
  • Scheduled SRM was performed with Q1 operated in unit resolution, Q3 in low resolution, a target scan time of 2 s, an average (minimal) dwell time of 151 ms (34 ms) and a retention time windows of ⁇ 3.25 min around the specific elution time.
  • SRM data were processed using the Skyline software (v2.6.0). Peaks were assigned manually after smoothing (Savitzky-Golay) and transition reports including information on background-reduced peak area of heavy and light peptides were exported as .xls file. For each peptide, peak areas of corresponding transitions were summed up for analysis. Peptides with unfavourable elution profile or interfering noise in the light transitions were excluded from further analysis. The ratio between the background reduced peak area of the light transition and the background reduced peak area of the heavy transition was calculated to correct for ionization or spray differences between runs.
  • gRNAs Guide/CRISPR RNAs
  • Zhang Lab http://crispr.mit.edu
  • Four distinct guide sequences were designed for each gene of interest and selected based on their respective "On-Target” and “Off-Target” scores (Table 6).
  • PlasmidSafe exonuclease treatment of ligation reaction to prevent unwanted recombination products (optional):
  • Murine ESCs were transfected with the purified targeting vectors (PX459 plasmid + gene-specific sgRNA) using the mouse ES cell nucleofector Kit (Lonza) and subsequently cultured on MEF feeder cells in ESC medium (1 .2). Cells were trypsinized and feeder cells were separated from cell suspension by plating into TPP tissue culture flasks for 30 minutes previous to nucleofection. Cells of the supernatant were washed once in PBS, collected and resuspended in 90 ⁇ Mouse ES Cell Nucleofector solution (Lonza) at RT. Meanwhile 5 pg of each plasmid used in the reaction were added to 10 ⁇ Mouse ES Cell Nucleofector solution.
  • PX459 plasmid + gene-specific sgRNA mouse ES cell nucleofector Kit
  • the cell suspension was added on top of the plasmid solution (100 ⁇ ), mixed by pipetting up and down three consecutive times followed by immediate transfer to an Amaxa cuvette. Electroporation was performed using the Amaxa Nucleofector I (Program A- 13). 500 ⁇ pre-warmed culture medium were added to the cuvette immediately prior to plating the cell mixture on puromycin-resistant MEFs (Stem Cell Technologies) in ESC medium for 24 h. For selection of positive clones, standard medium was replaced by puromycin-containing ESC medium (2 pg/ml) for 48h. Resistant clones (80-150) became visible after additional 5-7 days of culture in standard ESC medium.
  • the residual cell solution was lysed via addition of Proteinase K (Qiagen) and subsequently used in a PCR, flanking the target sequence (primers see Table 7) to confirm potential mutations (INDELs) induced by the error-prone non-homologous end joining (NHEJ) repair pathway after target-specific double-stranded DNA cleavage (Cas9).
  • INDELs error-prone non-homologous end joining
  • Cas9 target-specific double-stranded DNA cleavage
  • Hematopoietic differentiation potential of individual KO clones has been assessed via in vitro EB differentiation (2.3).
  • Differentiated cells (D6) have been FACS analysed based on the following antibody panel (for details see Table 5) representative for early hematopoietic differentiation:
  • cDNA was synthesized according to the protocol established by Simon Haas (Haas et al., 2015). Sorted 96-Well plated were centrifuged shortly at 4°C (300 g) and subsequently transferred to a PCR cycler. Designed target gene primers were used in reverse transcriptase and following qPCR reaction (Table 8).
  • Synthesized cDNA was used directly in a standard qPCR protocol or stored at -20°C.
  • Hematopoietic stem cells the paradigmatic tissue-specific stem cell. Am J Pathol 169, 338-346.
  • Nanog a new recruit to the embryonic stem cell orchestra. Cell 113, 551-552.
  • Runxl is required for the endothelial to haematopoietic cell transition but not thereafter. Nature 457, 887-891.
  • Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells. Nat Cell Biol 15, 916-925.
  • pilosebaceous unit implications for follicular stem cells, hair cycle, and skin
  • IFNalpha activates dormant haematopoietic stem cells in vivo. Nature 458, 904-908.
  • Fgd5 identifies hematopoietic stem cells in the murine bone marrow. J Exp Med 211, 1315-1331.
  • Tissue-resident macrophages originate from yolk- sac-derived erythro-myeloid progenitors. Nature 518, 547-551.
  • Hoxb4-YFP reporter mouse model a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis. Blood 117, 3521- 3528.
  • GFI1 and GFI1 B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment. Blood 120, 314-322.
  • the haemangioblast generates haematopoietic cells through a haemogenic endothelium stage. Nature 457, 892-895.
  • the Er71 is an important regulator of hematopoietic stem cells in adult mice. Stem Cells 29, 539-548.
  • ER71 acts downstream of BMP, Notch, and Wnt signalling in blood and vessel progenitor specification.
  • hematopoiesis is required in cells that express Tek. Blood 107, 106-110.
  • CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway.
  • phosphorylation are coordinately downregulated in human diabetes. Nature genetics 34, 267-273.
  • ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients. Development 118, 1343-1351. Muller et al. (1994). Development of hematopoietic stem cell activity in the mouse embryo. Immunity 7, 291-301.
  • the gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol Cell Biol 79, 5453-5465.
  • AML1 the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis.
  • Hematopoiesis an evolving paradigm for stem cell biology. Cell 732, 631-644.
  • Oshima et al. (201 1 ). Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic stem and progenitor cells developing from embryonic stem cells. Blood 777, e142-150.
  • T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86, 47-57.
  • RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells. Development 132, 1 117-1 126.
  • Hematopoietic Stem Cells Lessons from Development. Cell Stem Cell 18, 707-720.
  • BubbleGUM automatic extraction of phenotype molecular signatures and comprehensive visualization of multiple Gene Set Enrichment
  • CD97 is differentially expressed on murine hematopoietic stem- and progenitor-cells. Haematologica 93, 1 137-1 44.
  • Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une nouvelle approche pour l'identification, la caractérisation et l'isolement de cellules souches et progénitrices hématopoïétiques (HSPCs), et l'utilisation de marqueurs de surface cellulaire particuliers dans de tels procédés. L'invention concerne en outre de nouvelles populations de HSPCs, de nouveaux anticorps et de nouveaux kits.
PCT/EP2017/071372 2016-08-24 2017-08-24 Procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques WO2018037091A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP16001856 2016-08-24
EP16001856.0 2016-08-24

Publications (1)

Publication Number Publication Date
WO2018037091A1 true WO2018037091A1 (fr) 2018-03-01

Family

ID=56802201

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/071372 WO2018037091A1 (fr) 2016-08-24 2017-08-24 Procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques

Country Status (1)

Country Link
WO (1) WO2018037091A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110372786A (zh) * 2019-06-24 2019-10-25 南昌大学 用于制备mPRα单克隆抗体的免疫原
US11591390B2 (en) 2018-09-27 2023-02-28 Celgene Corporation SIRP-α binding proteins and methods of use thereof
US12084499B2 (en) 2018-09-27 2024-09-10 Celgene Corporation SIRP-α binding proteins and methods of use thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061584A1 (fr) * 1998-05-29 1999-12-02 Thomas Jefferson University Compositions et methodes utilisees pour modifier des populations de cellules souches hematopoietiques chez des mammiferes
WO2013028684A1 (fr) * 2011-08-23 2013-02-28 Wisconsin Alumni Research Foundation Cellules progénitrices angio-hématopoïétiques
US20140148351A1 (en) * 2010-09-30 2014-05-29 The Board Of Trustees Of The Leland Stanford Junior University Prediction of Clinical Outcome in Hematological Malignancies Using a Self-Renewal Expression Signature
WO2014165131A1 (fr) * 2013-03-13 2014-10-09 Wisconsin Alumni Research Foundation Méthodes et matériaux pour la différenciation de cellules souches pluripotentes humaines en cellules endothéliales et hématopoïétiques dans des conditions définies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061584A1 (fr) * 1998-05-29 1999-12-02 Thomas Jefferson University Compositions et methodes utilisees pour modifier des populations de cellules souches hematopoietiques chez des mammiferes
US20140148351A1 (en) * 2010-09-30 2014-05-29 The Board Of Trustees Of The Leland Stanford Junior University Prediction of Clinical Outcome in Hematological Malignancies Using a Self-Renewal Expression Signature
WO2013028684A1 (fr) * 2011-08-23 2013-02-28 Wisconsin Alumni Research Foundation Cellules progénitrices angio-hématopoïétiques
WO2014165131A1 (fr) * 2013-03-13 2014-10-09 Wisconsin Alumni Research Foundation Méthodes et matériaux pour la différenciation de cellules souches pluripotentes humaines en cellules endothéliales et hématopoïétiques dans des conditions définies

Non-Patent Citations (163)

* Cited by examiner, † Cited by third party
Title
ADAMO ET AL.: "Biomechanical forces promote embryonic haematopoiesis", NATURE, vol. 459, 2009, pages 1131 - 1135
AKASHI ET AL.: "A clonogenic common myeloid progenitor that gives rise to all myeloid lineages", NATURE, vol. 404, 2000, pages 193 - 197, XP002239809, DOI: doi:10.1038/35004599
AMABILE ET AL.: "In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells", BLOOD, vol. 121, 2013, pages 1255 - 1264, XP055063362, DOI: doi:10.1182/blood-2012-06-434407
ANTONCHUK, J.; SAUVAGEAU, G.; HUMPHRIES, R.K.: "HOXB4-induced expansion of adult hematopoietic stem cells ex vivo", CELL, vol. 109, 2002, pages 39 - 45, XP002275833, DOI: doi:10.1016/S0092-8674(02)00697-9
ARAI ET AL.: "Tie2/angiopoietin-1 signalling regulates hematopoietic stem cell quiescence in the bone marrow niche", CELL, vol. 118, 2004, pages 149 - 161
BELL, J.J.; BHANDOOLA, A.: "The earliest thymic progenitors for T cells possess myeloid lineage potential", NATURE, vol. 452, 2008, pages 764 - 767
BIGAS, A.; GUIU, J.; GAMA-NORTON, L.: "Notch and Wnt signalling in the emergence of hematopoietic stem cells", BLOOD CELLS MOL DIS, vol. 51, 2013, pages 264 - 270, XP028749632, DOI: doi:10.1016/j.bcmd.2013.07.005
BIGAS, A.; ROBERT-MORENO, A.; ESPINOSA, L.: "The Notch pathway in the developing hematopoietic system", INT J DEV BIOL, vol. 54, 2010, pages 1175 - 1188
BOISSET, J.C.; ROBIN, C.: "On the origin of hematopoietic stem cells: progress and controversy", STEM CELL RES, vol. 8, 2012, pages 1 - 13, XP028113472, DOI: doi:10.1016/j.scr.2011.07.002
BORGES ET AL.: "A critical role for endoglin in the emergence of blood during embryonic development", BLOOD, vol. 119, 2012, pages 5417 - 5428
BORTIN, M.M.: "A compendium of reported human bone marrow transplants", TRANSPLANTATION, vol. 9, 1970, pages 571 - 587
BRUNS I. ET AL.: "Multiple myeloma-related deregulation of bone marrow-derived CD34+ hematopoietic stem and progenitor cells", BLOOD, vol. 120, no. 13, 27 September 2012 (2012-09-27), pages 2620 - 2630, XP055417371 *
BRYDER, D.; ROSSI, D.J.; WEISSMAN, I.L.: "Hematopoietic stem cells: the paradigmatic tissue-specific stem cell", AM J PATHOL, vol. 169, 2006, pages 338 - 346, XP002476779, DOI: doi:10.2353/ajpath.2006.060312
BÜCHLER M. ET AL.: "Identification and characterization of novel functional markers of EHT", EXP. HEMATOL., vol. 53, no. suppl., 3217, September 2017 (2017-09-01), pages s121, XP085160543 *
CABEZAS-WALLSCHEID N. ET AL.: "Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis", CELL STEM CELL, vol. 15, no. 4, 2 October 2014 (2014-10-02), pages 507 - 522, XP055417327 *
CAVALERI, F.; SCHOLER, H.R.: "Nanog: a new recruit to the embryonic stem cell orchestra", CELL, vol. 113, 2003, pages 551 - 552
CERDAN, C.; BHATIA, M.: "Novel roles for Notch, Wnt and Hedgehog in hematopoesis derived from human pluripotent stem cells", INT J DEV BIOL, vol. 54, 2010, pages 955 - 963
CHEN ET AL.: "Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool", BMC BIOINFORMATICS, vol. 14, 2013, pages 128, XP021145036, DOI: doi:10.1186/1471-2105-14-128
CHEN ET AL.: "Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche", NATURE, vol. 530, 2016, pages 223 - 227
CHEN ET AL.: "Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter", NATURE, vol. 457, 2009, pages 887 - 891, XP055267427, DOI: doi:10.1038/nature07619
CHENG ET AL.: "Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast", DEVELOPMENT, vol. 135, 2008, pages 3447 - 3458
CHESHIER, S.H.; PROHASKA, S.S.; WEISSMAN, I.L.: "The effect of bleeding on hematopoietic stem cell cycling and self-renewal", STEM CELLS DEV, vol. 16, 2007, pages 707 - 717
CHOI ET AL.: "A common precursor for hematopoietic and endothelial cells", DEVELOPMENT, vol. 125, 1998, pages 725 - 732, XP002125755
CHOI ET AL.: "Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells", NAT PROTOC, vol. 6, 2011, pages 296 - 313, XP055342800, DOI: doi:10.1038/nprot.2010.184
CHOONG M.L. ET AL.: "MicroRNA expression profiling during human cord blood-derived CD34 cell erythropoiesis", EXP. HEMATOL., vol. 35, no. 4, 28 March 2007 (2007-03-28), pages 551 - 564, XP005929124 *
CLARKE ET AL.: "The expression of Sox17 identifies and regulates haemogenic endothelium", NAT CELL BIOL, vol. 15, 2013, pages 502 - 510
CONG ET AL.: "Multiplex genome engineering using CRISPR/Cas systems", SCIENCE, vol. 339, 2013, pages 819 - 823, XP055300065, DOI: doi:10.1126/science.1231143
COPLEY ET AL.: "The Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells", NAT CELL BIOL, vol. 15, 2013, pages 916 - 925
COTSARELIS ET AL.: "Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis", CELL, vol. 61, 1990, pages 1329 - 1337, XP024246262, DOI: doi:10.1016/0092-8674(90)90696-C
DA CUNHA ET AL.: "Bioinformatics construction of the human cell surfaceome", PROC NATL ACAD SCI U S A, vol. 106, 2009, pages 16752 - 16757, XP008164427, DOI: doi:10.1073/pnas.0907939106
DE BRUIJN ET AL.: "Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo", EMBO J, vol. 19, 2000, pages 2465 - 2474
DOU ET AL.: "Medial HOXA genes demarcate haematopoietic stem cell fate during human development", NAT CELL BIOL, vol. 18, 2016, pages 595 - 606
D'SOUZA ET AL.: "SCLfTal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development", BLOOD, vol. 105, 2005, pages 3862 - 3870
DZIERZAK, E.; SPECK, N.A.: "Of lineage and legacy: the development of mammalian hematopoietic stem cells", NAT IMMUNOL, vol. 9, 2008, pages 129 - 136
EASTERBROOK ET AL.: "Concise review: programming human pluripotent stem cells into blood", BRITISH JOURNAL OF HAEMATOLOGY, vol. 173, 2016, pages 671 - 679
EILKEN, H.M.; NISHIKAWA, S.; SCHROEDER, T.: "Continuous single-cell imaging of blood generation from haemogenic endothelium", NATURE, vol. 457, 2009, pages 896 - 900
ENDOH ET AL.: "SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation", EMBO J, vol. 21, 2002, pages 6700 - 6708
ESPIN-PALAZON ET AL.: "Proinflammatory signalling regulates hematopoietic stem cell emergence", CELL, vol. 159, 2014, pages 1070 - 1085
ESSERS ET AL.: "IFNalpha activates dormant haematopoietic stem cells in vivo", NATURE, vol. 458, 2009, pages 904 - 908
FEHLING ET AL.: "Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation", DEVELOPMENT, vol. 130, 2003, pages 4217, XP002393327, DOI: doi:10.1242/dev.00589
FORRESTER, L.M.; JACKSON, M.: "Mechanism of action of HOXB4 on the hematopoietic differentiation of embryonic stem cells", STEM CELLS, vol. 30, 2012, pages 379 - 385
GATTI ET AL.: "Immunological reconstitution of sex-linked lymphopenic immunological deficiency", LANCET, vol. 2, 1968, pages 1366 - 1369
GAZIT ET AL.: "Fgd5 identifies hematopoietic stem cells in the murine bone marrow", J EXP MED, vol. 211, 2014, pages 1315 - 1331
GENTLES A.J. ET AL.: "Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia", JAMA, vol. 304, no. 24, 22 December 2010 (2010-12-22), pages 2706 - 2715, XP055403732 *
GIAMPAOLO ET AL.: "HOXB gene expression and function in differentiating purified hematopoietic progenitors", STEM CELLS, vol. 13, no. 1, 1995, pages 90 - 105
GOMEZ PERDIGUERO ET AL.: "Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors", NATURE, vol. 518, 2015, pages 547 - 551
GORDON-KEYLOCK ET AL.: "Induction of hematopoietic differentiation of mouse embryonic stem cells by an AGM-derived stromal cell line is not further enhanced by overexpression of HOXB4", STEM CELLS DEV, vol. 19, 2010, pages 1687 - 1698
HAAS ET AL.: "Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors", CELL STEM CELL, vol. 17, 2015, pages 422
HARRISON ET AL.: "Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations", EXP HEMATOL, vol. 21, 1993, pages 206 - 219, XP000568703
HARRISON, D.E.; ZHONG, R.K.: "The same exhaustible multilineage precursor produces both myeloid and lymphoid cells as early as 3-4 weeks after marrow transplantation", PROC NATL ACAD SCI U S A, vol. 89, 1992, pages 10134 - 10138
HE, Q.; ZHANG, C.; WANG, L.; ZHANG, P.; MA, D.; LV, J.; LIU, F.: "Inflammatory signalling regulates hematopoietic stem and progenitor cell emergence in vertebrates", BLOOD, vol. 125, 2015, pages 1098 - 1106
HILLS D. ET AL.: "Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis", BLOOD, vol. 117, no. 13, 31 March 2011 (2011-03-31), pages 3521 - 3528, XP055416571 *
HILLS ET AL.: "Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis", BLOOD, vol. 117, 2011, pages 3521 - 3528
HOGGATT, J.; PELUS, L.M.: "Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment", STEM CELL RES THER, vol. 2, 2011, pages 13
IRION ET AL.: "Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells", DEVELOPMENT, vol. 137, 2010, pages 2829 - 2839, XP055069663, DOI: doi:10.1242/dev.042119
IVANOVS ET AL.: "Identification of the niche and phenotype of the first human hematopoietic stem cells", STEM CELL REPORTS, vol. 2, 2014, pages 449 - 456
JONES ET AL.: "Two phases of engraftment established by serial bone marrow transplantation in mice", BLOOD, vol. 73, 1989, pages 397 - 401
KANJI, S.; POMPILI, V.J.; DAS, H.: "Plasticity and maintenance of hematopoietic stem cells during development", RECENT PAT BIOTECHNOL, vol. 5, 2011, pages 40 - 53
KAUFMAN ET AL.: "Hematopoietic colony-forming cells derived from human embryonic stem cells", PROC NATL ACAD SCI U S A, vol. 98, 2001, pages 10716 - 10721, XP002308712, DOI: doi:10.1073/pnas.191362598
KELLER ET AL.: "Hematopoietic commitment during embryonic stem cell differentiation in culture", MOL CELL BIOL, vol. 13, 1993, pages 473 - 486
KIM, S.J.; LETTERIO, J.: "Transforming growth factor-beta signalling in normal and malignant hematopoiesis", LEUKEMIA, vol. 17, 2003, pages 1731 - 1737
KINGSLEY ET AL.: "Yolk sac-derived primitive erythroblasts enucleate during mammalian embryogenesis", BLOOD, vol. 104, 2004, pages 19 - 25
KOBARI ET AL.: "Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model", HAEMATOLOGICA, vol. 97, 2012, pages 1795 - 1803
KULESHOV ET AL.: "Enrichr: a comprehensive gene set enrichment analysis web server 2016 update", NUCLEIC ACIDS RES., 2016
KYBA ET AL.: "HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors", CELL, vol. 109, 2002, pages 29 - 37, XP055004824, DOI: doi:10.1016/S0092-8674(02)00680-3
LANCRIN ET AL.: "Blood cell generation from the hemangioblast", J MOL MED (BERL, vol. 88, 2010, pages 167 - 172, XP019791480
LANCRIN ET AL.: "GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment", BLOOD, vol. 120, 2012, pages 314 - 322
LANCRIN ET AL.: "The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage", NATURE, vol. 457, 2009, pages 892 - 895, XP055047769, DOI: doi:10.1038/nature07679
LAWRENCE ET AL.: "The role of HOX homeobox genes in normal and leukemic hematopoiesis", STEM CELLS, vol. 14, 1996, pages 281 - 291, XP002094040
LEDRAN ET AL.: "Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches", CELL STEM CELL, vol. 3, 2008, pages 85 - 98
LEE ET AL.: "ER71 acts downstream of BMP, Notch, and Wnt signalling in blood and vessel progenitor specification", CELL STEM CELL, vol. 2, 2008, pages 497 - 507, XP002717071, DOI: doi:10.1016/j.stem.2008.03.008
LEE, D.; KIM, T.; LIM, D.S.: "The Er71 is an important regulator of hematopoietic stem cells in adult mice", STEM CELLS, vol. 29, 2011, pages 539 - 548
LENSCH, M.W.: "An evolving model of hematopoietic stem cell functional identity", STEM CELL REV, vol. 8, 2012, pages 551 - 560, XP035063408, DOI: doi:10.1007/s12015-012-9347-x
LI ET AL.: "Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells", STEM CELLS DEV, vol. 14, 2005, pages 44 - 54
LI, Z.; CHEN, M.J.; STACY, T.; SPECK, N.A.: "Runx1 function in hematopoiesis is required in cells that express Tek", BLOOD, vol. 107, 2006, pages 106 - 110
LI, Z.; LI, L.: "Understanding hematopoietic stem-cell microenvironments", TRENDS BIOCHEM SCI, vol. 31, 2006, pages 589 - 595, XP025132590, DOI: doi:10.1016/j.tibs.2006.08.001
LIAKHOVITSKAIA ET AL.: "Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive hematopoietic stem cells and extends life of Runx1 knockout animals until birth", STEM CELLS, vol. 27, 2009, pages 1616 - 1624
LIN ET AL.: "CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway", PLOS BIOL, vol. 9, 2011, pages e1001148
LIZAMA ET AL.: "Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition", NAT COMMUN, vol. 6, 2015, pages 7739
MARIANO ET AL.: "Adult stem cells in neural repair: Current options, limitations and perspectives", WORLD J STEM CELLS, vol. 7, 2015, pages 477 - 482
MATSUBARA ET AL.: "Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development", J EXP MED, vol. 202, 2005, pages 1483 - 1492, XP055103110, DOI: doi:10.1084/jem.20051325
MATSUMOTO ET AL.: "Stepwise development of hematopoietic stem cells from embryonic stem cells", PLOS ONE, vol. 4, 2009, pages e4820
MATSUOKA ET AL.: "Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aorta-gonad-mesonephros region-derived stromal cells", BLOOD, vol. 98, 2001, pages 6 - 12, XP001155761, DOI: doi:10.1182/blood.V98.1.6
MCGRATH ET AL.: "Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo", CELL REP, vol. 11, 2015, pages 1892 - 1904
MCGRATH ET AL.: "Expression of homeobox genes, including an insulin promoting factor, in the murine yolk sac at the time of hematopoietic initiation", MOL REPROD DEV, vol. 48, 1997, pages 145 - 153
MCKINNEY-FREEMAN ET AL.: "Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells", BLOOD, vol. 114, 2009, pages 268 - 278
MCKINNEY-FREEMAN ET AL.: "The transcriptional landscape of hematopoietic stem cell ontogeny", CELL STEM CELL, vol. 11, 2012, pages 701 - 714
MENDELSON, A.; FRENETTE, P.S.: "Hematopoietic stem cell niche maintenance during homeostasis and regeneration", NAT MED, vol. 20, 2014, pages 833 - 846, XP055283934, DOI: doi:10.1038/nm.3647
MITJAVILA-GARCIA ET AL.: "Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells", DEVELOPMENT, vol. 129, 2002, pages 2003 - 2013
MOOTHA ET AL.: "PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes", NATURE GENETICS, vol. 34, 2003, pages 267 - 273
MORRISON ET AL.: "Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization", PROC NATL ACAD SCI U S A, vol. 94, 1997, pages 1908 - 1913
MORRISON, S.J.; SPRADLING, A.C.: "Stem cells and niches: mechanisms that promote stem cell maintenance throughout life", CELL, vol. 132, 2008, pages 598 - 611
MULLER ET AL.: "Development of hematopoietic stem cell activity in the mouse embryo", IMMUNITY, vol. 1, 1994, pages 291 - 301, XP024247802, DOI: doi:10.1016/1074-7613(94)90081-7
MULLER, A.M.; DZIERZAK, E.A.: "ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients", DEVELOPMENT, vol. 118, 1993, pages 1343 - 1351
NICHOLS ET AL.: "Validated germline-competent embryonic stem cell lines from nonobese diabetic mice", NAT MED, vol. 15, 2009, pages 814 - 818, XP055033151, DOI: doi:10.1038/nm.1996
NISHIMOTO ET AL.: "The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2", MOL CELL BIOL, vol. 19, 1999, pages 5453 - 5465, XP002155941
NOTTA ET AL.: "Distinct routes of lineage development reshape the human blood hierarchy across ontogeny", SCIENCE, vol. 351, 2016, pages aab2116
NWAJEI, F.; KONOPLEVA, M.: "The bone marrow microenvironment as niche retreats for hematopoietic and leukemic stem cells", ADV HEMATOL, 2013, pages 953982
OGAWA ET AL.: "Expression and function of c-kit in hemopoietic progenitor cells", J EXP MED, vol. 174, 1991, pages 63 - 71, XP000700180, DOI: doi:10.1084/jem.174.1.63
OH, I.H.; KWON, K.R.: "Concise review: multiple niches for hematopoietic stem cell regulations", STEM CELLS, vol. 28, 2010, pages 1243 - 1249
OKUDA ET AL.: "AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis", CELL, vol. 84, 1996, pages 321 - 330
ORKIN, S.H.: "Diversification of haematopoietic stem cells to specific lineages", NAT REV GENET, vol. 1, 2000, pages 57 - 64
ORKIN, S.H.; ZON, L.I.: "Hematopoiesis: an evolving paradigm for stem cell biology", CELL, vol. 132, 2008, pages 631 - 644
OSHIMA ET AL.: "Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic stem and progenitor cells developing from embryonic stem cells", BLOOD, vol. 117, 2011, pages e142 - 150
PEARSON ET AL.: "The sequential expression of CD40 and Icam2 defines progressive steps in the formation of blood precursors from the mesoderm germ layer", STEM CELLS, vol. 28, 2010, pages 1089 - 1098
PEARSON ET AL.: "The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF", DEVELOPMENT, vol. 135, 2008, pages 1525 - 1535, XP008092701, DOI: doi:10.1242/dev.011767
PEREIRA ET AL.: "Hematopoietic Reprogramming In Vitro Informs In Vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells", DEV CELL, vol. 36, 2016, pages 525 - 539
PETIT-COCAULT ET AL.: "Dual role of Mpl receptor during the establishment of definitive hematopoiesis", DEVELOPMENT, vol. 134, 2007, pages 3031 - 3040
PINEAULT ET AL.: "Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny", EXP HEMATOL, vol. 30, 2002, pages 49 - 57, XP002989802, DOI: doi:10.1016/S0301-472X(01)00757-3
PORCHER ET AL.: "The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages", CELL, vol. 86, September 1996 (1996-09-01), pages 47 - 57
POTTEN, C.S.; BOOTH, C.; PRITCHARD, D.M.: "The intestinal epithelial stem cell: the mucosal governor", INT J EXP PATHOL, vol. 78, 1997, pages 219 - 243
PULECIO ET AL.: "Conversion of human fibroblasts into monocyte-like progenitor cells", STEM CELLS, vol. 32, 2014, pages 2923 - 2938
RAMOS-MEJIA ET AL.: "HOXA9 promotes hematopoietic commitment of human embryonic stem cells", BLOOD, vol. 124, 2014, pages 3065 - 3075
RAN, F.A.; HSU, P.D.; WRIGHT, J.; AGARWALA, V.; SCOTT, D.A.; ZHANG, F.: "Genome engineering using the CRISPR-Cas9 system", NAT PROTOC, vol. 8, 2013, pages 2281 - 2308, XP009174668, DOI: doi:10.1038/nprot.2013.143
RIDDELL ET AL.: "Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors", CELL, vol. 157, 2014, pages 549 - 564, XP055329010, DOI: doi:10.1016/j.cell.2014.04.006
ROBERT-MORENO, A.; ESPINOSA, L.; POMPA, J.L.; BIGAS, A.: "RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells", DEVELOPMENT, vol. 132, 2005, pages 1117 - 1126
ROBIN ET AL.: "Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development", CELL STEM CELL, vol. 5, 2009, pages 385 - 395, XP009135553, DOI: doi:10.1016/j.stem.2009.08.020
ROSSI ET AL.: "Less is more: unveiling the functional core of hematopoietic stem cells through knockout mice", CELL STEM CELL, vol. 11, 2012, pages 302 - 317
ROWE, R.G.; MANDELBAUM, J.; ZON, L.I.; DALEY, G.Q.: "Engineering Hematopoietic Stem Cells: Lessons from Development", CELL STEM CELL, vol. 18, 2016, pages 707 - 720, XP029567594, DOI: doi:10.1016/j.stem.2016.05.016
RUIZ-HERGUIDO ET AL.: "Hematopoietic stem cell development requires transient Wnt/beta-catenin activity", J EXP MED, vol. 209, 2012, pages 1457 - 1468
RYBTSOV ET AL.: "Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region", J EXP MED, vol. 208, 2011, pages 1305 - 1315
SANDLER ET AL.: "Reprogramming human endothelial cells to haematopoietic cells requires vascular induction", NATURE, vol. 511, 2014, pages 312 - 318, XP055328630, DOI: doi:10.1038/nature13547
SAUVAGEAU ET AL.: "Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells", PROC NATL ACAD SCI U S A, vol. 91, 1994, pages 12223 - 12227, XP001145771, DOI: doi:10.1073/pnas.91.25.12223
SAWAMIPHAK ET AL.: "Interferon gamma signalling positively regulates hematopoietic stem cell emergence", DEV CELL, vol. 31, 2014, pages 640 - 653, XP029111600, DOI: doi:10.1016/j.devcel.2014.11.007
SCHIEDLMEIER ET AL.: "HOXB4's road map to stem cell expansion", PROC NATL ACAD SCI U S A, vol. 104, 2007, pages 16952 - 16957
SCHLENNER ET AL.: "Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus", IMMUNITY, vol. 32, 2010, pages 426 - 436
SCHROEDER ET AL.: "Stem cells for spine surgery", WORLD J STEM CELLS, vol. 7, 2015, pages 186 - 194
SHIBATA ET AL.: "Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype", STEM CELLS, vol. 27, 2009, pages 183 - 190
SHIVDASANI ET AL.: "Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL", NATURE, vol. 373, 1995, pages 432 - 434
SINGBRANT ET AL.: "Two new routes to make blood: Hematopoietic specification from pluripotent cell lines versus reprogramming of somatic cells", EXP HEMATOL, vol. 43, 2015, pages 756 - 759, XP029260418, DOI: doi:10.1016/j.exphem.2015.05.007
SLUKVIN I.I.: "Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells", CELL CYCLE, vol. 12, no. 5, 1 March 2013 (2013-03-01), pages 720 - 727, XP055128571 *
SOLAIMANI KARTALAEI ET AL.: "Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation", J EXP MED, vol. 212, 2015, pages 93 - 106
SPINELLI ET AL.: "BubbleGUM: automatic extraction of phenotype molecular signatures and comprehensive visualization of multiple Gene Set Enrichment Analyses", BMC GENOMICS, vol. 16, 2015, pages 814, XP021229230, DOI: doi:10.1186/s12864-015-2012-4
SUBRAMANIAN ET AL.: "Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles", PROC NATL ACAD SCI U S A, vol. 102, 2005, pages 15545 - 15550, XP002464143, DOI: doi:10.1073/pnas.0506580102
SUZUKI ET AL.: "Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation", MOLECULAR THERAPY : THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY, vol. 21, 2013, pages 1424 - 1431, XP055213298, DOI: doi:10.1038/mt.2013.71
SZABO ET AL.: "Direct conversion of human fibroblasts to multilineage blood progenitors", NATURE, vol. 468, 2010, pages 521 - 526
SZKLARCZYK ET AL.: "STRING v10: protein-protein interaction networks, integrated over the tree of life", NUCLEIC ACIDS RES, vol. 43, 2015, pages 447 - 452
TAKAHASHI ET AL.: "Induction of pluripotent stem cells from adult human fibroblasts by defined factors", CELL, vol. 131, 2007, pages 861 - 872, XP008155962, DOI: doi:10.1016/j.cell.2007.11.019
TAKAKURA ET AL.: "Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis", IMMUNITY, vol. 9, 1998, pages 677 - 686, XP000907291, DOI: doi:10.1016/S1074-7613(00)80665-2
TAKAYANAGI ET AL.: "Genetic marking of hematopoietic stem and endothelial cells: identification of the Tmtsp gene encoding a novel cell surface protein with the thrombospondin-1 domain", BLOOD, vol. 107, 2006, pages 4317 - 4325
TERSKIKH ET AL.: "Gene expression analysis of purified hematopoietic stem cells and committed progenitors", BLOOD, vol. 102, 2003, pages 94 - 101
TESAR, P.J.: "Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos", PROC NATL ACAD SCI USA, 2005, pages 8239 - 8244
THAMBYRAJAH ET AL.: "GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1", NAT CELL BIOL, vol. 18, 2016, pages 21 - 32
TILL, J.E.; MC, C.E.: "A direct measurement of the radiation sensitivity of normal mouse bone marrow cells", RADIAT RES, vol. 14, 1961, pages 213 - 222
VAN PEL ET AL.: "CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells", HAEMATOLOGICA, vol. 93, 2008, pages 1137 - 1144
VO, L.T.; DALEY, G.Q.: "De novo generation of HSCs from somatic and pluripotent stem cell sources", BLOOD, vol. 125, 2015, pages 2641 - 2648
WANG, Y.; YATES, F.; NAVEIRAS, O.; ERNST, P.; DALEY, G.Q.: "Embryonic stem cell-derived hematopoietic stem cells", PROC NATL ACAD SCI U S A, vol. 102, 2005, pages 19081 - 19086
WAREING ET AL.: "ETV2 expression marks blood and endothelium precursors, including hemogenic endothelium, at the onset of blood development", DEV DYN, vol. 241, 2012, pages 1454 - 1464
WEISEL ET AL.: "Stromal cell lines from the aorta-gonado-mesonephros region are potent supporters of murine and human hematopoiesis", EXP HEMATOL, vol. 34, 2006, pages 1505, XP025017598, DOI: doi:10.1016/j.exphem.2006.06.013
WESSEL, D.; FLUGGE, U.I.: "A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids", ANAL BIOCHEM, vol. 138, 1984, pages 141, XP024764511, DOI: doi:10.1016/0003-2697(84)90782-6
WILSON ET AL.: "Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair", CELL, vol. 135, 2008, pages 1118 - 1129
YAMANE ET AL.: "Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development", PROC NATL ACAD SCI U S A, vol. 106, 2009, pages 8953 - 8958
YING ET AL.: "The ground state of embryonic stem cell self-renewal", NATURE, vol. 453, 2008, pages 519 - 523, XP055033153, DOI: doi:10.1038/nature06968
YODER ET AL.: "In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus", PROC NATL ACAD SCI U S A, vol. 94, 1997, pages 6776 - 6780
YOKOMIZO ET AL.: "Runx1 is involved in primitive erythropoiesis in the mouse", BLOOD, vol. 111, 2008, pages 4075 - 4080
YOKOTA ET AL.: "The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice", BLOOD, vol. 113, 2009, pages 2914 - 2923
YOSHIHARA ET AL.: "Thrombopoietin/MPL signalling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche", CELL STEM CELL, vol. 1, 2007, pages 685 - 697, XP055072923, DOI: doi:10.1016/j.stem.2007.10.020
ZAMBIDIS ET AL.: "Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development", BLOOD, vol. 106, 2005, pages 860 - 870
ZHANG ET AL.: "Expression of podocalyxin separates the hematopoietic and vascular potentials of mouse embryonic stem cell-derived mesoderm", STEM CELLS, vol. 32, 2014, pages 191 - 203
ZHANG ET AL.: "High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 118, 2008, pages 1502 - 1510
ZHONG ET AL.: "Distinct developmental patterns of short-term and long-term functioning lymphoid and myeloid precursors defined by competitive limiting dilution analysis in vivo", J IMMUNOL, vol. 157, 1996, pages 138 - 145
ZHOU ET AL.: "A gene regulatory network in mouse embryonic stem cells", PROC NATL ACAD SCI U S A, vol. 104, 2007, pages 16438 - 16443
ZOVEIN ET AL.: "Fate tracing reveals the endothelial origin of hematopoietic stem cells", CELL STEM CELL, vol. 3, 2008, pages 625 - 636, XP002613103, DOI: doi:10.1016/j.stem.2008.09.018

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11591390B2 (en) 2018-09-27 2023-02-28 Celgene Corporation SIRP-α binding proteins and methods of use thereof
US12084499B2 (en) 2018-09-27 2024-09-10 Celgene Corporation SIRP-α binding proteins and methods of use thereof
CN110372786A (zh) * 2019-06-24 2019-10-25 南昌大学 用于制备mPRα单克隆抗体的免疫原
CN110372786B (zh) * 2019-06-24 2021-08-24 南昌大学 用于制备mPRα单克隆抗体的免疫原

Similar Documents

Publication Publication Date Title
Ng et al. Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros
Ratajczak et al. Adult murine bone marrow-derived very small embryonic-like stem cells differentiate into the hematopoietic lineage after coculture over OP9 stromal cells
US20230323303A1 (en) Haematopoietic stem/progenitor cells
Kubaczka et al. Direct induction of trophoblast stem cells from murine fibroblasts
Fong et al. Human Wharton’s jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells
US11261430B2 (en) Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells
Golan-Mashiach et al. Design principle of gene expression used by human stem cells; implication for pluripotency
US12146162B2 (en) Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells by episomal plasmid gene transfer
Kucia et al. Adult marrow-derived very small embryonic-like stem cells and tissue engineering
WO2018037091A1 (fr) Procédés d'identification et d'isolement de cellules souches et progénitrices hématopoïétiques
US20210355441A1 (en) Generation of hoxa-expressing hemogenic endothelium with enhanced t cell potential from hpscs
US20130309209A1 (en) Formation of hematopoietic progenitor cells from mesenchymal stem cells
US9523074B2 (en) Sub-totipotent stem cell product and apparent hereditary modifying label thereof
Creamer The role of CDX4 during patterning of definitive hemogenic mesoderm
Creamer et al. CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential
WO2023169076A1 (fr) Cellules souches potentielles totipotentes induites, leurs procédés de fabrication et d'utilisation
Fernandez The Role of Retinoic Acid Signaling in Development of the Human Embryonic Hematopoietic System
Era Pluripotent stem cell as a source of mesenchymal stem cell
Pantier et al. TET knockout cells transit between pluripotent states and exhibit precocious germline entry
Marcoux RET-altered Induced Pluripotent Stem Cells (iPSCs) for modeling Hematopoiesis and Non-Small Cell Lung Cancer (NSCLC)
Frobel DNA methylation changes in hematopoietic development and iPSC-derived model systems
Crosse Spatial transcriptome analysis of the human developmental haematopoietic stem cell niche
Garcia Defining the Role of SOX17 in Human Hematopoietic Development
Ding Polycomb protein Bmi1 in ES cell hematopoiesis and generation of iPSC from Flt3+ hematopoietic stem cells
Lange Mixl1 regulatory networks in development and disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17757541

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17757541

Country of ref document: EP

Kind code of ref document: A1