Methods for generating myeloid cells

Abstract

Normal hematopoiesis relies on a dynamic, interactive network of stromal cell types that secrete a variety of growth factors and cytokines. Two of the most intensely studied molecules implicated in the interaction between HSPCs and stromal cells is the CXCL12 / CXCR4 signaling pair. Mutations resulting in impaired desensitization and enhanced CXCR4 signaling have been identified in the WHIM Syndrome (WS). Hereby, we use a mouse model harboring a WS-linked Cxcr4 gain-of-function mutation (CXCR4 C1013G ), along with a unique and robust co-culture model between primary MSCs and lymphoid-primed MPP (or MPP4). We report functional alterations in MSCs lacking Cxcr4 desensitization, affecting their interaction with MPP4 and interfering with their lymphoid / myeloid specification. In addition, using a human MSC cell line transduced with another WS-linked CXCR4 gain-of-function mutation (CXCR4 R334X ), we show that these genetically engineered MSCs also promoted the myeloid differentiation of human primary HSPCs.

Description

[0001]METHODS FOR GENERATING MYELOID CELLS FIELD OF THE INVENTION: The present invention is in the field of medicine and in particular haematology and immunology. BACKGROUND OF THE INVENTION: Hematopoiesis is a highly regulated process of life-long blood cell generation, that begins with hematopoietic stem and progenitor cells (HSPCs). HSPCs reside in the bone marrow (BM) within a supportive microenvironment, also called a “niche”, that controls their survival and behavior. The BM niche is a complex interactive network of several stromal cell types, growthfactors, cell adhesion molecules and extracellular components, which can influence directly andindirectly HSPC survival and fate. However, despite remarkable technical improvements, suchas in situ imaging and single-cell sequencing approaches, the nature of the BM niche and thepositioning of HSPCs in the BM remain debatable. Among the highly studied BM stromal cells are the Skeletal Stromal / Stem Cells (MSCs), which have attracted great attention over the years and have been referred to by several interchangeable terms for decades, such as Bone Marrow Stromal Cells (BMSCs) and “Mesenchymal Stem Cells” (MSCs). Recently, the term “MSC” has been used to refer to a more homogeneous and immature multipotent stem cell population found in the “skeletal” tissues, residing at the top of the differentiation hierarchy and giving rise to cartilage, bone, fat and hematopoiesis-supporting stroma1–4. One of the basic chemokines through which HSPCs interact with their BM niche is thechemokine CXCL12, which acts via its highly conserved G protein-coupled receptor CXCR4expressed by both hematopoietic and non-hematopoietic cells. The CXCL12 / CXCR4 chemokine axis governs a wide range of physiological and cellular processes, includinghematopoietic stem cell (HSC) retention in the BM and immune cell trafficking5,6. Cxcr4- andCxcl12-KO mice die perinatally, due to cardiac malformations and hematopoietic defects, illustrating the vital role of this axis in organ growth and physiological activities7,8. The CXCL12 / CXCR4 signaling axis is tightly regulated by a negative feedback mechanism, called “desensitization”, which occurs to restrict G-protein coupling and receptor signaling upon Cxcl12 binding. This process starts with the phosphorylation of the receptor’s C-terminus domain by G-protein coupled Receptor Kinases (GRK), followed by the recruitment of β- arrestins, which will drive G-protein uncoupling and CXCR4 internalization, resulting in either degradation or recycling to the membrane9. Defective CXCR4 desensitization, leading to receptor gain-of-function, has been reported in a rare genetic immunodeficiency, called the WHIM Syndrome (WS)10,11. Most WS patients have autosomal dominant mutations in the region coding for the CXCR4 C-terminus part, resulting in a lack of desensitization and an exacerbation of G-dependent signaling in response to CXCL12 stimulation10,12. To further understand the physiological and cellular consequences of CXCR4 gain-of-function, we generated a knock-in mouse model carrying a WS-linked heterozygous Cxcr4 mutation (CXCR4C1013G)13. Cxcr4+ / 1013mice phenocopy certain features observed in affected individuals, including impaired Cxcr4 desensitization upon Cxcl12 binding, failure in receptor internalization and profound lympho-neutropenia13. We recently observed in the BM of Cxcr41013-bearing mice a reduction in the number of lymphoid progenitors, whereas the number of myeloid precursors remains intact, accompanied by changes in HSC self-renewal, quiescence and multipotency14. Moreover, our recent findings showed that exacerbated Cxcr4 signaling causes major intrinsic and extrinsic alterations in multipotent progenitors (MPPs), which represent the final step of the divergence between myeloid and lymphoid lineages, and in particular MPP415. Lymphoid-primed MPP4 carrying the Cxcr41013mutation underwentprofound epigenetic, transcriptomic and metabolic modifications, all of which promote theirskewing toward myeloid cell production with diminished lymphoid differentiation capacity15. Aside from causing intrinsic dysregulations in MPP4, defective Cxcr4 desensitization also results in abnormal localization in the BM, potentially affecting MPP4 access to niche factors that shape their maintenance and fate15. Simultaneously, we provided evidence that a lack of Cxcr4 desensitization interferes with the bone stromal compartment as well, especially MSCs16. We showed that proper regulation of Cxcr4 signaling is critical for bone homeostasis, and that excessive signaling disrupts bone microarchitecture, along with changes in the bone formation / resorption balance, consequently leading to osteopenia, a hallmark of osteoporosis16.However, how Cxcr4 gain of function affects MSC supportive capacities towards MPP hasnever been investigated. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to methods for generating myeloid cells. DETAILED DESCRIPTION OF THE INVENTION: Main definitions: As used herein, the term "population" refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest. As used herein, the term “isolated" refers to a product (e.g. a cell or a population of cells), compound, or composition that is separated from at least one other product, compound, or composition with which it is associated in its naturally occurring state, whether in nature or as made synthetically. As used herein, the terms “expressing”, “positive”, or “+” and “not expressing”, “negative”, or “-” are well-known in the art and refer to the expression level of a cell marker of interest, in that the expression level of the cell marker corresponding to “+” is high, intermediate or low (i.e., the cell marker is expressed or present at the cell surface), and the expression level of the cell marker corresponding to “-” is null (i.e., the cell marker is not expressed, or is absent, at the cell surface). As used herein, the term “myeloid cell” refers to a family of immune cells comprising monocytes, macrophages, myeloid dendritic cells (mDCs), granulocytes, and mast cells thatoriginate from a common myeloid progenitor (CMP) in the bone marrow. Examples of myeloidlineage markers include pan-myeloid markers CD11b, CD206 for M2-type macrophages, CD15for neutrophils. While some markers are unique to each cell class, often a combinatorial analysis of multiple markers is required to assess the true phenotype of the myeloid cell lineages. As used herein, the term “mesenchymal stromal cell” or “MSC” refers to cells that meet the definition set in 2006 by The International Society for Cellular Therapy (ISCT): (1) adherence to plastic, (2) expression of CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and (3) ability to differentiate to osteogenic, chondrogenic and adipogenic lineage (Dominici et al. (2006) Cytotherapy, 8:315-317). MSC have been traditionally defined as spindle-shaped or fibroblast-like plastic adherent cells. Although originally isolated from bone marrow, MSC have now been isolated from a variety of tissues including bone periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle, dental pulp and cord blood. MSCs can be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, and placenta. In particular, the term includes cells that are CD34 positive upon initial isolation from tissue but satisfy the ISCT criteria after expansion. The term also includes cells that are isolated from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof, and satisfy the ISCT criteria either before or after expansion. More particularly, the term includes cells described in the literature as bone marrow stromal stem cells (BMMSC), marrow- isolated adult multipotent inducible cells (MIAMI) cells, multipotent adult progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), MULTISTEM® (Athersys, Inc., Cleveland, Ohio), PROCHYMAL® (Osiris Therapeutics, Inc., Columbia, Md.), remestemcel-L, Mesenchymal Precursor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX- PAD, ALLOSTEM® (Allosource, Centennial, Colo.), ASTROSTEM® (Osiris Therapeutics,Inc., Columbia, Md.), Ixmyelocel-T, MSC-NTF, NurOwn™ (Brainstorm Cell TherapeuticsInc., Hackensack, N.J.), STEMEDYNE™-MSC (Stemedica Cell Technologies Inc., San Diego, Calif.), STEMPEUCEL® (Stempeudics Research, Bangalore, India), StempeucelCLI, StempeucelOA, HiQCell, Hearticellgram-AMI, REVASCOR® (Mesoblast, Inc., Melbourne, Australia) CARDIOREL® (Reliance Life Sciences, Navi Mumbai, India), CARTISTEM® (Medipost, Rockville, Md.), PNEUMOSTEM® (Medipost, Rockville, Md.), PROMOSTEM® (Medipost, Rockville, Md.), Homeo-GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Regenerative Cells (ERC), adipose-derived stem and regenerative cells (ADRCs) obtained with the CELUTION® System (Cytori Therapeutics, Inc., San Diego, Calif.), HS-5cells (i.e. CRL-3611 ™ that is an ATCC manufactured and accessioned progeny of ATCCCRL-11882 cited in US Pat. No. 5,879,940), perivascular-derived cells, and pericyte-derived cells. Finally, the term includes cells that only satisfy one or more of the ISCT criteria when cultured under one set of conditions but satisfy the full set of ISCT criteria when cultured on plastic tissue culture flasks in the presence of tissue culture medium containing 10% fetal bovine serum. As used herein, the term “Bone Marrow-Derived Mesenchymal Stromal Cell” has its general meaning in the art and refers to the population of mesenchymal stromal cells (MSCs) that residein the bone marrow (BM). Said cells offer physical support and regulate hematopoieticstem / progenitor cell (HSPC) homeostasis. In vitro, they are positive for the expression of specific surface markers, classification determinant (CD)105, CD90, and CD73, whereas they do not express hematopoietic (CD34, CD45) and endothelial markers (CD31). They express human leukocyte antigen (HLA) class I but they are negative for HLA class II. In the human BM, MSCs are localized around the blood vessels, where they offer physical support to HSPCsand differentiate into osteoprogenitors to guarantee a functional remodelling of the BM niche.Importantly, BM-MSCs control HSPC homeostasis by direct contact and in a paracrine manner through the secretion of soluble factors. The term encompasses skeletal stromal cells. As used herein, the term “skeletal stem cell” or “skeletal stromal cell” or “MSC” has itsgeneral meaning in the art and refers to a more homogeneous and immature multipotent stemcell population found in the “skeletal” tissues, residing at the top of the differentiation hierarchyand giving rise to cartilage, bone, fat and hematopoiesis-supporting stroma1–4. Human MSCsmay be characterized by their cell surface markers. Human MSCs are negative for theexpression of CD45, CD235, Tie2, and CD31; and positively express podoplanin (PDPN), CD164 and CD73 (ref: Chan et al., Cell 2018). Murine MSCs are identified as CD45-, TER119-, CD31-, CD51+ (or Integrin alpha-V) and Sca-1+ (for stem cells antigen-1), Pdgfra+ (for Platelet derived growth factor receptor alpha). As used herein, the term “hematopoietic stem and progenitor cell” or “HSPC” has its general meaning in the art and refers to a cell identified by the presence of the antigenic marker CD34 (CD34+) and are therefore characterized as CD34+ cells, and populations of such cells. In particular, the term "HSPC" refers to a cell identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (Lin) markers and are therefore characterized as CD34+ / Lin(-) cells, and populations of such cells. It is recognized that the population of cells comprising CD34+ and / or Lin(-) cells also includes hematopoietic progenitor cells. The term encompasses multipotent progenitor.As used herein, the term “multipotent progenitor” or “MPP” refers to a cell that is theimmediate progeny of a hematopoietic stem cell, and is distinguished by a restricted lineagedifferentiation potential and a reduced self-renewal capacity. These cells then differentiate intoeither CMP or common lymphoid progenitor (CLP). Both CMPs and CLPs are types ofoligopotent progenitor cells (progenitor cells that differentiate into only a few cell types).Recent studies in mouse models have shown that the MPP compartment is heterogeneous andcan be divided at least into four distinct subsets with different lineage fates (Ref: Pietras et al.,Cell stem cell, 2015. Sommerkamp et al., Blood, 2021). MPP1 are identified as LSK (Lin- forlineage markers, Sca-1+ for stem cell antigen-1 and c-Kit+ for the tyrosine-protein kinase Kit)CD150+CD48-CD135-, that represent the immediate progeny of hematopoietic stem cells(HSCs) and characterized by a slow cycling and short-term multilineage differentiationcapacities. At steady state, they presumably give rise to functionally distinct lineage-biasedMPPs that are more proliferative and defined as megakaryocyte / erythroid (ME)-biased MPP2(LSKCD150+CD48+CD135-), granulocyte / macrophage (GM)-biased MPP3 (LSKCD150-CD48+CD135-), and lymphoid-biased MPP4 (LSKCD150-CD48+CD135+), which differentiateessentially into mature lymphoid cells, with a limited myeloid potential (Ref: Pietras et al., Cellstem cell, 2015). As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides whendiscussed in the context of gene therapy, refer to the respective intact polypeptide, or anyfragment or genetically engineered derivative thereof that retains the desired biochemicalfunction of the intact protein. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after the assembly of the polymer. The termpolynucleotide, as used herein, refers interchangeably to double- and single-strandedmolecules. Unless otherwise specified or required, any embodiment of the invention describedherein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identicalpositions / total number of positions x 100), considering the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair-wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequenceor nucleic acid sequence are identical, irrespective of any chemical and / or biologicalmodification. According to the invention, a first amino acid sequence having at least 90%identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence. As used herein, the term “CXCR4” has its general meaning in the art and refers to the C-X-Cchemokine receptor type 4 (CXCR4), also known as Fusin or CD184. CXCR4 is an alpha-chemokine receptor specific for C-X-C motif chemokine 12 (CXCL12) also called stromal-derived factor-1 (SDF-1). An exemplary amino acid sequence is represented by SEQ ID NO:1and an exemplary nucleic acid sequence for CXCR4 is represented by SEQ ID NO:2. TheCXCL12 / CXCR4 signaling axis is tightly regulated by a negative feedback mechanism, called “desensitization”, which occurs to restrict G-protein coupling and receptor signaling upon Cxcl12 binding. This process starts with the phosphorylation of the receptor’s C-terminus domain by G-protein coupled Receptor Kinases (GRK), followed by the recruitment of β-arrestins, which will drive G-protein uncoupling and CXCR4 internalization, resulting in eitherdegradation or recycling to the membrane9. Defective CXCR4 desensitization, leading to receptor gain-of-function, has been reported in a rare genetic immunodeficiency, called the WHIM Syndrome (WS)10,11. SEQ ID NO:1 >sp|P61073|CXCR4_HUMAN C-X-C chemokine receptor type 4 OS=Homo sapiens OX=9606 GN=CXCR4 PE=1 SV=1 MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVI LVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNL YSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEA DDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKT TVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPI LYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS SEQ ID NO:2 > Homo sapiens C-X-C motif chemokine receptor 4 (CXCR4), transcript variant 1, mRNA. 1 cttccctcta gtgggcgggg cagaggagtt agccaagatg tgactttgaa accctcagcg 61 tctcagtgcc cttttgttct aaacaaagaa ttttgtaatt ggttctacca aagaaggata 121 taatgaagtc actatgggaa aagatgggga ggagagttgt aggattctac attaattctc 181 ttgtgccctt agcccactac ttcagaattt cctgaagaaa gcaagcctga attggttttt 241 taaattgctt taaaaatttt ttttaactgg gttaatgctt gctgaattgg aagtgaatgt 301 ccattccttt gcctcttttg cagatataca cttcagataa ctacaccgag gaaatgggct 361 caggggacta tgactccatg aaggaaccct gtttccgtga agaaaatgct aatttcaata 421 aaatcttcct gcccaccatc tactccatca tcttcttaac tggcattgtg ggcaatggat 481 tggtcatcct ggtcatgggt taccagaaga aactgagaag catgacggac aagtacaggc 541 tgcacctgtc agtggccgac ctcctctttg tcatcacgct tcccttctgg gcagttgatg 601 ccgtggcaaa ctggtacttt gggaacttcc tatgcaaggc agtccatgtc atctacacag 661 tcaacctcta cagcagtgtc ctcatcctgg ccttcatcag tctggaccgc tacctggcca 721 tcgtccacgc caccaacagt cagaggccaa ggaagctgtt ggctgaaaag gtggtctatg 781 ttggcgtctg gatccctgcc ctcctgctga ctattcccga cttcatcttt gccaacgtca 841 gtgaggcaga tgacagatat atctgtgacc gcttctaccc caatgacttg tgggtggttg 901 tgttccagtt tcagcacatc atggttggcc ttatcctgcc tggtattgtc atcctgtcct 961 gctattgcat tatcatctcc aagctgtcac actccaaggg ccaccagaag cgcaaggccc 1021 tcaagaccac agtcatcctc atcctggctt tcttcgcctg ttggctgcct tactacattg 1081 ggatcagcat cgactccttc atcctcctgg aaatcatcaa gcaagggtgt gagtttgaga 1141 acactgtgca caagtggatt tccatcaccg aggccctagc tttcttccac tgttgtctga 1201 accccatcct ctatgctttc cttggagcca aatttaaaac ctctgcccag cacgcactca 1261 cctctgtgag cagagggtcc agcctcaaga tcctctccaa aggaaagcga ggtggacatt 1321 catctgtttc cactgagtct gagtcttcaa gttttcactc cagctaacac agatgtaaaa 1381 gacttttttt tatacgataa ataacttttt tttaagttac acatttttca gatataaaag 1441 actgaccaat attgtacagt ttttattgct tgttggattt ttgtcttgtg tttctttagt 1501 ttttgtgaag tttaattgac ttatttatat aaattttttt tgtttcatat tgatgtgtgt 1561 ctaggcagga cctgtggcca agttcttagt tgctgtatgt ctcgtggtag gactgtagaa 1621 aagggaactg aacattccag agcgtgtagt gaatcacgta aagctagaaa tgatccccag 1681 ctgtttatgc atagataatc tctccattcc cgtggaacgt ttttcctgtt cttaagacgt 1741 gattttgctg tagaagatgg cacttataac caaagcccaa agtggtatag aaatgctggt 1801 ttttcagttt tcaggagtgg gttgatttca gcacctacag tgtacagtct tgtattaagt 1861 tgttaataaa agtacatgtt aaacttaaaa aaaaaaaaaa aaaa As used herein, the term “mutation” has its general meaning in the art and refers to asubstitution, deletion, or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is insertedinto the same position. Within the specification, the mutations are referenced according to thestandard mutation nomenclature. In particular, the term “mutation” encompasses “naturally-occurring mutations” and “non-naturally occurring mutations”. As used herein, the term“naturally occurring mutation” refers to any mutation that can be found in the naturally occurring variants of a polypeptide (e.g. CXCR4). As used herein, the term “non-naturally occurring mutation” refers to any mutation that is genetically inserted in a polypeptide (e.g.CXCR4). The nomenclature for designing mutations is well known in the art (see e.g. Ogino S,Gulley ML, den Dunnen JT, Wilson RB; Association for Molecular Patholpogy Training and Education Committtee. Standard mutation nomenclature in molecular diagnostics: practicaland educational challenges. J Mol Diagn. 2007 Feb;9(1):1-6.). For instance, “c.123A>G”indicates that on cDNA level, the nucleotide A at position 123 is substituted by G, and“p.P252R” indicates on protein level, that proline (P) at position 252 is substituted by arginine(R). In particular, the symbol “X” indicated a stop codon, and the symbol “fsX” indicated aframe shift. Thus, the S338X mutation denotes that the amino acid residue at position 338(Serine, S) is changed to a stop codon. As used herein, the term “CXCR4C1013G”or “CXCR41013” refers to a naturally occurring variantof CXCR4 wherein the nucleotide C at position 1013 is substituted by a nucleotide G in SEQID NO:2. Said mutation leads to the deletion of the 15 last amino acid residues in SEQ ID NO:1.(S338X). As used herein, the term “Cxcr4+ / 1013 SCC” refers to a SCC wherein at least oneCXCR4 allele harbors the CXCR41013 mutation. As used herein, the term “Cxcr41013 / 1013 MSC”refers to a SCC wherein both CXCR4 alleles harbor the CXCR41013 mutation.Method of the present invention:The object of the present invention relates to an in vitro method for generating myeloid cellscomprising the steps of i) co-culturing a population of hematopoietic stem and progenitor cells(HSPCs) with a population of mesenchymal stromal cells (MSCs) that naturally or exogenouslyexpress a CXCR4 polypeptide with an enhanced receptor signalling, in a culture medium for asufficient time, without the addition of exogenous cytokines, for allowing myeloid differentiation and ii) harvesting the generated myeloid cells.Typically, HSPCs and MSCs are isolated from the bone marrow. Thus, in some embodiments,the population of MSCs is a population of Bone Marrow-Derived Mesenchymal Stromal Cells. In some embodiments, the population of HSPCs is a population of CD34+cells. Methods for isolating HSPCs and MSCs are well known in the art and include, without limitation, methodsas described in the EXAMPLE. In some embodiments, CD34+ cells are isolated from cordblood.In some embodiments, the present invention relates to an in vitro method for generating murinemyeloid cells comprising the steps of i) co-culturing a population of murine primary lymphoid-primed multipotent progenitors (MPP4s) and a population of murine skeletal stem cell (MSCs that express a CXCR4 polypeptide with an enhanced receptor signalling in a culture mediumfor a sufficient time, without the addition of exogenous cytokines, for allowing myeloiddifferentiation and ii) harvesting the generated myeloid cells.In some embodiments, the MSCs of the present invention express a CXCR4 polypeptide withan enhanced receptor signalling. In some embodiments, the MSCs of the present inventionexpress a CXCR4 polypeptide having a shorter C-terminus tail that results in impaireddesensitization and enhanced receptor signalling. In some embodiments, the MSCs of thepresent invention express a CXCR4 polypeptide wherein the 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 last amino acid residues of said polypeptide are deleted.In some embodiments, the MSCs of the present invention express a CXCR4 polypeptide thatconsists of an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 333 in SEQ ID NO:1.In some embodiments, the MSCs of the present invention express a CXCR4 polypeptide thatconsists of an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 334 in SEQ ID NO:1.In some embodiments, the MSCs of the present invention express a CXCR4 polypeptide thatconsists of an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 337 in SEQ ID NO:1.In some embodiments, the MSCs of the present invention express a CXCR4 polypeptide thatconsists of an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 342 in SEQ ID NO:1.In some embodiments, the MSCs of the present invention harbour at least one gain-of-functionmutation, i.e. one mutation that results in enhanced receptor signalling. In some embodiments,the MSCs of the present harbour at least one mutation that leads to the shortening of the C-terminus tail of CXCR4, resulting in impaired desensitization and enhanced receptor signalling.In some embodiments, the MSCs of the present invention harbour at least one mutation thatleads to the deletion of the 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 last amino acid residues inthe CXCR4 polypeptide. In some embodiments, the mutation is selected from the groupconsisting of R334X, G335X, S338X, S339fs342X, and E343X. In some embodiments, saidmutation is CXCR4C1013G (i.e. S338X mutation). In some embodiments, the mutation ishomozygous or heterozygous. In some embodiments, the MSCs of the present invention are CXCR4+ / 1013or CXCR41013 / 1013.In some embodiments, the MSCs of the present invention are cells that naturally harbor onemutation leading to the shortening of the C-terminus tail of CXCR4. Typically, said cells canbe isolated from the bone marrow of a patient suffering from the WHIM syndrome (WS).In some embodiments, the MSCs of the present invention are engineered to express one CXCR4polypeptide with an enhanced receptor signalling. As used herein, the term “engineered cell” refers to a cell that has been subjected to manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to anappropriate reference cell, such as otherwise identical cell that has not been so manipulated. Insome embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the cell prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the cell prior to the manipulation; and / or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the cell prior to the manipulation. In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a polynucleotide, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. Thoseof ordinary skill in the art will appreciate that the reference to an “engineered cell” herein may,in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such a cell. Thus, in some embodiments, the method of the invention further comprises a step of introducinginto the population of MSCs a polynucleotide that encodes a CXCR4 polypeptide having ashorter C-terminus tail that results in impaired desensitization and enhanced receptor signalling. In some embodiments, the method of the invention comprises a step of introducing into thepopulation of MSCs a polynucleotide that encodes a CXCR4 polypeptide wherein the 10, 11,12, 13, 14, 15, 16, 17, 18, or 20 last amino acid residues of said polypeptide are deleted. In some embodiments, the method of the invention comprises a step of introducing into thepopulation of MSCs a polynucleotide that encodes a CXCR4 polypeptide that consists of anamino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 333 in SEQ ID NO:1. In some embodiments, the method of the invention comprises a step of introducing into thepopulation of MSCs a polynucleotide that encodes a CXCR4 polypeptide that consists of anamino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 334 in SEQ ID NO:1. In some embodiments, the method of the invention comprises a step of introducing into thepopulation of MSCs a polynucleotide that encodes a CXCR4 polypeptide that consists of anamino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 337 in SEQ ID NO:1. In some embodiments, the method of the invention comprises a step of introducing into thepopulation of MSCs a polynucleotide that encodes a CXCR4 polypeptide that consists of anamino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 342 in SEQ ID NO:1.It is contemplated that a polynucleotide can be introduced into the MSCs as naked DNA or ina suitable vector. Naked DNA generally refers to the DNA contained in a plasmid expressionvector in the proper orientation for expression. Physical methods for introducing apolynucleotide construct into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, nucleofection, and the like. Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. In some embodiments, the polynucleotide isintroduced into the MSC by a viral vector that is an adeno-associated virus (AAV), a retrovirus,lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus. Typically, the vector of the present invention includes "control sequences'", which refer collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control sequences need always be present, so long as the selectedcoding sequence is capable of being replicated, transcribed and translated in an appropriate hostcell. For instance, to increase the expression, polynucleotides of the present invention may beoperably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK),β-actin, ubiquitin, and simian virus 40 (SV40) / CD43 composite promoter, elongation factor(EF)-1a and the spleen focus-forming virus (SFFV) promoter.In some embodiments, the MSCs of the present invention are edited to express one CXCR4polypeptide with an enhanced receptor signalling. In some embodiments, the MSCs of thepresent invention are edited to express a CXCR4 polypeptide having a shorter C-terminus tailthat results in impaired desensitization and enhanced receptor signalling. In some embodiments,the MSCs of the present invention are edited to express one CXCR4 polypeptide wherein the10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 last amino acid residues of said polypeptide are deleted. As used herein, the term "editing", "edit", "edition", or "edited" refers to a method of alteringthe nucleic acid sequence of a polynucleotide (e.g., a naturally occurring wild-type nucleic acidsequence or a naturally-occurring mutated nucleic acid sequence) by introducing a change to aspecific genomic target; the genomic target may include a chromosomal region, a coding polynucleotide (e.g., a gene), a promotor, a non-coding polynucleotide, or any nucleic acid sequence. The changes to a nucleic acid may include deletion, addition and other changes tothe nucleic acid sequence in the genome. Examples of methods of gene editing that may be usedin the present invention include, but are not limited to, methods based on engineered nucleases,methods based on recombinant Adeno- Associated Virus (or AAV), methods based ontransposons (e.g., Sleeping Beauty transposon system), methods based on homologous recombination, conditional targeting using site-specific recombinases (e.g., Cre-LoxP and Flp- FRT systems), and Multiplex Automated Genomic Engineering (MAGE). Other examples of methods of gene editing that may be used in the present invention include, but are not limited to, methods based on nickases. Non-limiting examples of engineered nucleases include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR) transcriptionactivator like effector nuclease (TALEN), zinc finger endonuclease (ZFN), meganuclease (mn, also known as homing endonuclease), or megaTAL (combining a TAL effector with a mn cleavage domain). Other non-limitative examples of engineered nucleases are base editors or prime editors. In some embodiments, the edition is performed with a baseediting enzyme that typically consists of a fusion protein comprising a defective CRISPR / Casnuclease linked to a deaminase polypeptide. Two classes of base-editing enzymes--cytosine base-editing enzymes (CBEs) and adenine base-editing enzymes (ABEs)--can be used to generate single-base pair edits without double-stranded breaks.In some embodiments, before step i), the MSCs are expanded and seeded in a solid support (e.g.96-well plate) with the appropriate culture medium for a time sufficient for allowing cellattachment on the solid support. Then, the HSPCs (e.g. CD34+ cells) are cultured directly onthe generated layer of MSCs.As used herein, the term “culture medium” has its general meaning in the art and refers to amedium generally used in the culturing of mammalian, particularly human or murine cells,including hematopoietic progenitors and stromal cells in the context of the invention. Such amedium is preferably based on Dulbecco's Modified Eagle Medium (DMEM) or MinimumEssential Medium (MEM), supplemented with fetal bovine serum (FBS) and additionalcompounds such as 2-mercaptoethanol, which enhances MSC proliferation.Typically for generating myeloid cells, the cells are co-cultured for at least 3, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days, preferably 7days.In some embodiments, the HSPCs (e.g. CD34+ cells) are engineered. In some embodiments,an exogenous nucleic acid sequence expressing a gene of interest is introduced into the HSPCs (e.g. CD34+ cells), preferably before step i) of the methods. In some embodiments, thetransduction or transfection of HSPCs (e.g. CD34+ cells) is carried out before step i) of themethods of the present invention. In some embodiments, the exogenous nucleic acid sequence to be introduced into the cells encodes a chimeric antigenic receptor (CAR). A CAR is a cell surface protein that recognizes an antigen, such as, for example, a cell surface protein specifically expressed by the target cells (e.g. expressed by cancer cells or infected cells). In some embodiments, the exogenous nucleic acid sequence to be introduced into the cells encodes a protein selected from the group comprising or consisting of cytokines or cytokinesreceptors or variants thereof (such as, for example, variants of cytokines or cytokines receptorswith increased stability). In some embodiments, the exogenous nucleic acid sequence encodes a chimeric cytokine receptor or orthogonal cytokine-receptor pairs. In some embodiments, the genetic modification step(s) is / are a gene disruption step, aiming at decreasing or abolishing the expression of specific genes. Examples of genes that can be deleted include, but are not limited to, genes from the group comprising or consisting of PD1, TIGIT, LAG-3, TIM-3, TGFB2, cytokine-induced STAT inhibitor (CIS) and signal regulatory protein a (SIRPa). Myeloid cells of the present invention: A further object of the present invention is an isolated population of myeloid cells susceptible to be obtained, or obtained, by the in vitro method of the invention. In some embodiments, the myeloid cells of the present invention express a CAR. In some embodiments, the myeloid cells of the present invention express a protein selected from the group comprising or consisting of cytokines or cytokines receptors or variants thereof (such as, for example, variants of cytokines or cytokines receptors with increased stability). In some embodiments, the myeloid cells of the present invention express a chimeric cytokine receptor or orthogonal cytokine-receptor pairs. In some embodiments, the myeloid cells of the present invention express IL-15 or a variant thereof. In some embodiments, the myeloid cells of the present invention express CD16 or a variant thereof (such as, for example, cleavage resistant variant of CD16). In some embodiments, the myeloid cells of the present invention do not express at least one gene selected from the group comprising or consisting of PD1, TIGIT, LAG-3, TIM-3, Cytokine induced STAT inhibitor (CIS) and Signal regulatory protein alpha (SIRPa). Therapeutic uses and pharmaceutical compositions:A further object of the present invention is a population of myeloid cells as described herein,for increasing or for use in increasing the number of said cells in a subject in need thereof.A further object of the present invention is a population of myeloid cells as described herein foruse in therapy, such as, for example, in “off-the-shelf” therapy.A further object of the present invention is a population of myeloid cells as described herein foruse as a medicament.The present invention further relates to the use of a population of myeloid cells as describedherein, for the manufacture of a medicament for increasing the number of said cells in a subject. The present invention further relates to a method for increasing the number of myeloid cells in a subject in need thereof, comprising administering to the subject a myeloid cell population asdescribed herein (in particular, a therapeutically effective amount of myeloid cells as describedherein). The present invention further relates to a composition comprising, consisting essentially of orconsisting of a population of myeloid cells according to the present invention.In some embodiments, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient. Consequently, the present invention further relates to a pharmaceutical composition.In some embodiments, the pharmaceutical composition comprises, consists essentially of, orconsists of a population of myeloid cells according to the present invention and at least onepharmaceutically acceptable excipient. Pharmaceutically acceptable excipients that may be used in the pharmaceutical composition of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as, for example, human serum albumin, buffer substances such as, for example, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as, for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes,polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.In some embodiments, the composition of the present invention is, or is for use as, a medicament. Consequently, the present invention further relates to a medicament. In some embodiments, the medicament comprises, consists essentially of or consists of apopulation of myeloid cells according to the present invention.As used herein, the term “consisting essentially of”, with reference to a composition,pharmaceutical composition, or medicament, means that the myeloid cells of the invention arethe only therapeutic agents or agents with biological activity within said composition,pharmaceutical composition or medicament. In some embodiments, the population of myeloid cells, composition, pharmaceuticalcomposition or medicament of the invention is for treating cancer or an infectious disease.The present invention thus relates to a population of myeloid cells, a composition,pharmaceutical composition or medicament as disclosed herein for treating cancer or aninfectious disease.The present invention further relates to the use of a population of myeloid cells as disclosedherein for the manufacture of a medicament for treating cancer or an infectious disease. The present invention further relates to a method for treating cancer or an infectious disease in a subject in need thereof, comprising administering to the subject a population of myeloid cells as described herein. In some embodiments, the subject is a human. In some embodiments, the subject is affected, preferably diagnosed with cancer. Examples of cancers include, but are not limited to leukemia (e.g., acute myeloid leukemia, B cell acute lymphoblastic leukemia (B-ALL), T cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia), lymphoma (e.g., B lymphomas, peripheral T cell lymphoma), non-Hodgkin lymphoma, glioblastoma, neuroblastoma, multiple myeloma, cervical cancer, breast cancer (e.g., Triple-negative breast cancer), ovarian cancer, bladder cancer, prostate cancer, pancreatic cancer, gastric cancer, thyroid cancer, melanoma, uterine cancer, kidney cancer, liver cancer (e.g., hepatocellular cancer), sarcoma, carcinoma e.g., renal cell carcinoma, breast carcinoma), small cell lung cancer, non-small cell lung cancer, pediatric solid tumor, CD 133+ cancer stem cells, NKGDL+ cancer cells, PD-L1+ cancer cells, oral and oropharyngeal cancer (e.g., tongue cancer, esophageal cancer, laryngeal cancer, pharyngeal cancer) methylcholanthrene-induced sarcomas and colorectal cancer. In some embodiments, the subject suffers from a myelodysplastic syndrome (MDS), a myeloproliferative neoplasm (MPN), a clonal cytopenia of uncertain significance (CCUS), or a clonal hematopoiesis of indetermined potential (CHIP). In some embodiments, the subject is affected, preferably is diagnosed, with an infectious disease. In some embodiments, the subject is affected, preferably is diagnosed, with a viral persistent infection caused by a virus selected from the group comprising or consisting of human immunodeficiency virus (HIV), herpesvirus (e.g., herpes simplex virus-1, cytomegalovirus (CMV)) influenza, retroviruses, human papillomavirus (HPV), enteroviruses e.g., coxsackie B3 virus). In some embodiments, the subject is affected, preferably is diagnosed, with a parasitic infection. Examples of parasitic infections include, but are not limited to, toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. In some embodiments of the invention, the subject to be treated is administrated at least once with the therapeutically effective amount of the composition as described above.In some embodiments, a single dose of cells from the present invention is administered to thesubject. In another embodiment, a plurality of doses of cells of the present invention are administered over a period of time. In some embodiments, a therapeutically effective amount of cells is administered, is for administration or is to be administered to the subject to be treated. In some embodiments, the therapeutically effective amount ranges from about 0.5x107to about 3x107cells / kg body weight. In some embodiments, the population of cells, the composition, the pharmaceutical composition or the medicament of the invention is administered (or is to be administered or is for administration) by intravesical administration, intravaginal administration, intraosseous administration, intraperitoneal administration, intrauterine administration, intraocular administration, intradermal administration, intraarterial administration, intracerebral administration, intranasal administration, enteral administration, buccal administration, intranasal administration, oral administration, rectal administration, or by inhalation. In some embodiments, the population of cells, the composition, the pharmaceutical composition or the medicament of the invention is administered (or is to be administered or is for administration) by injection, including, without limitation, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intra-sternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1: (A) Schematic representation of the co-culture assay between WT MPP4 and WT, + / 1013 or 1013 / 1013 MSCs. Seven days after the co-culture, the differentiation of MPP4 intomyeloid cells (CD11b+ CD11c+) was evaluated by flow cytometry. (B) Schematicrepresentation of the assay (left) and absolute numbers of CD11b+Cd11c+cells produced after 7 days of co-culture of WT MPP4 with either WT or mutant MSCs, with or without the addition of AMD3100 to the co-culture media (right). Data (means + SEM) are from three independent experiments with six mice per group. (C-D) Expression levels of lymphoid (Flt3, IL7R, Ikzf1) (C) and myeloid (Mpo and Irf8) genes (D) in WT MPP4 sorted after 2 days of coculture with WT or mutant MSCs determined by single cell qPCR. Data are from two independent experiments, with two mice per group, each sample was run in duplicate and standardized forβ-actin expression, with >50 cells analyzed for each condition. (E) Schematic diagram of theexperimental procedure for the generation of BM chimeras with WT MPP4 co-cultured with either WT or mutant MSCs for 2 days. (F) Proportions of donor WT CD45.2+B cells (B220+CD19+) and myeloid cells (CD11b+) detected in the BM of CD45.1.2+recipients 14 days following the transplantation. Data are from one experiment with 3 mice per group. *P <0.05 and **P<0.005 compared to WT MSCs.§P <0.05 and§§P<0.005 compared to non-treated groups.Figure 2: (A) Additional myeloid (F4 / 80+ and Ly6c+) cell markers after 7 days of MPP4 co-culture with MSCs. (B) Percentage of surviving WT MPP4 at days 2 and 4 (D2 and D4) of co-culture with WT MSCs in a Transwell indirect system analyzed by flow cytometry, compared to the direct co-culture assay. Data are from three independent experiments with six mice per group. (C) Representative flow cytometry plot showing the frequencies of B cells (CD11b- B220+) and myeloid cells (CD11b+B220-) generated from MPP4 after 7 days of culture withOP9 / IL7 stromal cells. ***P<0.005 compared to direct coculture condition.Figure 3: (A) Schematic representation of the experimental protocol: HS5 cells were transduced with lentiviral particles encoding WT hCXCR4 and GFP, R334X mutant hCXCR4 and GFP or GFP only. GFP+ HS5 cells were sorted and co-cultured with CD34+ hematopoietic stem and progenitor cells from cord blood. Seven days after the co-culture, the differentiation of CD34+ cells into granulocyte macrophage progenitors (GMP, gated as: CD45+ CD38-CD10- CD45RA+) or common lymphoid progenitors (CLP, gated as: CD45+ CD38- CD10+CD45RA+) was evaluated by flow cytometry. (B) Absolute numbers of GMP and CLPproduced after 7 days of co-culture of hCD34+ cells with either control-GFP, WT CXCR4-GFP or R334X CXCR4-GFP transduced HS5 cells. Data (means + SEM) are from two biological replicates and are representative of two independent experiments. EXAMPLE: Material & Methods Mice and genotyping All mice were bred as previously mentioned13. Heterozygous Cxcr4+ / 1013(+ / 1013) mice were developed by a knock-in strategy13. Cxcr41013 / 1013mice (1013 / 1013) were generated by crossing heterozygous mice. WT littermate mice served as controls. All mice were littermates and were between 8 and 12 weeks old. Adult Boy / J (CD45.1 / 2) (Charles River) mice were used as transplant recipients. All experiments followed the European Union guide for the care and use of laboratory animals and have been reviewed and approved by an institutional review committee (CE1-21, Animal Care and Use Committee, Villejuif, France and Comité d’éthique Paris-Nord N°121, Paris, France). Daily observation was conducted to make sure that no mouse endured any kind of pain or discomfort throughout the experimentation. Sample isolation in miceFor MSC and MPP4 extraction, the tibia, hips and femurs were isolated and centrifuged toseparate the marrow and bone fractions. Flushed bones were chopped into small pieces, followed by enzymatic digestion with 2.5 U / mL collagenase type I (Thermofisher) for 45 min at 37 °C with agitation. Cells were then filtered with a 70 μm nylon strainer and washed with PBS containing 2% fetal bovine serum (FBS)36. The marrow fraction was collected in Phosphate-Buffered Saline solution (PBS) with 2% FBS and filtered with a 70 μm nylon strainer to remove remaining fat and debris. Flow cytometry experiments Flow cytometry staining was performed as previously described16. Samples were acquired on an LSRII Fortessa flow cytometer (BD Biosciences) and cells were sorted using the FACSAria III cell sorter (BD). Data were collected using the FACS Diva software version 7 (BD). FLOWJO software v10.7 (BD) was used for the analysis of all flow cytometric data. Co-culture of primary MSCs and MPP4 Sorted MSCs were expanded for seven days and then seeded in 96-well plates (1 x 103MSCs / well) in “complete MEMα medium” (MEMα medium enriched with 10% FBS, 1% P / S and 50 μM 2-mercaptoethanol), followed by an incubation of 24 hours for cell attachment.Next, the MSC growth medium was replaced with “complete DMEM medium” (DMEMmedium supplemented with 10% FBS, 1% P / S and 50 μM 2-mercaptoethanol) either with or without the addition of 10 μM of AMD3100, and MPP4 were subsequently sorted directly onthe MSC monolayer (0.5 x 103 MPP4 / well). The co-culture was maintained for seven days at37°C and 5% CO2.For the Transwell-based assay, MSCs were seeded in complete MEMα medium, into the lowerchamber of a 96-well Transwell plate (0.4 µm pore size, CLS3381 SIGMA) (1 x 103MSCs / well). After 24 hours, the medium was replaced with complete DMEM medium, and 0.5 x 103MPP4 per well were added to the top chambers in complete DMEM medium. Transduction and selection of HS-5 The human HS-5 MSC cell line was transduced with lentiviral vectors (pLenti-P2A-tGFP, Origene) encoding: 1) GFP only, 2) human CXCR4 WT and GFP, 3) human CXCR4 R334X and GFP. Transduced cells expressing the GFP were then sorted on a FACSAria III cell sorter(BD) and used for subsequent analyses.Co-culture of transduced HS-5 with human CD34+ HSPCCD34+ HSPC were isolated from cord blood through Ficoll gradient and magnetic enrichment (CD34+ enrichment Kit, Miltenyi). 104CD34+ HSPC were cultured with 104HS-5 (control-GFP, WT hCXCR4-GFP or R334X hCXCR4-GFP) for 7 days in MEMalpha supplementedwith FCS 10% and penicillin / streptomycin 1%. Statistics Prism software (GraphPad Software) was used for all statistical analysis. The significance between two groups was assessed using the Unpaired two-tailed Student t test. Results: EXAMPLE 1 : Cxcr41013-bearing MSCs promote the differentiation of MPP4 into myeloid cells We focused on the relationship between MSCs and MPP4, and the impact of Cxcr4 gain-of-function on this “HSPC- niche cell” interaction. To do so, we developed a novel in vitro co-culture model between primary sorted MSCs and MPP4, without requiring the addition of exogenous cytokines (Figure 1A). Seven days following the co-culture, we reported efficient differentiation of MPP4 into myeloid cells (CD11b+CD11c+, F4 / 80+and Ly6clowcells) by flow-cytometry analyses (Figures 1A, 2A), proving that MSCs alone are able to provide a properenvironment and support for in vitro expansion and differentiation of MPP4. Co-culture usinga Transwell system failed to support MPP4 survival and expansion, suggesting the essentialrequirement for direct physical contact with MSCs in vitro (Figure 2B). Strikingly, we onlyobserved myeloid specification of lymphoid-biased MPP4. This discrepancy could be explained by the persistence of some myeloid potential within this normally lymphoid-biased cell subset,evidenced by co-expression of different lineage-related genes15,25. The in vitro lymphoiddifferentiation capacity of sorted MPP4 (CD11b- B220+cells) has been successfully demonstrated when cultured on OP9 / IL-7 stromal cells (Figure 2C) Next, to assess whether MPP4 fate could be influenced, extrinsically, by MSCs carrying the Cxcr41013mutation, WT MPP4 were cultured with either WT, + / 1013 or 1013 / 1013 MSCs for seven days (Figure 1A). The number of CD11b+CD11c+generated was significantly increased after co-culture with mutant MSCs compared to WT, and in an allele dose-dependent manner (Figure 1B). The addition of AMD3100 entirely blocked MPP4 differentiation in the WT-WT co-culture, highlighting the critical role played by the Cxcr4 / Cxcl12 signaling axis in the interaction between MSCs and MPP4. In the presence of + / 1013 or 1013 / 1013 MSCs, myeloidcell production from MPP4 was normalized or significantly reduced by AMD3100, respectively(Figure 1B). In addition, single-cell microfluidic-based qRT-PCR performed on MPP4 after two days of co-culture with mutant MSCs, showed a downregulation of their lymphoid geneexpression (Flt3, Il7r and Ikzf1) mirrored by an increase in the expression of the myeloid genes(Mpo and Irf8) (Figures 1C, 1D). As such, our results revealed that exacerbated Cxcr4 signaling in MSCs could affect MPP4 destiny, stimulating their myeloid differentiation potential by altering their myeloid-lymphoid gene expression profile. We next examined the effect of Cxcr41013-bearing MSCs on in vivo repopulating activity of MPP4. Co-cultured WT CD45.2+MPP4 were isolated after 2 days and transplanted into sub- lethally irradiated WT CD45.1.2+recipient mice, and their lineage output was evaluated 14 dayspost-transplant (Figure 1E). MPP4 collected from WT MSC cultures gave rise to bothlymphoid (B220+CD19+) and myeloid (CD11b+) cells (Figure 1F). However, MPP4 co- cultured with 1013 / 1013 MSCs were less efficient at reconstituting lymphoid cells, while their myeloid output in the BM was increased, unraveling that Cxcr41013-carrying MSCs drive an imbalance in MPP4 lymphoid-myeloid reconstitution capacity (Figure 1F). Collectively, these data imply that Cxcr41013-bearing MSCs promote MPP4 myeloid skewing, possibly through the production of secreted factors. Discussion: Using our unique primary co-culture model and in vivo approach, we demonstrated that Cxcr41013–bearing MSCs cause a myeloid skewing in WT lymphoid-primed MPP4, a phenotype seen in mutant mice15, linked with transcriptional reprogramming of MPP4 lineage- specific genes. Indeed, the overproduction of Il6 by the surrounding feeder-cell may partially explain this observed lineage skew, as increased Il6 signaling was previously correlated to the age-associated myeloid bias of HSPCs26. Moreover, our study showed that an excess of secreted Cxcl12 may act, in a paracrine manner, as a potential driver of MPP4 impaired differentiation capacity.Hence, we showed here an increase in the myelopoietic supportive ability of Cxcr41013-bearingMSCs, which were previously reported to have a diminished potential to differentiate into bone- forming osteoblasts16. Interestingly, this observed alteration in the “osteogenic fate / myelopoiesis support” balance of Cxcr41013-bearing MSCs aligns with earlier studies revealing that aged BMSCs display decreased osteogenic potential along with enhanced myeloid-promoting activity27–29. Likewise, Tikhonova et al. identified, at steady state, a subset of BMSCs primed transcriptionally for adipocytic specification with an enrichment of its pro- myeloid niche factors19. Thus, further studies may be conducted to assess whether Cxcr41013- bearing MSCs exhibit an aged phenotype. In conclusion, the coculture of MSCs, especially MSCs having a CXCR4 with an enhanced activity, and MPP4 is particularly interesting for generating myeloid cells. EXAMPLE 2 : Human HS5 cells were transduced with lentiviral particles encoding WT hCXCR4 and GFP, R334X mutant hCXCR4 and GFP or GFP only, without addition of exogenous cytokines. GFP+ HS5 cells were sorted and co-cultured with CD34+ hematopoietic stem and progenitor cellsisolated from cord blood (Figure 3A). Seven days after the co-culture, the differentiation ofCD34+ cells into granulocyte macrophage progenitors (GMP) or common lymphoid progenitors (CLP) was evaluated by flow cytometry. The results revealed an increase of GMP after co-culture of hCD34+ cells with R334X hCXCR4-GFP HS5 cells compared to GFP orWT hCXCR4-GFP HS5 cells (Figure 3B). Then, no significant difference was observedbetween CLP number generated from hCD34+ cells cultured with either R334X hCXCR4-GFPor WT hCXCR4-GFP HS5 cells. The coculture of human mesenchymal stem cells (MSCs),particularly those with enhanced CXCR4 activity, alongside human hematopoietic stem and progenitor cells, is thus effective for generating myeloid cells. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.1. Chan, C. K. F. et al. Identification and Specification of the Mouse Skeletal Stem Cell.Cell 160, 285–298 (2015).2. Bianco, P. & Robey, P. G. Skeletal stem cells. Development 142, 1023–1027 (2015).3. Serowoky, M. A., Arata, C. E., Crump, J. 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Claims

CLAIMS:

1. An in vitro method for generating myeloid cells comprising the steps of i) co-culturinga population of hematopoietic stem and progenitor cells (HSPCs) with a population of mesenchymal stromal cells (MSCs) that naturally or exogenously express a CXCR4 polypeptide with an enhanced receptor signalling, in a culture medium for a sufficient time, without the addition of exogenous cytokines, for allowing myeloid differentiation and ii) harvesting the generated myeloid cells.

2. The method of claim 1 for generating murine myeloid cells comprising the steps of i)co-culturing a population of murine primary lymphoid-primed multipotent progenitors (MPP4s) and a population of murine skeletal stem cell (MSCs) that express a CXCR4polypeptide with an enhanced receptor signalling in a culture medium for a sufficient time, without the addition of exogenous cytokines, for allowing myeloid differentiation and ii) harvesting the generated myeloid cells.

3. The method according to claim 1 or 2 wherein the MSCs express a CXCR4 polypeptidewith an enhanced receptor signalling, more particularly a CXCR4 polypeptide having a shorter C-terminus tail that results in impaired desensitization and enhanced receptor signalling, even more particularly a CXCR4 polypeptide wherein the 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 last amino acid residues of said polypeptide are deleted.

4. The method according to any one of claims 1 to 3 wherein the MSCs express:- a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 333 in SEQ ID NO:1, or -a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 334 in SEQ ID NO:1, or -a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 337 in SEQ ID NO:1, or- a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 342 in SEQ ID NO:1.

5. The method according to any one of claims 1 to 4 wherein the MSCs harbour at leastone gain-of-function mutation, more particularly at least one mutation that leads to theshortening of the C-terminus tail of CXCR4, resulting in impaired desensitization and enhanced receptor signalling, even more particularly harbour at least one mutation that leads to the deletion of the 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 last amino acid residues in the CXCR4 polypeptide.

6. The method of claim wherein the mutation is selected from the group consisting ofR334X, G335X, S338X, S339fs342X, and E343X.

7. The method according to any one of claims 1 to 6 that comprises a step of introducinginto the population of MSCs a polynucleotide that encodes: -a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 333 in SEQ ID NO:1, or -a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 334 in SEQ ID NO:1, or -a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 337 in SEQ ID NO:1, or -a CXCR4 polypeptide that consists of an amino acid sequence having at least 90%of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 342 in SEQ ID NO:1.

8. The method according to any one of claims 1 to 6 wherein the MSCs are edited toexpress one CXCR4 polypeptide with an enhanced receptor signalling, more particularly express one CXCR4 polypeptide having a shorter C-terminus tail thatresults in impaired desensitization and enhanced receptor signalling, even moreparticularly a CXCR4 polypeptide wherein the 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20last amino acid residues of said polypeptide are deleted.

9. The method according to any one of claims 1 to 8 wherein before step i), the MSCs areexpanded and seeded in a solid support (e.g.96-well plate) with the appropriate culture medium for a time sufficient for allowing cell attachment on the solid support, then, the HSPCs (e.g. CD34+ cells) are cultured directly on the generated layer of MSCs.

10. The method according to any one of claims 1 to 9 wherein the cells are co-cultured forat least 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days, preferably 7 days.

11. The method according to any one of claims 1 to 10 that further comprises a geneticmodification in HSPCs step that consists to a gene disruption step, a gene correctionstep or a gene addition step, preferably a gene addition step.

12. The method of claim 11 wherein an exogenous nucleic acid sequence expressing a geneof interest is introduced into the HSPCs, preferably before step i) of the methods.

13. The method of claim 12 wherein the exogenous nucleic acid sequence to be introducedinto the cells encodes a Chimeric Antigenic Receptor (CAR).

14. An isolated population of myeloid cells susceptible to be obtained, or obtained, by thein vitro method according to any one of claims 1 to 13.

15. A pharmaceutical composition that comprises the isolated population of cells accordingto claim 14.

16. A method of therapy in a subject in need thereof comprising administering to the subjecta therapeutically effective amount of the population of cells according to claim 14.

17. The method of claim 16 wherein the subject suffers from a cancer or an infectiousdisease.

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