WO2018037091A1

WO2018037091A1 - Methods for the identification and isolation of hematopoietic stem and progenitor cells

Info

Publication number: WO2018037091A1
Application number: PCT/EP2017/071372
Authority: WO
Inventors: Michael Dennis MILSOM; Paul KASCHUTNIG
Original assignee: HI-STEM GGMBH
Current assignee: HI-STEM GGMBH
Priority date: 2016-08-24
Filing date: 2017-08-24
Publication date: 2018-03-01
Anticipated expiration: 2019-02-24

Abstract

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.

Description

METHODS FOR THE IDENTIFICATION AND ISOLATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS

FIELD OF THE INVENTION

[0001] 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.

BACKGROUND OF THE INVENTION

[0002] Adult 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).

[0003] Transplantation of tissue-specific stem cells, aiming to replace damaged or aged cells, holds great promise for the treatment of various malignancies. In fact, hematopoietic stem cells (HSCs) have been used in transplantation-based treatment of hematologic diseases including distinct forms of leukaemia, lymphoma and myeloma since the late 1960s (Bortin, 1970; Gatti et al., 1968). Besides the transplantation of HSCs, which represents by far the most commonly used stem cell- based therapy approach, epidermal skin stem cells are clinically used to grow sheets of new skin for severe burn patients. Other potential therapy approaches involve the use of mesenchymal stem cells for cartilage and bone repair as well as the repair of blood vessels after heart attacks and ischemic strokes. Moreover the Food and Drug Administration (FDA) recently approved two clinical trials using neural stem cells in patients that suffer from Parkinson's disease and spinal cord injury (Mariano et al., 2015; Schroeder et al., 2015). Hence, the areas for stem cell-based therapy are diverse and the field has grown dramatically over the last years and will continue to do so.

[0004] Despite their great therapeutic potential, all 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.

[0005] To date, adult bone marrow, mobilized peripheral blood and the neonatal umbilical cord represent the main sources of HSCs routinely used in clinical transplantation therapy. However, finding HLA-matched donors and attaining sufficient numbers of LT-HSCs in the graft significantly hinders large-scale HSC therapy. The lack of compatible cells for therapeutic application could be alleviated via the efficient generation of autologous HSCs from a patient's own cells. The resulting scalable source of 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. Without the ability to maintain and expand HSCs in vitro, two complementary strategies of blood cell derivation have emerged, one of them being the hematopoietic differentiation of pluripotent stem cells (PSCs) and the other one the directed conversion of mature somatic cells into hematopoietic progenitors.

[0006] Additionally, several approaches for an in vivo differentiation of pluripotent cells into HSCs have been pursued. The first approach puts pluripotent cells (PSCs) like embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into the spotlight. These cell types are capable of differentiating into nearly every cell type of the organism, including blood 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).

[0007] Most protocols currently used for hematopoietic differentiation of PSCs rely on simulating the environment of embryonic HSC differentiation by step-wise addition of cytokines during differentiation of PSCs in three-dimensional structures known as embryoid bodies (EBs) as well as co-culture on supporting stromal cells (Kaufman et al., 2001 ; Ledran et al., 2008; Zambidis et al., 2005). Although some success in producing mature blood cells (macrophages, dendritic cells and erythrocytes) has been demonstrated, to date the efficient in vitro generation of robust multi-lineage hematopoietic progenitors remains elusive (Choi et al., 201 1 ; Kobari et al., 2012).

[0008] In-depth kinetic analysis of ESC-derived blood cell formation revealed that the developmental steps in EBs largely recapitulated primitive hematopoiesis generating cells that lack robust and durable repopulation capacity (Keller et al., 1993). This deficiency is believed to reflect an immature developmental state of the in vitro differentiated cells, a hypothesis that was further supported by the fact that primitive embryonic blood progenitors can acquire definitive engraftment potential upon transplantation into neonates or culturing on AGM-derived stromal cells (Matsuoka et al., 2001 ; Muller and Dzierzak, 1993; Yoder et al., 1997).

[0009] Reports of several groups on variable hematopoietic differentiation potential of different PSCs under identical culture conditions shifted the scientific focus to the analysis of the intrinsic cellular signalling of developing hematopoietic cells. It was hypothesized that potential developmental barriers during PSC-derived specification into definitive HSCs could be broken by manipulation of identified master regulatory transcription factors (TFs).

[0010] 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). Despite the fact that the donor grafts demonstrated significant myeloid skewing, indicating that HOXB4 expression alone is not sufficient to fully convert ESC-derived progenitors, these seminal studies marked the starting point for the induction of HSCs by manipulation of transcriptional regulators. In line with this, recent studies confirmed a lack of retinoic acid signalling-induced expression of Hoxa cluster genes in ESC-derived hematopoietic progenitors indicating that members of the Hoxa gene family (e.g. Hoxa5, Hoxa7 & Hoxa9) might play an even more important role during the switch to definitive hematopoiesis (Ramos-Mejia et al., 2014) (Dou et al., 2016).

[0011] Over the last years, various groups used different strategies including ectopic expression of single individual TFs and more recently also combinatorial sets of TFs in order to enhance hematopoietic differentiation from PSCs (Table 1) (Figure 6) (Vo and Daley, 2015).

[0012] As an alternative route, the directed conversion of mature somatic cells into HSCs has been studied. Reprogramming of cells has most prominently been demonstrated by the conversion of fully differentiated somatic cells into iPSCs by the exogenous expression of just four TFs (Takahashi et al., 2007). Based on this and even earlier studies, TF-based cellular reprogramming has been used to produce hematopoietic progenitor cells from different starting cells.

[0013] Several studies in 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). Although these studies confirmed that reducing the epigenetic barrier is beneficial to the in vitro conversion process, definitive progenitors termed induced-HSCs (i-HSCs) were only obtained if the targeted cells where returned to the hematopoietic inductive microenvironment in vivo, highlighting the importance of extrinsic signalling during maturation of HSPCs (Riddell et al., 2014; Sandler et al., 2014). The requirement of complex experimental steps like ex vivo transcription factor delivery and subsequent in vivo expansion would make this type of strategy difficult to routinely replicate in the clinical setting (Easterbrook et al., 2016).

[0014] Recently, a completely different strategy to obtain hPSC-derived hematopoietic progenitors has 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). In a similar report 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). The thereby generated cells demonstrated long-term reconstitution potential over serial transplantations into irradiated mice. The variable generation of HSC-like cells from teratomas and the initial tumour formation from injection of PSCs are obvious limitations to this differentiation strategy. Nevertheless, these studies once more confirmed that definitive HSCs with multi-lineage differentiation potential can in principle be derived from pluripotent stem cells (Vo and Daley, 2015).

[0015] Conclusively, the most recent studies demonstrate the undoubted progress if the field of HSC specification. However it is equally obvious that we are still some way from achieving large-scale production of fully functional HSCs for therapeutic application in the clinics.

[0016] The lack of success in generating definitive PSC-derived HSCs is most likely rooted in a failure to precisely recapitulate conditions present during in vivo HSC development. Mammalian embryonic hematopoiesis occurs in a stepwise process across distinct developmental waves within different hematopoietic organs. The "immature" nature of in vitro differentiated hematopoietic cells may be due to them arising from the first or second hematopoietic wave in the yolk sac. A more comprehensive understanding of the characteristic extracellular and intracellular signalling during the definitive wave of hematopoiesis as well as the identification of cell surface markers on developing HSCs and their precursors might provide vital reference points for future in vitro HSC production (Ivanovs et al., 2014; Rybtsov et al., 201 1).

[0017] On a different note, many of the constitutively expressed TFs currently used in hematopoietic programming studies represent potent proto-oncogenes. The safe transfer of PSC-derived HSCs to the clinics requires the development of dynamic protocols based on inducible and transient TF expression. Precise orchestration of extrinsic and intrinsic signals mimicking the spatial, temporal and mechanical environment during mammalian embryogenesis must be achieved in regard to a prospective efficient in vitro HSC production (Easterbrook et al., 2016).

[0018] Thus, despite certain progress that has been made in the identification and generation of HSCs, there is still no robust way for identifying and isolating hematopoietic stem and progenitor cells (HSPCs). [0019] The solution to this problem, i.e. the identification of cell-surface markers that characterize hematopoietic stem and progenitor cells (HSPCs) and that can be used to identify and isolate hematopoietic stem and progenitor cells (HSPCs), are neither provided nor suggested by the prior art.

OBJECTS OF THE INVENTION

[0020] It was thus an object of the invention to provide a novel approach for the identification of markers on hematopoietic stem and progenitor cells (HSPCs) that can be used to detect such cells, to further characterize the cells, their generation and differentiation and the enrichment and/or isolation of such cells from cell populations comprising such cells.

SUMMARY OF THE INVENTION

[0021] Surprisingly it has been found that a number of cell-surface markers can be identified that are characteristic of hematopoietic stem and progenitor cells (HSPCs) and that define a particular subpopulation of such cells.

[0022] Thus, in one aspect, 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:

(a) 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 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; and, optionally,

(b) 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).

[0023] In a second aspect, the present invention relates to a method for the isolation of hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:

(a) identifying hematopoietic stem and progenitor cells (HSPCs) in a cell population by using the identification method of the present invention; and

(b) selecting hematopoietic stem and progenitor cells (HSPCs) that have been identified in step (a).

[0024] In a third aspect, the present invention relates to a method for isolating hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:

(a) culturing embryonic stem cells (ESCs) in vitro under hypoxic conditions (5% 0₂) for 2 to 3 days, in particular for 2.5 days;

(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);

(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

(d) performing one or more selection steps for cells expressing one or more 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 , 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 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, ΤΊΕ1 , IFITM1 and IFITM3; and, optionally,

(e) performing one or more selection steps for cells additionally expressing one or more cell surface proteins 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); and

(f) collecting the cells selected in steps (d) and optionally (e).

[0025] In a fourth aspect, 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 TYROBP; in particular 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, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; particularly characterized by the presence of at least at least six, particularly at least eight, particularly at least ten cell surface proteins selected from said list.

[0026] In a fifth aspect, 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 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3.

[0027] In a sixth aspect, 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, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; and wherein optionally, at least one antibody is directed against a cell surface protein 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).

FIGURES

[0028] 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).

[0029] 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).

[0030] 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).

[0031] 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 β-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).

[0032] 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. Abbreviations: 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 was taken from Orkin and Zon, 2008).

[0033] 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, and 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. (Figure was taken from Vo and Daley, 2015).

[0034] Figure 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). 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).

[0035] 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). (C) Southern blot of Seal-digested DNA probed with 3' probe (P3). Clone 30 is the primary targeted clone. Clones 30/7 and 30/8 were recovered after transient removal of the selection cassette from clone 30. The lower band in both blots represents the wild-type untargeted allele (BamHI, 4.4 kb; Seal, 9.7 kb). YFP in open box marks the Venus open reading frame. Bsd in open box marks the PGK/EM7 dual eukaryotic/prokaryotic promoter driven blasticidin selection cassette. B indicates BamHI; H, Hindlll; S, Seal. (Figure taken from Hills et al., 2011 ).

[0036] 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. 33 morulae were isolated from (E0.5) oviducts of pregnant females and plated out on MEF feeder cells under stem cell conditions (2i/LIF) (Nichols et al., 2009; Ying et al., 2008). 15 individual ES cell lines were subcloned. (C) Genotyping of individual ESC clones. The 170 bp and the 400 bp DNA bands represent the wild-type (WT) and the transgenic (TG) allele, respectively. As indicated, 10 heterozygous (+/TG), 2 homozygous (TG/TG) and 3 WT control (+/+) ESC lines were generated. (D) Histological analysis of teratomas derived from Hoxb4-YFP ES cell injection. Formation of all three germ layers could be observed. 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). ESC derivation and validation experiments performed by MSc. Irem Bayindir-Wagenknecht.

[0037] 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. (B) Representative FACS contour plots illustrating distribution of CD41 (ITGA2B) and CD1 17 (KIT) expressing cells on day 4-6 of EB differentiation (n = 3). (C) Mean distribution of in vitro differentiated cells across CD41 and KIT expression quadrants for individually tested transgenic Hoxb4-YFP (+/TG) and WT (+/+) ESC clones. Error bars depict mean ± SD (n = 3). (D) Representative FACS contour plots illustrating the increase in Hoxb4- YFP signal (fluorescence signal shift) between day 4 (D4) and day 5 (D5) of EB differentiation followed by a slight decrease in percentage of HOXB4 expressing cells on day 6 (D6). Gating based on non-reporter WT (+/+) ESC clone. (E) Mean percentages of HOX-YFP+ cells on day 4-6 of differentiation in comparison to WT (+/+) ESC controls. Error bars depict mean ± SD, n = 3. ^****P < 0.0001 ; ns = P > 0.05, not significant; by unpaired (two-tailed) t-test.

[0038] Figure 11 shows the gene expression analysis and functional characterization of in vitro specified HOXB4+ cells: (A) qRT-PCR-based analysis comparing relative hematopoietic gene expression levels of HOXB4+ and HOXB4- (normalized to 1) cells on day 6 (D6) of EB differentiation (n = 3). (B) Analyses of hematopoietic progenitor proliferation and differentiation potential of sorted HOXB4+ & HOXB4- cells, represented by number of colony-forming units (CFUs). Sorted cells were plated in cytokine-supplemented methylcellulose medium (100 K/ml) on EB differentiation stage D5 and D6 (n = 3). (C) Colony-forming unit (CFUs) count of cells sorted for additional hematopoietic marker proteins within the HOXB4+ cell fraction on D6 of EB differentiation (100 K/ml) (n = 3). (D) Number of hematopoietic colonies formed by 00K HOXB4+ and HOXB4- cells (D6) after co-culture on OP9 stromal cell feeder layers for 7 days (n = 3). (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..

[0039] Figure 12 shows the immunophenotypic kinetics of in vitro differentiated of HOXB4+ cells: (A) Percentages of differentiated cells consecutively expressing endothelial and hematopoietic cell surface markers across day 4-6 of in vitro EB differentiation frequently analysed by FACS (every 12 h). Error bars depict mean ± SD, n = 3. (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. Immunophenotypic correlation of each timepoint of differentiation is illustrated by overlap of white (Live cells; 7AAD-), green (HOXB4+) and individually coloured circles (CSMs) (n = 3). Circle size represents percentage of marker-positive cells. Overlap of HOXB4 and individual marker protein expression is additionally depicted in column graphs. SP = single positive cells; DP = double positive cells. Coloured frames represent consecutive developmental profiles during in vitro differentiation..

[0040] Figure 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. (B) Percentage of HOXB4+ cells after continued differentiation (24-72 hours) of individually sorted cell fractions, representing different stages of hematopoietic development. Error bars depict mean ± SD, n = 3. (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. (D-E) Clonogenic colony formation analysis (MethoCult & OP9) and gene expression analysis of developmentally consecutive Upstream, HOXB4+ and Downstream cells during in vitro EB differentiation. Error bars depict mean ± SD, n = 3. ^*P < 0.05; **P <0.01 ; **^*P < 0.001 ; by unpaired (two-tailed) t-test.

[0041] 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-). HOXB4hi cells represent the most pronounced definitive hematopoietic progenitor phenotype and comprise a subpopulation expressing the early AGM- and FL-HSC marker AA4.1 (1 % of total cells) (n = 3). (B) 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..

[0042] 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). (C) Microarray-based heat maps (Chipster v3.8) depicting relative expression values of individual genes expressed during hematopoietic HSPC development, TCR/BCR signalling and inflammatory signalling across distinct stages of hematopoietic in vitro differentiation including ESC, Upstream, HOXB4hi and HOXB4+ AA4.1 + cells (n = 3). Mean transcript expression levels are represented by colour gradient (red = high expression; blue = low expression). (E-H) Microarray-based expression kinetics (normalized expression values) of individual transcripts characteristic for definitive hematopoietic development, HSCs, TCR/BCR signalling and inflammatory signalling across the ESC, Upstream and HOXB4+ AA4.1 + double positive stages of in vitro EB differentiation. Error bars depict mean ± SD, n = 3..

[0043] Figure 16 shows the molecular profile of in vitro differentiated HOXB4+ AA4.1 + cells strongly resembles profile of AGM- and FL-HSCs: (A) Combined GSEA analysis (BubbleGum) demonstrated a significant overlap in HSC-, TCR/BCR- and inflammatory gene signature expression between in vitro differentiated HOXB4+ AA4.1 + and isolated HOXB4+ BM-HSCs in comparison to Up- and Downstream cells. 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. Statistical significance of 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. Statistical significance of GSEA was assessed using 1.000 permutations. NES, normalized enrichment score; FDR, false discovery rate. (C) Comparative gene list enrichment analysis (Enrichr^®) of HOXB4+ AA4.1 + cells against Upstream/Downstream cell populations. Ranking according to combined Enrichr^® score, representing a combination of calculated p-value and z-score (Chen et al., 2013).

[0044] Figure 17 shows the identification of CSM encoding genes coexpressed on HOXB4+ AA4.1+ cells: (A) Correlation plots illustrating differential gene expression between HOXB4+ AA4.1 + cells and corresponding Upstream and Downstream cell populations. 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. Subsequent screening for cell surface marker-encoding genes was conducted via the web-based Surfaceome database. (B) Schematic outline of CSM identification and validation workflow. Microarray data was analysed for cell surface protein encoding transcripts and ranked based on level of regulation (FC) and novelty. Validation of selected genes was carried out via qRT-PCR (mRNA level) and MRM mass spectrometry (protein level). (C) Heatmap generated with GENE-E software package (Broad Institute) based on normalized microarray expression values depicting HOXB4+ AA4.1 +- specific CSM encoding transcripts in alphabetical order (n = 3). High, medium and low levels of transcript expression represented by colour gradient (red = high expression, blue = low expression). (D) qRT-PCR validation of a selection of 42 novel CSM encoding transcripts excluding genes not yielding robust amplicons (CD276, Tyrobp, Tnfrsf13b) as well as genes already described during hematopoietic development (CD34, CD41 , Vecad, Eng, Kit, Mpl). HOXB4+ AA4.1 + expression levels normalized to 1. Error bars depict mean ± SD, n = 3. ^*P < 0.05; ^**P <0.01 ; ^**^*P < 0.001 ; *^***P < 0.0001 ; by unpaired one-way ANOVA. [0045] Figure 18 shows MRM mass spectrometry based proteomic verification of cell surface proteins co-expressed on HOXB4+ AA4.1 + cells: (A) Column diagrams depicting relative expression levels of Multiple Reaction Monitoring (MRM)-detected cell surface proteins on sorted subpopulations representative for the consecutive stages of in vitro HSPC specification. MRM mass spectrometry experiments were conducted by Dr. Sabrina Hanke and Dr. Wiebke Nadler. Protein-specific oligopeptide sequences used for protein identification are indicated. ND = not detected. (B) Table depicting relevant information of undetected proteins as well as proteins without clear regulation across the distinct cell populations.).

[0046] Figure 19 shows the whole Proteome analysis of differentiated cell populations representing consecutive stages of in vitro HSPC specification: (A) Schematic outline of Hyper Reaction Monitoring (HRM) whole proteome mass spectrometry workflow depicting sorted cell populations and spectral peptide library content. Sorted biological replicates (n = 3) of each sample group were pooled in order to obtain sufficient cell numbers (1.5 x 106). Protein lysate preparation was conducted by Dr. Sabrina Hanke and Dr. Wiebke Nadler. HRM data acquisition and spectral library design conducted by Biognosys AG (Switzerland). (B) Two dimensional unsupervised hierarchical clustering of cell populations based on the individual protein expression levels. High, medium and low protein intensity values are represented by colour code (yellow = high expression, blue = low expression). (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).

[0047] Figure 20 shows the functional knockout of EVI2A and LYVE1 proteins result in severe hematopoietic differentiation defects: (A) Cross-correlation analysis of newly identified pre-selected cell surface proteins to whole transcriptome data encompassing HSC gene expression profiles throughout murine embryogenesis and adulthood (McKinney-Freeman et al., 2012). Marker proteins either predominantly expressed on early-stage HSCs (red box = YS, AGM, FL12.5) or strong overall expression during HSC development (green box) were selected for knockout studies. Heatmap (Gene-E) based on normalized microarray expression values of individual developmental stages (McKinney-Freeman et al., 2012). (B) 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. (D) Mean hematopoietic differentiation potential of knockout ES clones across multiple biological replicates assessed by FACS analysis (Percentage of CD41 + KIT+ cells). Error bars depict mean ± SD, n = 3. ^*P < 0.05; ^**P <0.01 ; ^****P < 0.0001 ; by unpaired (two-tailed) t-test.

[0048] 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. (B) Mean percentage as determined by FACS analysis and representative FACS contour plots CD41 + CD144+ expressing HECs after hematopoietic differentiation of EVI2A and LYVE1 KO ES clones in comparison to control cell populations. (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.

[0049] Figure 22 shows the developmental arrest in EVI2A and LYVE1 KO ES clones during EHT transition. [0050] 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.

[0051] 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+.

[0052] Figure 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.

[0053] 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.

[0054] 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.

[0055] 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.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0057] Thus, in one aspect, 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:

(b) 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).

[0058] In the context of the present invention, the term "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.

[0059] Within the hematopoietic system one can distinguish between two distinct types of hematopoietic stem cells defined by a slightly different immunophenotypic marker set. Upon transplantation into lethally irradiated mice, long-term HSCs (LT- HSCs) can reconstitute and sustain the hematopoietic system during the entire life span of the organism, whereas their direct cellular descendants, the short-term reconstituting HSCs (ST-HSCs), can maintain hematopoietic homeostasis only for several weeks post-transplantation (Harrison and Zhong, 1992; Jones et al., 1989; Zhong et al., 1996). HSCs represent a very rare cell population with an estimated frequency of 1 in 100.000 murine bone marrow (BM) cells (Harrison et al., 1993).

[0060] Further downstream of the 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. This includes the multipotent progenitor cells (MPPs), retaining full range differentiation capacity and giving rise to the lineage-restricted progenitors: common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), megakaryocyte/erythrocyte progenitors (MEPs) and granulocyte/macrophage progenitors (GMPs) (Akashi et al., 2000; Bell and Bhandoola, 2008; Schlenner et al., 2010).

[0061] However, the described hierarchical view on hematopoiesis, which is based on the assumption that the various blood cell lineages arise progressively through a series of multipotent, oligopotent and eventually unilineage progenitors, was recently challenged by studies demonstrating a more gradual path of blood cell differentiation with a multipotent HSC compartment at the top differentiating into unipotent progenitor cell populations without going through an intermediate oligopotent cell stage (Figure 1 ) (Notta et al., 2016). [0062] In the context of the present invention, the term "comprises" or "comprising" means "including, but not limited to". The term is intended to be open-ended, to specify the presence of any stated features, elements, integers, steps or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. The term "comprising" thus includes the more restrictive terms "consisting of" and "consisting essentially of".

[0063] In certain embodiments, 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).

[0064] In a particular embodiment, 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.

[0065] In a particular embodiment, 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.

[0066] In a particular embodiment, said population of cells is a cell population selected from the list of:

(a) a population of adult bone marrow cells;

(b) a population of cells from mobilized peripheral blood;

(c) a population of cells from the neonatal umbilical cord;

(d) a population of cells obtained by differentiating pluripotent cells in vitro, in particular embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); (e) a population of cells obtained by directed conversion of mature somatic cells; and

(f) a population of cells obtained from teratomas in vivo.

[0067] In a second aspect, the present invention relates to a method for the isolation of hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:

[0068] In a particular embodiment, said step of selecting is performed by fluorescence-activated cell sorting (FACS).

[0069] In a particular embodiment of the methods of the present invention, in said identification steps (a), and optionally (b), and/or in said selecting step one or more antibodies directed against said one or more cell surface proteins are used.

[0070] In a third aspect, the present invention relates to a method for isolating hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:

(a) culturing embryonic stem cells (ESCs) in vitro under hypoxic conditions (5% O2) for 2 to 3 days, in particular for 2.5 days;

(d) performing one or more selection steps for cells expressing one or more 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 TYROBP; in particular selected from the list of: ADAM 5, ALOX5AP, CD9, DARC, EVI2A, F2RL3, FZD10, GJA4, GPR97, HEPH, ICAM1 , IFITM1 , IFITM3, IGNGR1 , LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; and, optionally,

(e) performing one or more selection steps for cells additionally expressing one or more cell surface proteins 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 ); and

(f) collecting the cells selected in steps (d) and optionally (e).

[0071] Several alternative ways of achieving the differentiation of ESCs, or of induced pluripotent stem cells (iPSCs), are available. 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. 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). Kinetic blast colony formation analysis has proven that the very first markers acquired by the hemangioblast cells upon differentiation are of endothelial origin, such as TIE2, VECAD and CD31. Furthermore, the cells at that stage are capable of forming endothelial networks in 3D culture (Lancrin et al., 2009). Importantly, some of these endothelial-like cells acquire the hematopoietic marker CD41 and are subsequently able to generate primitive and definitive hematopoietic cells. These findings support a model in which the hemangioblast precursors generate hematopoietic cells through an intermediate hemogenic endothelium stage (Figure 3) (Lancrin et al., 2010; Rowe et al., 2016).

[0072] The molecular and cellular mechanisms regulating the choice between differentiation into primitive or definitive hematopoietic lineages remains largely unknown. Wnt signalling has been described to promote primitive erythropoiesis, whereas activation of the Notch pathway inhibits this developmental program (Cheng et al., 2008). The two pathways seem to counteract one another, suggesting an important role for Notch signalling (e.g. Notchl ) during the switch from primitive to definitive hematopoiesis (Figure 3).

[0073] Recent studies indicate that Wnt signalling upstream of the Notch pathway is required for hematopoietic development prior to AGM formation (Bigas et al., 2013; Ruiz-Herguido et al., 2012). It has been demonstrated that active β-catenin is restricted to few non-hematopoietic cells of the endothelial layer closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Genetic deletion of β-catenin from the embryonic endothelium (using VE-cadherin-Cre recombinase) precluded the progression of mutant cells towards the hematopoietic lineage. These findings indicate that Wnt/p-catenin activity is needed for the emergence but not the maintenance of HSCs (Figure 4).

[0074] A complex network of transcription factors (TFs) orchestrates the progressive specification of blood progenitors. TFs involved in 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).

[0075] Targeting studies in mice identified 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). Furthermore, it has been shown that Scl is required during the formation of VECAD+ cells from FLK1 + cells, demonstrating that Scl is not required for the formation of the hemangioblast but is critical for the development of endothelial and hemogenic endothelial cells thereafter (D'Souza et al., 2005; Endoh et al., 2002).

[0076] Another crucial transcription factor specifically required for the development of definitive hematopoietic progenitors is Runt-related transcription factor 1 (Runxl ) (Figure 5). In contrast to Scl, primitive erythropoiesis is only slightly affected by the absence of Runxl , while the generation of definitive hematopoietic progenitors and hematopoietic stem cells is completely abolished (Okuda et al., 1996; Yokomizo et al., 2008). 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). Hence, 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; Liakhovitskaia et al., 2009).

[0077] Recently, 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). The fact that Gfi1 and Gfilb alone cannot confer full hematopoietic potential to Runxl-/- HECs implicates other potential effectors of hematopoietic activation downstream of Runxl Upon lineage commitment, the cellular phenotype of surface markers correlates with a subset of hematopoietic TFs expressed in committed progenitors and precursors. Among the factors important for lineage differentiation, GATA-1 and PU.1 play a role in erythroid/megakaryocytic/eosinophil as well as myeloid differentiation (Figure 5). Within the lymphoid branch of lineage commitment, PAX5 has been described as factor necessary for proper B cell development while T cell development depends on active signalling through Notch and GATA-3 (Figure 5) (Bryder et al., 2006; Orkin, 2000).

[0078] A plethora of additional TFs have been described to be essential during distinct stages of hematopoietic development including Lmo2, Gata2, Sox17, Pu.1 , Etv2, Evil , Cdx4 as well as various members of the homeobox gene families Hoxa and Hoxb.

[0079] 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 ).

[0080] Besides the already described TFs crucial to hematopoietic development, homeobox genes involved in HSC cell self-renewal and proliferation have drawn much attention over the last years, since expression of these master regulatory genes facilitates ex vivo expansion of adult HSCs. The expanded cells retained their multipotency and did not lead to any leukemic malformations in mice (Antonchuk et al., 2002). The expression of the Hoxa and Hoxb gene families is largely restricted to stem and precursor populations and downregulated upon lineage commitment, which also makes them potential markers for the most potent LT-HSCs (Pineault et al., 2002; Sauvageau et al., 994).

[0081] In particular embodiments, in step (d) two or more of said selection steps, or three or more of said selection steps, are performed. In particular embodiments, said selection steps are performed by fluorescence-activated cell sorting (FACS).

[0082] In particular embodiments, in said selection steps (d), and optionally steps (e), one or more antibodies directed against said one or more cell surface proteins are used.

[0083] In a fourth aspect, 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 TYROBP; in particular 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, and ΤΊΕ1; more particularly selected from the list of: EVI2A, LYVE1, PTPRE, TIE1, IFITM1 and IFITM3; particularly characterized by the presence of at least at least six, particularly at least eight, particularly at least ten cell surface proteins selected from said list.

[0084] In a fifth aspect, 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, LAPTM5, LRMP, LTC4S, LYVE1, MRC1, ORAM, PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1; more particularly selected from the list of: EVI2A, LYVE1, PTPRE, TIE1, IFITM1 and IFITM3.

[0085] In a sixth aspect, 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, ICAM1, IFITM1, IFITM3, IGNGR1, LAPTM5, LRMP, LTC4S, LYVE1, MRC1, ORAM, PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1; more particularly selected from the list of: EVI2A, LYVE1, PTPRE, TIE1, IFITM1 and IFITM3; and wherein optionally, at least one antibody is directed against a cell surface protein selected from the list of: CD34, CD41, CD93 (AA4.1), EMCN, ENG, ESAM, ICAM2, JAM1/F11R, THSD1, and VECAD; in particular CD93 (AA4.1). [0086] In a particular embodiment, 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).

EXAMPLES

Introduction: Hoxb4-YFP reporter mouse model

[0087] As described previously, 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.

A wide range of HOXB4 target genes associated with numerous cellular processes have been identified indicating multiple modes of action for this transcription factor.

[0088] The first large scale study of HOXB4 target genes was 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). A large number of the target genes are involved in cellular signalling including factors of the FGF, TGF-β, Wnt, Hedgehog and Notch signalling cascades (Forrester and Jackson, 2012). [0089] In a different study, 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). Gata2, Runxl and Scl were shown to be direct targets of HOXB4 indicating that the TF might act by reprogramming cells into definitive HSPCs by simultaneously modulating the expression of various hematopoietic regulators. Accordingly, another study confirmed that HOXB4 expression results in an upregulation of CD34 and MPL, the receptor for thrombopoietin during the transition of pre-HSCs to embryonic HSCs (Matsumoto et al., 2009).

[0090] These findings indicate that 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. Additionally, 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).

[0091] The importance of the Notch, Wnt, and Hedgehog signalling that has been highlighted by these HOXB4 target analyses is consistent with studies investigating their role directly during ESC differentiation (Cerdan and Bhatia, 2010). For instance, 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). Treatment with the gamma secretase inhibitor, an inhibitor of Notch signalling, reduced the hematopoietic inductive effects associated with both HOXB4 overexpression, and AGM stromal co-culture suggesting that some molecular mechanisms might be shared between the two inductive strategies (Gordon-Keylock et al., 2010).

[0092] Despite the identification of potential downstream signalling targets of HOXB4 not much is known about its precise mode of action. In order to gain a better understanding of the physiologic role of HOXB4 within the hematopoietic system, the group of Medvinsky et al. generated a Hoxb4-yellow fluorescent protein (YFP) mouse model harbouring a knock-in of the enhanced YFP reporter gene, Venus, in the first exon of the Hoxb4 locus. (Figure 8) (Hills et al., 201 1 ; Lawrence et al., 1996).

[0093] Using this model, the group was able to show that BM LSK cells expressed Hoxb4-YFP and demonstrated in a functional transplantation assay that all definitive hematopoietic stem cells with long-term reconstitution potential express Hoxb4-YFP, indicating that this master regulatory TF is intrinsically expressed in HSCs. This observation was confirmed for adult BM-HSCs (LSK CD150+ CD48-), (E14.5) FL- HSCs (LSK CD150+ CD48-), (E12.5) FL-HSCs (LSK Mac1 +) and the earliest emerging HSCs enriched within the CD45+ VECAD+ cell population of the (E .5) AGM region. Of specific interest was the observation that the HECs (CD45- VECAD+), the 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.

[0094] 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+). In addition, virtually all HSCs from BM and FL (E 13.5) expressed high levels of Hoxb4 (Figure 9A). This results are in line with the already described Hoxb4-YFP mouse model characterisation of the Medvinsky group, which confirmed YFP expression within hemogenic endothelial cells (HECs) as well as in early and late-stage hematopoietic stem cells (HSCs) of the AGM, FL and BM compartment, respectively (Hills et al., 201 1 ). Hence, we hypothesized that ESCs derived from these mice would facilitate the identification and subsequent analysis of in vitro generated HECs and HSCs based on their YFP expression levels. [0095] By isolation and subsequent plating of (E 0.5) morulae cells on MEF feeders, Bayindir-Buchhalter and colleagues were able to successfully derive 12 transgenic Hoxb4-YFP ES cell lines, of which 10 lines are heterozygous (+ TG) and 2 homozygous (TG/TG), as well as 3 wild-type (+/+) littermate control cell lines (Figure 9B & 9C). Pluripotency of the generated ESC lines was assessed at the molecular level via qRT-PCR and immunohistochemistry (data not shown) targeting characteristic pluripotency genes/proteins (Nanog, Sox2, Oct4). Functional validation was performed via Teratoma assays (Figure 9D).

[0096] Using the generated Hoxb4-YFP ES cell lines in EB-differentiation assays facilitates the in-depth analysis of hematopoietic cells intrinsically expressing HOXB4, which in turn could result in the identification of signalling components and cell surface proteins characteristic for cells of the definitive wave of hematopoiesis. Hence, our model represents a powerful platform that facilitates the comprehensive analysis of the sequential steps of hematopoiesis during in vitro specification of definitive hematopoietic progenitors expressing HOXB4.

Example 1 : In vitro differentiation of Hoxb4-YFP reporter ESCs into hematopoietic cells

[0097] Directed in vitro differentiation of ESCs into hematopoietic cells was facilitated via the combination of induced EB formation in liquid suspension culture and additive cytokine signalling.

[0098] Hoxb4-YFP ESCs were cultured under hypoxic conditions (5% 0₂) 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). The sequential or combined application of these cytokines has been described to result in the production of highly enriched hematopoietic progenitors (Pearson et al., 2008).

[0099] 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). As described previously, this immunophenotypically defined double positive cell compartment contains all functional ES cell-derived HSPCs (Irion et al., 20 0; McKinney-Freeman et al., 2009). Over the course of in vitro EB differentiation, a small percentage of KIT+ cells (≤ 15%) was already observed on day 4 of differentiation followed by the developmental transition into CD41 + KIT+ cells (Figure 10B). The size of this double positive cell fraction increased significantly within the next 48 h peaking at > 40% on day 6 (D6); a result that was confirmed on all tested transgenic Hoxb4-YFP reporter ES cell lines (n = 3) and non-reporter control ES cell lines (n = 3) (Figure 2B & 2C).

[00100] Furthermore, a significant percentage of differentiated reporter ES cells expressed Hoxb4-YFP during EB differentiation (Figure 10D & 10E). Continuous FACS analysis demonstrated a rapid increase in the YFP signal starting at day 4.5, while the highest percentage of Hoxb4-YFP+ cells was observed between day 5 and day 5.5 of EB differentiation (>20%) (n = 3). Gating of HOXB4+ cells was based on a non-reporter ES cell line (+/+) that served as Hoxb4-YFP negative control during FACS analysis.

Example 2: Hematopoietic Gene expression analysis and functional characterization of in vitro specified HOXB4+ cells

HEMATOPOIETIC GENE EXPRESSION ANALYSIS OF HOXB4-YFP⁺ CELLS

[00101] 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). The strong expression of genes described to be required at the onset of definitive HSPC formation indicates that the Hoxb4-YFP reporter knock-in cassette marks in vitro differentiated cells with HSPC characteristics similar to the Hoxb4-YFP marked cells described within the respective mouse model (Hills et al., 201 1).

COLONY FORMATION ASSAYS DEMONSTRATE ENRICHED CLONOGENIC PROGENITOR POTENTIAL OF IN VITRO SPECIFIED HOXB4⁺ CELLS

[00102] 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.

[00103] 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).

[00104] In addition to MethoCult-based colony formation analysis, differentiated 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).

[00105] Functional analysis of HOXB4+ cells based on colony formation assays revealed enriched hematopoietic progenitor proliferation and differentiation potential within this cell fraction in accordance with the characteristic expression of HSPC genes described previously (3.2.1 ). Hence, the combined results of gene expression analysis and colony formation assays underline the HSPC-like characteristics of in vitro specified HOXB4+ cells.

Example 3: Immunophenotypic kinetics of in vitro differentiated HOXB4+ cells

IMMUNOPHENOTYPIC MARKER EXPRESSION ON DIFFERENTIATED HOXB4 EXPRESSING CELLS

[00106] Extended cellular analysis of the immunophenotypic kinetics during in vitro EB differentiation indicated a remarkable resemblance to embryonic HSPC development, confirmed by the expression of various cell surface proteins typically associated with early hematopoietic development during murine embryogenesis (Figure 12A).

[00 07] On day 4.5 of EB differentiation, the vast majority of differentiated cells (> 85%) expressed the early endothelial marker FLK1 (KDR), followed by a significant decrease of FLK1 + endothelial cells (< 30%) within the next 48 h. Simultaneously to the decrease of endothelial cells starting at day 5, we observed an increase of cells expressing KIT (-80%), HOXB4 (≤ 20%) as well as TIE2 (> 80%) and VECAD (> 40%), two surface markers predominantly expressed on HECs. The developmental transition from initially abundant FLK1+ endothelial cells to cells expressing hemogenic endothelial markers (TIE2, VECAD) represents a typical hallmark of EHT, a process crucial for hematopoietic stem and progenitor cell (HSPC) emergence during hematopoietic development in mammals. After day 5, we observed a strong increase in expression of the hematopoietic marker protein CD41 (> 60%) with a subset of CD41+ cells also expressing KIT. On day 6 of EB differentiation, up to 40% of the cells were double positive for CD41 and KIT, as described previously (3.1 )

[00108] Cross-correlation of the individual marker proteins with HOXB4 expression levels facilitated a better resolution of the cell surface marker (CSM) kinetics and the consecutive stages HOXB4+ cells passage during in vitro hematopoietic development (Figure 12B). The first HOXB4+ cells observed at day 4.5 of EB differentiation were only present at low frequency (< 5%) and almost entirely expressed FLK1 + (-75%) with a subset of cells already expressing KIT (-55%) as well as the HEC markers VECAD (-40%) and TIE2 (-20%) (grey box) (Figure 12B). On day 5, these developmental events were followed by a numerical increase of HOXB4+ cells (up to 20%) and the simultaneous transition to a more pronounced HEC-like expression profile with a significant percentage of HOXB4+ cells expressing VECAD+ (-60%), TIE2+ (-65%) and KIT+ (-80%) (orange box) (Figure 12B). At later stages of differentiation (D6) nearly all HOXB4+ cells expressed a marker set characteristic for definitive hematopoietic progenitors (red box) including CD41 (> 85%), KIT (> 85%) and TIE2 (> 80%) (Figure 12B)

[00109] Taken together, immunophenotypic analysis confirmed that cytokine- induced hematopoietic differentiation of Hoxb4-YFP ESCs results first in the formation of endothelial cells (FLK1 +), followed the formation of HECs (HOXB4+ VECAD+) and subsequent EHT transition into HOXB4+ definitive hematopoietic progenitor cells expressing CD41 , KIT and TIE2. These results demonstrate that during in vitro differentiation, expression of Hoxb4-YFP marks a subpopulation of HECs and subsequently arising definitive hematopoietic progenitor cells (HPCs) as already described in the HOB4-YFP reporter mouse model (Hills et al., 201 1 )

HOXB4-YFP ES CELLS TRANSIENTLY EXPRESS HOXB4 DURING HEMATOPOIETIC IN VITRO DIFFERENTIATION

[001 10] To further improve our understanding of the developmental kinetics of Hoxb4-YFP+ cells during in vitro differentiation, we dissociated EBs of day 5 and subsequently sorted the cells based on their distinct antigen (CSMs) expression profile, hypothesizing that the individually sorted cell populations represent different stages of hematopoietic development. Next, sorted cells were reagreggated in liquid suspension culture and the continued differentiation process was monitored by frequent FACS analysis (Figure 13A).

[001 11] Reaggregation analysis allowed us to demonstrate that the expression of Hoxb4-YFP within the sorted hematopoietic HOXB4+ compartment (HOXB4+ CD41+ KIT+) decreased dramatically (> 90%) after continued differentiation for 24 h post FACS sorting, indicating that the fast-cycling and differentiating cells express the HSPC-specific transcription factor only for a very short timeframe during in vitro culture (Figure 13B). In contrast, a significant proportion (10-30%) of sorted HOXB4- cells expressing an endothelial-like immunophenotype (HOXB4- CD41- KIT+ FLK1 +) started to express Hoxb4-YFP and CD41 after continued differentiation (Figure 13B). This observation confirmed that within our cytokine-induced differentiation system, a subset of endothelial-like cells undergo EHT, similar to embryonic HSPC development. The fact that FACS sorted hematopoietic HOXB4- CD41 + KIT+ cells were not able to form any HOXB4+ cells after reaggregation, further confirms that this subpopulation represents already committed hematopoietic cells arising at a later stage of in vitro differentiation downstream of the transient HOXB4 expressing stage (Figure 13B & 13C).

[00112] Clonal colony formation (MethoCult) and OP9 co-culture assays confirmed that the highest clonogenic progenitor output originates from the HOXB4- CD41+ KIT+ cell population, enriched for lineage-restricted hematopoietic progenitors (CFR: 1 :250). As expected, differentiated HOXB4- CD41- KIT+ FLK1 + endothelial- like cells did not form hematopoietic colonies at all, while the HOXB4+ cell population developed lineage-defined hematopoietic output to a lower extend than the HOXB4- CD41+ KIT+ cells confirming their more primitive state of development (CFR: 1 :500) (Figure 13D).

[00113] Gene expression analysis (qRT-PCR) of the three distinct cell populations confirmed the enrichment of characteristic developmental stage-specific transcripts during in vitro differentiation. 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). Differentiated 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). In line with previous functional data, 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).

[001 14] Hence, we functionally confirmed that HOXB4- CD41- KIT+ FLK1 + endothelial-like cells (Upstream) undergo endothelial to hematopoietic transition into short-lived (max. 24 h) definitive hematopoietic progenitors (HOXB4+ CD41 + KIT+). The cytokine supplemented suspension culture system results in rapid differentiation of HOXB4+ cells into more committed HOXB4- CD41 + KIT+ hematopoietic cells (Downstream), which cannot differentiate into HSPC-like HOXB4+ cells anymore (Figure 13C).

Example 4: Immunophenotypic analysis identifies differentiated cells expressing early HSC marker AA4.1 within HoxB4hi cell fraction

IDENTIFICATION OF IN VITRO SPECIFIED HOXB4⁺ AA4.1⁺ CELLS

[001 15] Comprehensive FACS analysis of in vitro differentiated cells demonstrated a strong correlation between Hoxb4-YFP expression levels and HEC/HSPC marker protein expression, meaning cells with the highest HOXB4 protein levels (HOXB4hi) also expressed VECAD, TIE2, KIT and CD41 at a significant higher rate than HOXB4low and HOXB4- negative cells. Furthermore, we identified a subpopulation of cells (1 % of total cells) expressing CD34 and AA4.1 , representing two early AGM and FL-HSC markers, exclusively within the HOXB4hi cell fraction (Figure 14A & 14B) (Yamane et al., 2009). These observations are in line with studies that detected highest HOXB4 expression levels in the most primitive hematopoietic progenitors - the HSCs (Giampaolo et al., 1995; Sauvageau et al., 1994).

GENE EXPRESSION ANALYSIS CONFIRMS HOXB4-YFP EXPRESSION AS IMPORTANT SELECTION MARKER FOR DEFINITIVE HSPCs WITHIN AA4.1 + CELL FRACTION

[00116] 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).

[001 17] Comparison of sorted AA4.1 + HOXB4+ and AA4.1 + HOXB4- cells via gene set enrichment analysis (GSEA) revealed a significant enrichment of HSPC, inflammatory and lymphoid gene signatures in the double positive cell compartment (Figure 14D). These findings are in line with recent reports demonstrating that proinflammatory signalling regulates definitive HSPC emergence in developing vertebrate embryos (Espin-Palazon et al., 2014; He et al., 2015; Sawamiphak et al., 2014). The expression of lymphoid genes signatures might be an additional indicator for early definitive hematolymphoid differentiation potential of in vitro specified HOXB4+ AA4.1 + HSC-like cells.

[001 18] In contrast, 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-β 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).

[00119] Hence, 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

[00120] Global gene expression profiling (lllumina Mouse WG-6 v2.0) revealed that during in vitro differentiation, cells undergo characteristic transcriptomic shifts, strongly resembling endothelial to hematopoietic transition and HSPC development during murine embryogenesis (Figure 15A-15C). [00121] As expected, high expression of pluripotency factor transcripts including Sox2, Nanog, Oct4, and Esrrb were observed in the undifferentiated ESCs (Cavaleri and Scholer, 2003; Nishimoto et al., 1999; Zhou et al., 2007). Microarray analysis once more confirmed the expression of Podxl, Etv2 and Eng on the FLK1 expressing endothelial Upstream cells. High transcript levels of these factors exclusively mark mesodermal cells with definitive hematopoietic differentiation potential (as described in 3.3.2) (Borges et al., 2012; Wareing et al., 2012; Zhang et al., 2014). In addition, 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).

[00122] 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.

[00123] In accordance to previous microarray data-based GSEA (3.4.2), 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).

[00124] Combined GSEA analysis including comparisons between ESC, Upstream, HOXB4+ AA4.1 +, Downstream and BM-HSC cells via the BubbleGum software confirmed the strong similarities between HOXB4+ AA4.1+ cells and isolated BM-HSCs from HOXB4 reporter mice (LSK CD150+ CD48- HOXB4+) (Figure 16A & 16B). In accordance with previous expression data, both cell populations show significant enrichment (red circles) of inflammatory-, lymphoid- and HSPC-specific gene sets in comparison to ESCs, Upstream and Downstream cell populations. 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).

[00125] In addition, significantly upregulated transcripts of the HOXB4+ AA4.1 + cell fraction were analysed via the gene list enrichment analysis tool Enrichr^®. Pathway analysis (KEGG, WikiPathways) again listed HSC differentiation, BCR/TCR signalling and inflammatory signalling within the top regulated candidates (Figure 16C). The ChlP-based transcription factor analysis tool of Enrichr^®, identified Mecom (Evil), Gatal , Gata2, Runxl , Nfkbl , Scl and Lmo2 as some of the defining transcription factors of HOXB4+ AA4.1 + cells (Figure 16C).

[00126] Taken together, the high expression of individual HSC-specific transcripts along with expression of lymphoid and inflammatory gene signatures recently described to be essential during HSC emergence and maintenance underlines that in vitro differentiated HOXB4+ AA4.1 + double positive cells share all the molecular characteristics of HSCs developing within the early hematopoietic organs like the AGM-region and the fetal liver (FL) (Espin-Palazon et al., 2014; He et al., 2015; Sawamiphak et al., 2014).

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

IDENTIFICATION & VALIDATION OF CELL SURFACE PROTEINS CO- EXPRESSED ON IN VITRO DIFFERENTIATED HSC-LIKE HOXB4⁺ AA4.1⁺ CELLS

[00127] 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. Relative gene expression values of the HOXB4+ AA4.1 + samples were compared to Up- and Downstream samples using R/Bioconductor through the graphical user interface Chipster (v3.8) (n = 3). Upregulated genes (log2 fold change > 1) of both individual comparisons were further examined by Venn analysis (Venny v2.1.0) in order to identify common regulated genes exclusively expressed in HOXB4+ AA4.1 + cells (Figure 17A & 17B). Screening for cell surface marker (CSM)-encoding genes was conducted via the web-based Surfaceome database (da Cunha et al., 2009).

[00128] Screening of the comprehensive microarray data set resulted in the identification of 50 CSM encoding genes exclusively expressed on HOXB4+ AA4.1 + cells. Although some of the marker genes within our list have recently been shown to be expressed on HSCs (CD9, Esam, Emcn, Gpr97, Thsdl , Icam2 and Robo4), the large majority of them has not yet been described in the context of HSC biology (Figure 17C) (Matsubara et al., 2005; Solaimani Kartalaei et al., 2015; Takayanagi et al., 2006; Yokota et al., 2009). Next, 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).

[00129] Multiple Reaction Monitoring (MRM) mass spectrometry was performed to validate the expression levels of 39 preselected marker-encoding genes at protein level. Differential protein regulation and relative expression levels were validated for 30 distinct cell surface markers, with 26 of them representing potential markers not yet described in the context of hematopoietic development (Figure 18A). In accordance with the microarray and qRT-PCR data, the majority of the analysed proteins demonstrated peak expression in the HOXB4+ AA4.1 + cell population. No clear protein regulation was determined for 9 of the selected markers, which might not necessarily reflect the real biological state of these proteins, but could also be rooted in technical problems during the complex experimental procedure (Figure 18B). WHOLE PROTEOME ANALYSIS (HRM) OF DIFFERENTIATED CELL POPULATIONS REPRESENTING CONSECUTIVE STAGES OF IN VITRO HSPC SPECIFICATION

[00130] Besides focussing on the proteomic validation of CSM-encoding genes of HOXB4+ AA4.1 + cells we performed Hyper reaction monitoring (HRM) whole proteome mass spectrometry in order to gain more biological insights into the processes crucial to in vitro HSC specification.

[00131] In collaboration with Biognosys AG (Switzerland), we generated spectral peptide libraries of sorted in vitro differentiated cell populations, by measuring individual sample pools (3 biological replicates/pool) in shotgun mode. The resulting library consisted of 23,989 peptides and 3,334 protein groups and represented the basis for identification and quantification of potential HSPC-specific proteins (Figure 19A & 19B).

[00132] Focussing on the HOXB4+ AA4.1 + sample pool we observed strong expression of hematopoietic proteins, including HSPC-specific transcription factors (FGD5, RUNX1 , TAL1 , GFI1 , GFI1 B and LIN28B) and cell surface markers (ESAM, GPR56, CD34, CD41 , CD81 and CD97) (Copley et al., 2013; Gazit et al., 2014; Lin et al., 201 1 ; Solaimani Kartalaei et al., 2015; van Pel et al., 2008) (Figure 19C).

[00133] Additionally, enrichment analysis (Enrichr^®) 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).

[00134] 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.

Example 7: Crispr/Cas9 knockout screen reveals functional requirement of newly identified cell surface receptors during hematopoietic in vitro specification of HSPCS

SELECTION OF A SUBSET OF SURFACE RECEPTOR PROTEINS EXPRESSED ON HOXB4⁺ AA4.1⁺ CELLS FOR CRISPR/CAS9 KO SCREEN

[00135] A subset of the newly identified marker proteins expressed on HOXB4+ AA4.1 + cells was selected for CRISPR-based functional analysis. Different selection criteria were applied to select for the most promising protein candidates, potentially crucial for HSPC formation. In a first step, CSMs were ranked according to the combined results obtained from the microarray analyses and the subsequent validation experiments. Only CSMs demonstrating specifically high levels of expression in HOXB+ AA4.1 + cells that were further confirmed via qRT-PCR and targeted mass spectrometry (MRM) were selected. 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 MEDIATED KNOCKOUT DEMONSTRATES FUNCTIONAL REQUIREMENT OF EVI2A, LYVE1 , PTPRE AND TIE1 DURING IN VITRO HSPC SPECIFICATION

[00136] Cell surface proteins selected as described (3.7.1), were knocked out at the ES cell stage by the induction of gene-specific double strand breaks (DSBs) using the CRISPR/Cas9 system. Designed gene-specific guide RNA oligonucleotides (gRNA) are listed in Table 6. Transfected clones were selected via Puromycin for 48 h and subsequently validated by gel electrophoresis and Sanger sequencing (Figure 20B). Individual homozygous knockout (KO) clones (-/-) were differentiated according to our standard cytokine-induced EB differentiation protocol in order to assess potential defects during hematopoietic differentiation rooted in the induced lack of protein functionality.

[00137] 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). 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. As expected, RUNX1 -KO clones (n = 3) demonstrated an almost complete lack of CD41 + KIT+ double positive cells after differentiation (3% ± 2%) and served as positive controls throughout the functional KO screening (Figure 20C & 20D) (Chen et al., 2009; Li et al., 2006). Knockout of EVI2A (n = 8) and LYVE1 (n = 3) resulted in an equally strong phenotype with the almost complete loss of hematopoietic differentiation potential with only 3% ± 2% of cells expressing CD41 and KIT (Figure 20C & 20D). A hypomorphic hematopoietic phenotype was observed upon hematopoietic differentiation of PTPRE-KO (n = 4; 15% ± 8% CD41+ KIT+) and TIE1 -KO (n = 3; 20% ± 8% CD41 + KIT+) clones (Figure 12C & 12D). No significant hematopoietic differentiation defects were observed upon knockout of F2RL3, MRC1 , CD52, ADAM 15, FCER1 G, PAQR7 and SIRPA (data not shown) (n = 3) (Figure 20D). 3.7.3 EVI2A- AND LYVE1-KO CLONES DEMONSTRATE SIGNIFICANT DEFETCS

DURING IN VITRO EHT TRANSITION

[00138] Detailed immunophenotypic analysis of the 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. RUNX1-KO ES cells served as positive control (Figure 22). In accordance to the differentiation defect during EHT transition, no early HSC-like cells expressing the marker protein AA4.1 were observed during later-stages of EVI2A-KO and LYVE1-KO cell differentiation (EVI2A: 0%; LYVE1 : 0%; WT: 3-4%) (Figure 22). Hence, knockout of the cell surface receptor proteins EVI2A and LYVE1 results in a severe hematopoietic phenotype during HOX4-YFP ES cell differentiation, strongly resembling the knockout of the EHT master regulatory transcription factor RUNX1. These results suggest that EVI2A and LYVE1 represent key regulator proteins during EHT transition and the subsequent formation of definitive HSPC during in vitro specification and embryonic hematopoietic development.

Example 8: Human Array Data:

[00139] 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).

Example 9: IFITM1 and IFITM3 Expression Analyses

[00140] 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

[00141] 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).

[00142] Thus, it could be shown that the expression of these two markers is conserved in hematopoietic stem/progenitor precursors in human development as opposed to just in mouse.

Material & Methods

1 Cell culture reagents

1.1 BUFFERS

Cytokine reconstitution buffer

^■ 5 ml 2% BSA (0.1 g BSA in 5 ml PBS)

^■ 1 ml 1 M HEPES

^■ PBS to 100 ml 1.2 MEDIA & SUPPLEMENTS

MEF medium

^■ Dulbecco's Modified Eagle Medium (DMEM; Gibco)

^■ 10% Fetal Calf Serum (FCS; Gibco)

^■ 1 % Penicillin-Streptomycin-Glutamine (Gibco)

ESC medium

^■ Knockout DMEM (Gibco)

^■ 15% ES-FCS (Gibco)

^■ 1 % Penicillin-Streptomycin-Glutamine (Gibco)

^■ 1 % non-essential amino acids (Gibco)

^■ 50 μΜ β-mercaptoethanol (Gibco)

^■ 1000 U/ml mouse leukemia inhibitory factor (ESGRO LIF; Millipore)

^■ Two inhibitors (2i):

- 3 μΜ CHIR99021 (Stemgent)

- 1 μΜ PD0325901 (Stemgent)

EB differentiation medium

^■ Iscove's modified Dulbecco's medium (IMDM; Gibco)

^■ 15% FCS (Gibco/PAA Laboratories)

^■ 5% Protein free Hybridoma medium II (PFHM II; Gibco)

^■ 1 % Penicillin-Streptomycin-Glutamine (Gibco)

^■ 50 μg ml ascorbic acid (Sigma-Aldrich)

^■ 4.5 mM monothioglycerol (Sigma-Aldrich)

^■ 200 μg/ml bovine holo-transferrin (Sigma-Aldrich)

1.2 ADDITIONAL TISSUE CULTURE REAGENTS

Dissociation enzyme mix

^■ 500mg Collagenase (Invitrogen)

^■ 1 g Hyaluronidase (Sigma-Aldrich)

^■ 40.000 U DNase (Sigma-Aldrich)

^■ in 50 ml PBS (Sigma-Aldrich) 1.3 CYTOKINES & INHIBITORS

2 Cell lines & in vitro differentiation

2.1 GENERATION OF HOXB4-YFP REPORTER ES CELL LINES

[00143] Hoxb4-YFP reporter ES cell lines (ESCs) 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. 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 embryos were then stored in M2 droplets and each morula was transferred into a single well (96-Well plate, Corning) containing irradiated mouse embryonic fibroblast cells (CF-1 IRR) (GlobalStem, Rockville, MD). Attached morulae outgrowths (core/ring structure) were disaggregated and the resulting suspension transferred onto a fresh feeder layer. Resulting ESC colonies were propagated for three to four passages before cryopreservation in freezing medium (90% FCS (Gibco) and 10% DMSO (Sigma-Aldrich)).

2.2 MAINTENANCE OF ESC LINES

[00144] 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 10⁴ cells/cm². 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% C0₂.

2.3 IN VITRO EMBRYOID BODY DIFFERENTIATION OF HOXB4 REPORTER ESC LINES

[00145] 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.

Small differentiation reaction

^■ 7.5 x 10⁴ cells

^■ 7 ml EB medium

^■ T25 Ultra Low Attachment flask

Large differentiation reaction

^■ 2.5 x 10⁵ cells

^■ 21 ml EB medium

^■ T75 Ultra Low Attachment flask [00146] Cells were cultured for 60 h at 37°C and 5% CO₂ under hypoxic conditions (5% O₂). On day 2.5 of EB differentiation the cytokines BMP-4, Activin-A, VEGF and FGF-2 were added at a final concentration of 5 ng/ml each (BD Biosciences). Formed EBs were cultured for additional 84 h under hypoxic conditions. On day 6, EBs were transferred into 50ml Falcon tube and flasks were rinsed out once with PBS. After EBs settled to the bottom of the tube via gravity residual supernatant was aspirated and the tube was washed once with 50ml PBS. EBs dissociation was carried out by addition of 250 μΙ (T25) or 750 μΙ (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).

3 Characterization of in vitro differentiated HOXB4 expressing cells

3.1 COLONY FORMING UNIT ASSAY

[00147] In vitro differentiated ESCs were plated into cytokine-enriched semisolid media (Methocult). 300 μΙ 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% CO₂. Hematopoietic colonies were counted and characterized after 7 to 10 days of incubation.

3.2 QUANTITATIVE REAL-TIME PCR

[00148] Total RNA was extracted and purified using the miRNeasy kit (Qiagen) combined with RNase-Free DNase (Qiagen) treatment according to the manufacturer's protocol. cDNA was synthesized using the Superscript VILO cDNA Synthesis Kit (Invitrogen). Power SYBR Green PCR Master Mix (Life Technologies) in combination with gene-specific primers (Table 4) was used to acquire expression data with the ViiA 7 real-time PCR system (Applied Biotechnologies). qRT-PCR was performed according to the following cycler program: 50°C for 2 min, 95°C for 10 min, 95°C for 15 sec, 60°C for 1 min (40 cycles). 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.

3.3 FACS ANALYSIS

[00149] 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).

Table 5: FACS antibody panel

Table 4: List of primers used for quantitative real-time PCR

3.4 REAGGREGATION ASSAY

[00150] EBs were dissociated (as described in 2.3 above) on day 5 of differentiation and subsequently sorted according to the following immunophenotypic marker proteins:

^■ Upstream: HOXB4 CD41^" FLK1⁺ KIT⁺

^■ HE/HSPCs: HOXB4⁺ CD41 ^hi KIT^hi AA4.1 ⁺

^■ Downstream: HOXB4^" CD41⁺ KIT⁺

[00151] 5 x 103 - 1 x 104 sorted cells of each respective population were transferred to a single well (96 Well Ultra low attachment plate; Corning) containing EB differentiation media. Cells were monitored via microscopy for 96 hours. FACS analyses of the reaggregated colonies were performed every 24 h.

4. Global Gene expression profiling

[00152] Gene expression profiling was performed on independent biological triplicates of the following FACS-isolated populations: 4.1 SAMPLE PREPARATION

[00153] Total RNA was extracted and purified using the miRNeasy kit (Qiagen) combined with RNase-Free DNase (Qiagen) treatment according to the manufacturer's protocol. RNA quality was evaluated on the Agilent 2100 Bioanalyzer, using the Agilent RNA 6000 Pico Kit (Agilent Technologies). The resulting RNA Integrity Number (RIN) 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^®

■ Required genomic material - 100ng total cRNA

^■ > 700.000 oligonucleotides per bead/spot

■ 30x redundancy for each transcript

■ Expression level of 45.281 mouse transcripts, variants and EST clusters

Affymetrix Mouse 430 2.0 ^®

■ Required genomic material - 100ng total cRNA

^■ 45.000 probe sets

■ Expression level of 39.000 transcripts and variants

[00154] lllumina chips were laser-scanned via the lllumina iScan system®, Affymetrix chips with the GeneChip® Scanner 3000.

4.2 GENE EXPRESSION ANALYSIS

[00155] Differential gene expression analysis was carried out using R/Bioconductor through the graphical user interface Chipster (v3.8, chipstercsc.fi). Differentially regulated genes were called using two group tests (empirical Bayes). Differential expression thresholds were set to log2 fold change >1 (upregulated) or≤1 (down regulated) with an adjusted p-value of ≤ 0.05 (corrected by Benjamini Hochberg; BH). [00156]

[00157] Hierarchical Clustering & Heatmap design of regulated gene signatures was performed via the matrix visualization and analysis platform GENE-E (Broad Institute).

[00158] Gene set enrichment analysis (GSEA) is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states (e.g. phenotypes). GSEA was performed on quantile-normalized microarray data (Affymetrix 430.2 and lllumina Mouse WG v2.0) via the Broad Institute GSEA software (Mootha et al., 2003; Subramanian et al., 2005) using the following settings:

Expression dataset: Respective .get file

(Expression values of sample groups)

Gene sets database: (h) hallmark genesets

(c2) all curated genesets

(c5) all gene ontology (GO) gene sets

Number of permutations: 1000 (default)

Phenotype labels: Respective .cls file

(Phenotype labels of sample groups)

Collapse dataset to gene symbols: true (default)

Permutation type: gene_set

Chip platform: Respective .chip file

(Illumina/Affymetrix gene annotation data)

Enrichment statistic: weighted (default)

Metric for ranking genes: Signal2Noise (default)

Gene list sorting mode: descending (default)

Max size (exclude larger sets): 500 (default)

Min size (exclude smaller sets): 15 (default)

Collapsing mode for probe sets: Median_of_probes Normalization mode: meandiv (default)

Randomization mode: no_balance (default)

Omit features with no symbol match: true (default)

Make detailed gene set report: true (default)

Median for class metrics: false (default)

Number of Markers: 100 (default)

Plot graphs for the top sets: 20-100

Seed for permutation: timestamp (default)

Save random ranked lists: false (default)

Make zipped file with all reports: false (default)

[00159] Files indicated in red (.get, .cls, .gmt and .chip) were generated/downloaded manually according to the Broad Institute documentation guidelines.

[00160] Bubble GUM (GSEA Unlimited Map), a computational tool that allows performing GSEA between various samples was used to integrate multiple GSEA data (Spinelli et al., 2015). A combined .get file containing all lllumina expression samples was created and used as expression database during the subsequent GSEA analysis. All settings were adjusted as described before. The results were illustrated in enrichment bubble maps with a maximal Normalized Enrichment Score (NES) of 3.4. False Discovery Rate (FDR) range was adjusted from <0.01 (strong enrichment) to >1.0 (no significant enrichment).

[00161] 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.

[00162] Web-accessible STRING analysis (version 10) for differentially expressed genes was used to detect known and predicted protein-protein interactions including direct (physical) and indirect (functional) associations (Szklarczyk et al., 2015).

[00163] Selection for Cell-Surface protein encoding genes was carried out in two steps. First a Venn diagram analysis (Venny 2.1) for differentially expressed genes between the 3 sample groups (Upstream, HOXB+ AA4.1 +, Downstream) has been performed. The overlapping gene list was subsequently screened for cell surface protein encoding genes via the Surfaceome database (da Cunha et al., 2009).

5 Proteomics

5.1 MULTIPLE REACTION MONITORING (MRM) MASS SPECTROMETRY

[00164] 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. 250 pg protein per sample were precipitated in 50 pg aliquots with chloroform/methanol as described previously (Wessel and Flugge, 984). Briefly, the protein solution was diluted with 4 volumes of methanol, 1 volume chloroform, 3 volumes of water and centrifuged for 2 min at 15000 x g. The upper aqueous phase was discarded, 4 volumes of methanol were added and the proteins were pelletized for 2 min at 15000 x g. 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. 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. To determine the levels of the endogenous target peptides in the samples, heavy peptide standards (lysine (13C615N2) or arginine (13C615N4) at the C terminus) corresponding to their natural counterparts (light) (Intavis) were pooled and spiked into the digested and desalted samples to a final concentration of 0.5 pmol/μΙ per peptide.

[00166] 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 μηι x 15 cm, 130 A) (Waters). Samples were separated over 120 min at a flow rate of 6 μΙ/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.

[00167] For SRM method optimization and validation, 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.

[00168] 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.

[00169]

6 Knockout screen of marker proteins via crispr/cas9

6.1 GUIDE RNA DESIGN

[00170] Guide/CRISPR RNAs (gRNAs) were designed using the Optimized CRISPR design tool of the 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).

6.2 TARGET SEQUENCE CLONING

[00171] During the design process sticky end sequences were added to the 5' and 3' end of the respective sgRNA target sequences in order to facilitate cloning into the pSpCas9 (BB)-2A-Puro (PX459) v2.0 plasmid (Addgene plasmid # 62988) (Figure X) Cloning procedure followed the guidelines of the Zhang Lab CRISPR cloning protocol (Cong et al., 2013; Ran et al., 2013):

1. Digestion of plasmid (1 pg) with Bbs\ for

30 min at 37°C:

^■ 1 μΙ Plasmid

^■ 1 μΙ FastDigest Bbs\ (Fermentas)

^■ 1 μΙ FastAP (Fermentas)

^■ 2 μΙ 10X FastDigest Buffer

^■ X μΙ ddH₂O

20 μΙ total

2. Gel purification of digested plasmid using

QIAquick Gel Extraction Kit and elute in EB Phosphorylation and annealing of each pair of oligos:

Ligation reaction and incubation at room temperature

or (RT) 10 min:

PlasmidSafe exonuclease treatment of ligation reaction to prevent unwanted recombination products (optional):

Incubate reaction at 37°C for 30 min. Transformation

Annealing in thermocycler using the following parameters:

37°C 30 min

95°C 5 min and then ramp down to 25°C

(5°C/min) Table 6: List of designed Guide RNAs (crRNAs)

Sticky end sequences for plasmid cloning sgRNA sequence(s) 6.3 NUCLEOFECTION OF ES CELLS

[00172] 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 μΙ Mouse ES Cell Nucleofector solution (Lonza) at RT. Meanwhile 5 pg of each plasmid used in the reaction were added to 10 μΙ Mouse ES Cell Nucleofector solution. Next, the cell suspension was added on top of the plasmid solution (100 μΙ), 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 μΙ 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.

6.4 SCREENING OF PUROMYCIN RESITANT CLONES

[00173] 10 - 15 resistant clones per gene knockout (KO) were picked under microscope using a pipette. Clones were immediately transferred into a single well (96-Well plate) containing Trypsin-EDTA (50 μΙ) and mixed by pipetting up and down 3 times. 150 μΙ of ESC medium was added to the individual clones after 2-3 minutes incubation at 37°C. After thorough trituration, half of the reaction volume (100 μΙ) was transferred to a 96-Well plate containing 100 μΙ ESC medium per well for ESC cultivation. 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). [00174] Homozygous knockout clones were selected based on DNA banding patterns and expanded to 6-well plate format before cryopreservation.

6.5 HEMATOPOIETIC DIFFERENTIATION OF SELECTED KO CLONES

[00175] 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:

7 Single-cell qRT-PCR

7.1 SINGLE-CELL INDEX SORTING

[00176] 96-Well plate single cell sorting experiments were performed on the Aria I cytometer system (BD Biosciences) in collaboration with MSc. Lisa Becker and Dr. Simon Haas using the index-sorting feature of the BD FACSDiva software (v8.01). Dead cells were excluded by using 7-Aminoactinomycin (7AAD; Invitrogen).

7.2 SINGLE CELL cDNA SYNTHESIS AND PRE-AMPLIFICATION

[00177] 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).

Gene-specific single-cell reverse transcriptase cycler program:

^■ RT and cell lysis: 50°C for 60 min

^■ RT inactivation and

Tag Polymerase activation 95°C for 3min

^■ Pre-amplification 95°C -> 15 sec, 60°C -> 60sec (23 cycles)

^■ Final elongation 60°C -> 15 min

Synthesized cDNA was used directly in a standard qPCR protocol or stored at -20°C.

7.3 SINGLE CELL QPCR ANALYSIS

[00178] Hierarchical clustering, heatmap and PCA for single cell qRT-PCR experiment was generated using the following R code:

[00179]

### loading of libraries

> library(gplots)

> library(gdata) > library(RColorBrewer)

> library(FactoMineR)

> CTs_SecondSort.df <- read.xis ("AnalysisSecondSort.xIsx", sheet = 5, header = TRUE)

> All. CTs <- CTs_SecondSort.df

rownames(AII.CTs) <- CTs_SecondSort.df[,1]

AII.CTs<- AII.CTs [c(2:34)]

#### plotting of heatmap

# create color scale

> color.scale <-colorRampPalette(colors=c("red3","white","royalblue4"))(400)

# create heatmap

> heatmap. results <- heatmap.2(data.matrix(AII. CTs),

Rowv = T, Colv = TRUE,

dendrogram = 'both',

labRow = CTs_SecondSort.df[,1],

col = color.scale, trace = 'none',

key = TRUE,

keysize = 1.5,

scale="col",

cexRow=0.45,cexCol=1 ,

key.title = NA)

### Plot PCA for scPCR DATA

#### PCAs

### Perform PCA

> BA8.pca = PCA(CTs_SecondSort.df [,2:34],

scale.unit = FALSE,

ncp = 5,

graph = TRUE) Table 8: List of single-cell qRT-PCR primers

8 Statistical analysis

[00180] Statistical analysis and graphical representation of data was carried out via GraphPad Prism software (version 6.0b). All statistically evaluated in vitro experiments were performed with at least 3 biological replicates. Unless indicated otherwise in the figure, statistical analyses were always carried out in comparison to the control group. Unpaired nonparametric t-tests were used for two-group comparisons and one-way ANOVA for comparisons involving more than two groups. Statistical significance is indicated by ^*P < 0.05 or ^**P < 0.01. Error bars indicate the standard deviation (SD). For GSEA, a false discovery rate (FDR) of < 0.2 was considered statistically significant.

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Claims

1. A method for the isolation of hematopoietic stem and progenitor cells (HSPCs) from a cell population comprising the steps of:

(a) identifying cells characterized by the presence of one or more proteins on the cell surface selected from the list of: EVI2A, LYVE1 , PTPRE, ΤΊΕ1 , IFITM1 and IFITM3;

(b) optionally 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); and

(c) selecting hematopoietic stem and progenitor cells (HSPCs) that have been identified in step (a), and optionally in step (b).

2. The method of claim 1 , wherein 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.

3. The method of claim 1 or 2, wherein said population of cells is a cell population selected from the list of:

(a) a population of adult bone marrow cells;

(b) a population of cells from mobilized peripheral blood;

(c) a population of cells from the neonatal umbilical cord;

(d) a population of cells obtained by differentiating pluripotent cells in vitro, in particular embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs);

(e) a population of cells obtained by directed conversion of mature somatic cells; and

(f) a population of cells obtained from teratomas in vivo.

4. The method of any one of claims 1 to 3, wherein said step of selecting is performed by fluorescence-activated cell sorting (FACS).

5. A method for the identification of hematopoietic stem and progenitor cells (HSPCs) in a cell population comprising the steps of:

(a) identifying cells characterized by the presence of one or more proteins on the cell surface 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, ΤΊΕ1 , CD276, CD52, CNR2, F2R, FXYD5, MC5R, NRG1 , PDZK1 IP1 , PTPRB, ROB04, SLC39A4, TNFRSF13B, and TYROBP; in particular 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, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; and, optionally,

(b) 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 ).

6. The method of any one of claims 1 to 5, wherein in said identification steps (a), and optionally (b), and/or in said selecting step (c) one or more antibodies directed against said one or more cell surface proteins are used.

7. A method for isolating hematopoietic stem and progenitor cells (HSPCs) comprising the steps of:

(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); (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

(d) performing one or more selection steps for cells expressing one or more 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 TYROBP; in particular 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, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; and, optionally,

(f) collecting the cells selected in steps (d) and optionally (e).

8. The method of claim 7, wherein in step (d) two or more of said selection steps are performed.

9. The method of claim 8, wherein in step (d) three or more of said selection steps are performed.

10. The method of any one of claims 7 to 9, wherein said selection steps are performed by fluorescence-activated cell sorting (FACS).

1 1. The method of any one of claims 7 to 10, wherein in said selection steps (d), and optionally steps (e), one or more antibodies directed against said one or more cell surface proteins are used.

12. 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, 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, LAPTM5, LRMP, LTC4S, LYVE1, MRC1, ORAI1, PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1; more particularly selected from the list of: EVI2A, LYVE1, PTPRE, TIE1, IFITM1 and IFITM3; particularly characterized by the presence of at least at least six, particularly at least eight, particularly at least ten cell surface proteins selected from said list.

13. 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, ORAI1, 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, LAPTM5, LRMP, LTC4S, LYVE1, MRC1, ORAI1, PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1; more particularly selected from the list of: EVI2A, LYVE1, PTPRE, TIE1, IFITM1 and IFITM3.

14. 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, ICAM1, IFITM1, IFITM3, IGNGR1, LAPTM5, LRMP, LTC4S, LYVE1 , MRC1 , ORAM , PAQR7, PSEN2, PTPRE, SELPLG, SIRPA, and TIE1 ; more particularly selected from the list of: EVI2A, LYVE1 , PTPRE, TIE1 , IFITM1 and IFITM3; and wherein optionally, at least one antibody is directed against a cell surface protein 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 ).

15. The kit of claim 14, further comprising 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).