Use of artificial marrow organoid for generating NK cells and myeloid cells

Abstract

The human bone marrow (BM) microenvironment involves hematopoietic and non-hematopoietic cell subsets organized in a rather complex architecture. Tremendous efforts have been made to model it in order to analyse normal or pathological hematopoiesis and its stromal counterpart. Herein, the inventors report an original, fully-human in vitro 3D model of the BM microenvironment dedicated to study interactions taking place between mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC) during the hematopoietic differentiation. This fully-human artificial marrow organoid (AMO) model is highly efficient to recapitulate MSC support to myeloid differentiation and NK cell development from the most immature CD34+ HSPC to the most terminally differentiated CD15+ CD64+ polymorphonuclear neutrophils or NKG2A-KIR2D+CD57+ NK subset. Interestingly, mature NK cell's phenotype showed significant differences after AMO-based culture compared to a conventional 2D co-culture system in the expression of adhesion molecules and immune checkpoint receptors, suggesting that the 3D model is more suitable to study the BM immunosuppressive microenvironment. Thus, the present invention relates to the use of an artificial marrow organoid for generating NK cells and myeloid cells.

Description

[0001] USE OF ARTIFICIAL MARROW ORGANOID FOR GENERATING NK CELLS

[0002] AND MYELOID CELLS

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine and in particular haematology and immunology.

[0005] BACKGROUND OF THE INVENTION:

[0006] No perfect in vitro model has been established so far to study the complex human hematological cell biology. Such a model should be based on the use of primary cells and encompass within the appropriate local bone marrow (BM) microenvironment (BMM) including a functional immune system. Moreover, ideally such model should be easy to use, fast, reproducible and cheap. A wide range of in vivo models have been developed but all face some major drawbacks in term of insufficient transferability to human beings (no immunological context), the poor engraftment of hematopoietic stem and progenitor cells (HSPC) into recipient mice and high technical complexity (1,2). Therefore, 2D in vitro models are still widely used, due to their relative simplicity, cheapness, reproducibility but missing the spatial organization and biomechanical forces of the BMM, crucial for the cellular fate. To overcome this point, there has been a surge of interest for the development of several 3D models (2,3) that so far do not meet simultaneously the requirements of user-friendliness, reproducibility, low cost and system versatility.

[0007] In parallel, the field of Natural Killer (NK) cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation (4). NK-cells are a heterogeneous subset of cytotoxic lymphocytes, produced within the BM from the HSPCs, and functionally regulated by a repertoire of activating and inhibitory receptors. Through the production of cytokines (ZFN-y / TNF-a) and cytolytic proteins (perforin / granzymes) and the death receptors, NK-cells are very potent to eliminate cancer cells (5). Consequently, various strategies to generate NK-cells for adoptive immunotherapy have been developed, mostly relying on a combinations of cytokines with or without feeder cells (5-7), each with its specific advantages and disadvantages in regard of cell numbers, function and handling efforts. Yet, how the BMM can support, reshape and eventually interfere with the production and function of NK progenitor and mature cells remains largely unknown. SUMMARY OF THE INVENTION:

[0008] The present invention is defined by the claims. In particular, the present invention relates to the use of an artificial marrow organoid for generating NK cells and myeloid cells.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] The human bone marrow (BM) microenvironment involves hematopoietic and non- hematopoietic cell subsets organized in a rather complex architecture. Tremendous efforts have been made to model it in order to analyse normal or pathological hematopoiesis and its stromal counterpart. Herein, the inventors report an original, fully-human in vitro 3D model of the BM microenvironment dedicated to study interactions taking place between mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC) during the Natural Killer (NK) cell’s development. This artificial marrow organoid (AMO) model is highly efficient to support NK-cell development from the CD34+ HSPC to the terminally differentiated NKG2A- KIR2D+CD57+ NK subset. In addition, myeloid differentiation can be also recapitulated in this model. Interestingly, mature NK cell’s phenotype showed significant differences after AMO- based culture compared to a conventional 2D co-culture system in the expression of adhesion molecules and immune checkpoint receptors, suggesting that the 3D model is more suitable to study the BM immunosuppressive microenvironment. All these later makes the AMO model a perfect fully-human platform for drug testing, including immunomodulating molecules. To conclude, the AMO system constitutes a friendly and faithful experimental tool for studying cell-cell interactions and hematological ontogenesis taking place in human BM.

[0011] Main definitions:

[0012] As used herein, the term "about" encompasses a deviance of between 0.0001-5% from the indicated number or range of numbers. Further, it may encompass a deviance of between 1 - 10% from the indicated number or range of numbers. In addition, it may encompass a deviance of up to 25% from the indicated number or range of numbers.

[0013] 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 (e.g. a population of mesenchymal stromal cells comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the highly immune functions).

[0014] As used herein, the term “isolated" refers to a product (e.g. a cell or a population of cells), compound, or composition which 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.

[0015] 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 or 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).

[0016] 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 (BMSSC), 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 Therapeutics Inc., 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.), 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.

[0017] 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 "hematopoietic cell" refers to a continuum of cells ranging from a HSPC, to a fully differentiated blood cell, including all the cells of the myeloid and lymphoid lineages.

[0018] As used herein, the term “NK cell” or “natural killer cell” refers to a cytotoxic lymphocyte playing an important role in the innate immunity. NK cells are constantly in contact with other cells. NK cells express activating and inhibitory receptors at their cell surface. This mechanism allows NK cells to recognize if a cell is to be eliminated (such as, for example, a tumor cell or an infected cell) or not. In some embodiments, NK cells may be defined as “immature” (immature NK cells are generally defined by the absence of expression of CD 16 and of KIR receptors), or “mature” (mature NK cells are generally defined by the expression of CD16 and of KIR receptors). Mature NK cells include memory -like NK cells.

[0019] 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 that originate from a common myeloid progenitor in the bone marrow. Examples of myeloid lineage markers include pan-myeloid marker CDl lb, CD206 for M2 -type macrophages, CD68, and CD 15 for 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.

[0020] Methods of the present invention:

[0021] The first object of the present invention relates to an in vitro method for generating NK cells comprising the steps of i) aggregating hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs), ii) seeding and culturing the cell pellet obtained at step i) on a culture support at the air-fluid interface in a medium supplemented with appropriate cytokines for allowing NK differentiation and iii) harvesting the generated NK cells.

[0022] The second object of the present invention relates to an in vitro method for generating myeloid cells comprising the steps of i) aggregating hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs), ii) seeding and culturing the cell pellet obtained at step i) on a culture support at the air-fluid interface in a medium supplemented with appropriate cytokines for allowing myeloid differentiation and iii) harvesting the generated myeloid cells.

[0023] Mesenchymal stromal cells (MSCs):

[0024] Any type of isolated MSCs may be suitable for the purposes of the present invention. Such mesenchymal stromal cells may be isolated from a variety of organisms. Preferably the MSCs are isolated from murine or human sources. Most preferably, the MSCs are isolated from human sources. The MSCs may be isolated from a variety of tissue types. MSCs are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum. The presence of MSCs in culture colonies may be verified by specific cell surface markers which are identified with monoclonal antibodies. See U.S. Pat. Nos. 5,486,359 and 7,153,500. MSCs are characterized by their ability to adhere to plastic tissue culture surfaces (Friedenstein et. al., reviewed in Owen & Friedenstein, 1988), and by being an effective feeder layers for hematopoietic stem cells (Eaves et. al., 2001). Mesenchymal stem cells (MSCs) may be purified using methods known in the art (Wakitani et. al., 1995; Fukuda and Yuasa, 2006; Woodbury et al., 2000; Deng et. al., 2001; Kim et. al., 2006; Mareschi et. al., 2006; Krampera et. al., 2007).

[0025] Hematopoietic stem and progenitor cells (HSPCs):

[0026] In some embodiments, the HSPCs are isolated from cord blood cells.

[0027] In some embodiments, the HSPCs are recovered from an adult donor. In some embodiments, HSPCs are obtained from a bone marrow puncture of from peripheral blood from adult donors. As the number of HSPCs is low in an adult peripheral blood (i.e., 0,15% of the cells) HSPCs may be mobilized from the bone marrow to the periphery (e.g., the blood) to increase the number of HSPCs in the peripheral blood. In some embodiments, to mobilize HSPCs, adult donors may be treated with granulocyte colony-stimulating factor (G-CSF) and / or plerixafor, preferably with G-CSF. Other examples of mobilizing agents include, but are not limited to, agonists of CXCR2 (e.g., MGTA 145), and analogs of plerixafor.

[0028] In some embodiments, the HSPCs are isolated from mobilized peripheral blood.

[0029] Methods for isolating HSPCs are well known in the art and include, without limitation, methods using beads coated with an antibody recognizing CD34. In some embodiments, HSPCs are isolated using the indirect CD34 microbead kit (Miltenyi).

[0030] Aggregation:

[0031] According to the present invention, step i) can be performed by any cellular aggregating method well-known in the art and typically described. In particular, the aggregation method is a centrifugation-based methos which produces submersed cell pellets that subsequently aggregate in the medium in which they were generated (e.g. White, Andrea, et al. "Lymphotoxin a- dependent and-independent signals regulate stromal organizer cell homeostasis during lymph node organogenesis. "Blood, The Journal of the American Society of Hematology 110.6 (2007): 1950-1959). More particularly, the aggregation is performed according to the teaching of Sheridan JM, Taoudi S, Medvinsky A, Blackburn CC. A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations. Genesis. 2009 May;47(5): 346-51. Typically, a simple compaction reaggregation can be used by which MSCs are aggregated with HSPCs by centrifugation. Depending on the experiment, different amount from 8* 104to 1.5>< 105MSCs are suspended with 6x l03-7,5>< 103HSPCs. Then, the cell suspension is centrifuged at a relative centrifugal force that ranges from 100 to 400g, more preferably about 300g for a period that ranges from 2min to lOmin, preferably for about 5min.

[0032] Culture support:

[0033] Any culture support that allows the culture at the air-fluid interface may be suitable for performing step ii). Typically, the solid support is a semi-permeable synthetic membrane, such as, the culture insert as described in the EXAMPLE. In particular, one side of the support membrane is exposed to the culture medium.

[0034] Culture medium:

[0035] Any culture medium for allowing the culture of NK cells and / or myeloid cells can be suitable. Typically, the culture medium is selected from the group comprising RPMI Glutamax medium (Thermo Fischer, MA, USA), StemSpan serum free medium (Stem Cell Technologies, Vancouver, Canada), Serum-free CellGro SCGM medium (Bioz, CA, USA), CellGro DC medium (CellGenix, Freiburg, Germany), Glycostem Basal Growth medium (Clear Cell Technologies, Beernem, Belgium) and Alpha MEM (Thermo Fischer, MA, USA) preferably the RPMI Glutamax medium (Thermo Fischer, MA, USA).

[0036] Typically, the culture medium is changed completely every 2, 3, 4, or 5 days, preferably 3 days.

[0037] Cytokines for NK differentiation:

[0038] In some embodiments, the culture medium used at step ii) for the NK differentiation comprises at least 1, 2, 3, 4 or 5 cytokines selected from the group comprising or consisting of IL-7 (interleukin-7), SCF (stem cell factor), TPO (thrombopoietin), Flt3-L (Flt3 ligand), and IL-15 (interleukin-15). In some embodiments, the culture medium used at step ii) comprises at least at least 1, 2, 3, 4 or 5 cytokines selected from the group comprising or consisting of human IL- 7, human SCF, human TPO, human Flt3-L, and human IL-15. In some embodiments, the culture medium comprises IL-7 (preferably hIL-7), SCF (preferably hSCF), Flt3 -L (preferably hFlt3- L), TPO (preferably hTPO) and IL-15 (preferably hIL-15).

[0039] Said cytokines are well known in the art and are commercially available. For instance, an exemplary amino acid sequence for hlL-7 can correspond to uniprot accession number: P13232, an exemplary amino acid sequence for hSCF can correspond to the uniprot accession number: P21583, an exemplary amino acid sequence for hFlt3-L can correspond the uniprot accession number: P49771, an exemplary amino acid sequence for hTPO can correspond the uniprot accession number: P40225 and an exemplary amino acid sequence for hlL-15 can correspond to uniprot accession number: P40933.

[0040] In some embodiments, the concentration of hIL-7 ranges from about 2 to about 100 ng / mL, preferably from about 2 to about 20 ng / mL and more preferably is of about 10 ng / mL.

[0041] In some embodiments, the concentration of hSCF ranges from about 2 to about 100 ng / mL, preferably from about 2 to about 20 ng / mL and more preferably is of about 10 ng / mL.

[0042] In some embodiments, the concentration of hFlt3-L ranges from about 2 to about 100 ng / mL, preferably from about 2 to about 20 ng / mL and more preferably is of about 10 ng / mL.

[0043] In some embodiments, the concentration of hTPO ranges from about 2 to about 100 ng / mL, preferably from about 2 to about 20 ng / mL and more preferably is of about 10 ng / mL.

[0044] In some embodiments, the concentration of hIL-15 ranges from about 2 to about 100 ng / mL, preferably from about 10 to about 40 ng / mL and more preferably is of about 20 ng / mL.

[0045] In some embodiments, the culture medium used at step ii) comprises an antagonist of aryl hydrocarbon receptor (AHR). As used herein, the term “AHR antagonist” refers to any agent / compound that inhibits AHR activity, or to any agent / compound that down-regulates an activity and / or expression of AHR, or to any agent / compound that is a down-stream effecter of AHR pathway”. The AHR antagonist can include, for example, StemReginin-1 (SR-1) (Boitano et al. Science. 329, 1345-1348 (2010)), CH-223191 (Kim et al. Mol. Pharmacol. 69, 1871-1878 (2006)), GNF351 (Smith et al. J. Pharmacol. Exp. Ther. 338, 318-327 (2011)), and / or 6,2,4- trimethoxyflavone (Murray et al. Pharmacol. Exp. Ther. 332, 135-144 (2010)). In some embodiments, the antagonist is selected from the group consisting of StemRegeninl (SRI), PDM2, GNF351, CH-223191, BAY 2416964, PDM-11, or BAY-218. In some embodiments, a preferred AHR antagonist is StemReginin-1 (SR-1).

[0046] Cytokines for myeloid differentiation:

[0047] In some embodiments, the culture medium used at step ii) for the myeloid differentiation comprises at least 1, 2, 3, or 4 cytokines selected from the group comprising or consisting of IL-3 (interleukin-3), SCF (stem cell factor), TPO (thrombopoietin), and Flt3-L (Flt3 ligand). In some embodiments, the culture medium used at step ii) comprises at least at least 1, 2, 3, or 4 cytokines selected from the group comprising or consisting of human IL-3, human SCF, human TPO, and human Flt3-L. In some embodiments, the culture medium comprises IL-3 (preferably hIL-7), SCF (preferably hSCF), Flt3 -L (preferably hFlt3-L), and TPO (preferably hTPO).

[0048] Said cytokines are well known in the art and are commercially available. For instance, an exemplary amino acid sequence for hlL-3 can correspond to uniprot accession number: P08700, an exemplary amino acid sequence for hSCF can correspond to the uniprot accession number: P21583, an exemplary amino acid sequence for hFlt3-L can correspond the uniprot accession number: P49771, and an exemplary amino acid sequence for hTPO can correspond the uniprot accession number: P40225.

[0049] In some embodiments, the concentration of hIL-3 ranges from about 5 to about 50 UI / mL, preferably from about 5 to about 30 UI / mL and more preferably is of about 20 UI / mL.

[0050] In some embodiments, the concentration of hSCF ranges from about 10 to about 300 ng / mL, preferably from about 10 to about 200 ng / mL and more preferably is of about 100 ng / mL.

[0051] In some embodiments, the concentration of hFlt3-L ranges from about 10 to about 400 ng / mL, preferably from about 10 to about 300 ng / mL and more preferably is of about 200 ng / mL. In some embodiments, the concentration of hTPO ranges from about 2 to about 100 ng / mL, preferably from about 2 to about 20 ng / mL and more preferably is of about 10 ng / mL.

[0052] Duration:

[0053] Typically, for generating NK cells, the pellet is cultured for at least 2 weeks, preferably 3 weeks. Typically for generating myeloid cells, the pellet is cultured for at least 1 week, preferably 2 weeks.

[0054] Engineering o f the cells:

[0055] In some embodiments, the generated NK cells or myeloid cells are engineered. As used herein, the term “engineered” refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered cell” refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some 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. Those of ordinary skill in the art will appreciate that 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 cell.

[0056] In some embodiments, the methods of the invention further comprise a genetic modification step. In some embodiments, the genetic modification step(s) correspond(s) to a gene disruption step, a gene correction step or a gene addition step, preferably a gene addition step. In some embodiments, the genetic modification step(s) is / are carried out by a method selected from the group comprising, but not limited to, transfection, transduction or gene editing. Examples of methods of gene editing that may be used in 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 on Transposons (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.

[0057] 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.

[0058] In some embodiments, an exogenous nucleic acid sequence expressing a gene of interest is introduced into the HSPCs, preferably before step i) of the methods. In some embodiments, the transduction or transfection of HSPCs is carried out before step i) of the methods of the present invention.

[0059] 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).

[0060] 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 cytokines receptors or variant thereof (such as, for example, variants of cytokines or cytokines receptors with increased stability). In some embodiments, the exogenous nucleic acid sequence encodes a chimeric cytokine receptor or orthogonal cytokine-receptor pairs.

[0061] In some embodiments, the genetic modification step(s) is / are a gene disruption step, aiming at decreasing or abolishing 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).

[0062] NK cells of the present invention:

[0063] A further object of the present invention is an isolated population of NK cells susceptible to be obtained, or obtained, by the in vitro method of the invention.

[0064] In some embodiments, the NK cells of the invention are CD3- CD56+ NK cells.

[0065] In some embodiments, the CD3-CD56+ NK cells generated by the method of the invention have a purity of at least about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%, i.e., more than 60% of the cells recovered at step iii) are CD3-CD56+ NK cells.

[0066] In some embodiments, the NK cells susceptible to be obtained, or obtained, by the method of the invention express CD 16. In some embodiments, the NK cells susceptible to be obtained, or obtained, by the method of the invention express at least one activating receptor selected from the group comprising or consisting of NKp30, NKG2D and DNAM-1, and at least one inhibitory receptor selected from the group comprising or consisting of NKG2A and KIR molecules. In some embodiments, the NK cells susceptible to be obtained, or obtained, by the method of the invention express at least one further molecule selected from the group consisting of KIRDS1 / S2, KIR2DS4, KIR2DL4, CD94 / NKG2C, KIR3DL2, NKG2D, NCRs, 2B4, NTBA, NKp44, NKp46 and NKp80.

[0067] In some embodiments, the NK cells susceptible to be obtained, or obtained, by the method of the invention are thus mature NK cells. Mature NK cells may for example express CD 16 and / or KIR receptors.

[0068] In some embodiments, the NK cells of the present invention (that may for example be obtained by the method of the present invention) are functional, i.e., are capable of cytotoxic activity. The function of NK cells may be verified by conventional methods known in the art. Examples of such methods include, without limitation, cytotoxicity assays, measurement of the secretion of IFN-g or of TNF-a and measurement of the production (and capacity of degranulation) of perforin and granzyme. Examples of such methods are presented in EXAMPLE. In some embodiments, the NK cells of the present invention express a CAR. In some embodiments, the NK 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 NK cells of the present invention express a chimeric cytokine receptor or orthogonal cytokine-receptor pairs. In some embodiments, the NK cells of the present invention express IL-15 or a variant thereof. In some embodiments, the NK cells of the present invention express CD 16 or a variant thereof (such as, for example, cleavage resistant variant of CD 16). In some embodiments, the NK 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). In some embodiments, the NK 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, TGFB2, Cytokine induced STAT inhibitor (CIS) and Signal regulatory protein alpha (SIRPa).

[0069] Myeloid cells of the present invention:

[0070] 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.

[0071] In some embodiments, the myeloid cells of the invention are CD45+ CD 15+ myeloid cells.

[0072] In some embodiments, the CD45+ CD 15+ myeloid cells generated by the method of the invention have a purity of at least about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%, i.e., more than 60% of the cells recovered at step iii) are CD45+ CD 15+ myeloid cells.

[0073] In some embodiments, the myeloid cells susceptible to be obtained, or obtained, by the method of the invention can also express at one further phenotypic marker such as CD13 or CD33.

[0074] 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 CD 16 or a variant thereof (such as, for example, cleavage resistant variant of CD 16). 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). 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, TGFB2, Cytokine induced STAT inhibitor (CIS) and Signal regulatory protein alpha (SIRPa).

[0075] Therapeutic uses and pharmaceutical compositions:

[0076] A further object of the present invention is a cell population (i.e. a population of NK cells or 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

[0077] A further object of the present invention is a cell population (i.e. a population of NK cells or a population of myeloid cells) as described herein for use in therapy, such as, for example, in “off-the-shelf therapy.

[0078] A further object of the present invention is a cell population (i.e. a population of NK cells or a population of myeloid cells) as described herein for use as a medicament.

[0079] The present invention further relates to the use of a cell population (i.e. a population of NK cells or a population of myeloid cells) as described herein, for the manufacture of a medicament for increasing the number of said cells in a subject.

[0080] The present invention further relates to a method for increasing the number of NK cells in a subject in need thereof, comprising administering to the subject a NK cell population as described herein (in particular a therapeutically effective amount of NK cells as described herein). 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 as described herein (in particular a therapeutically effective amount of myeloid cells as described herein).

[0081] The present invention further relates to a composition comprising, consisting essentially of or consisting of a cell population (i.e. a population of NK cells or a population of myeloid cells) according to the present invention.

[0082] 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.

[0083] In some embodiments, the pharmaceutical composition comprises, consists essentially of or consists of a cell population (i.e. a population of NK cells or a population of myeloid cells) according to the present invention and at least one pharmaceutically acceptable excipient.

[0084] 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.

[0085] 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.

[0086] In some embodiments, the medicament comprises, comprises, consists essentially of or consists of a cell population (i.e. a population of NK cells or a population 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 NK cells or myeloid cells of the invention are the only one therapeutic agents or agents with a biologic activity within said composition, pharmaceutical composition or medicament.

[0087] In some embodiments, the cell population (i.e. a population of NK cells or a population of myeloid cells), composition, pharmaceutical composition or medicament of the invention is for treating cancer or an infectious disease.

[0088] The present invention thus relates to a cell population (i.e. a population of NK cells or a population of myeloid cells), a composition, pharmaceutical composition or medicament as disclosed herein for treating cancer or an infectious disease.

[0089] The present invention further relates to the use of a cell population (i.e. a population of NK cells or a population of myeloid cells) as disclosed herein for the manufacture of a medicament for treating cancer or an infectious disease.

[0090] 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 cell population (i.e. a population of NK cells or a population of myeloid cells) as described herein.

[0091] In some embodiments, the subject is a human.

[0092] In some embodiments, the subject is affected, preferably diagnosed with a 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).

[0093] 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.

[0094] 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 here above.

[0095] In some embodiments, a single dose of cells of the present invention is administered to the subject. In another embodiment, a plurality of doses of cells of the present invention are administered over a period of time.

[0096] 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 0.5xl07to about 3xl07cells / kg body weight.

[0097] 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-stemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0098] 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.

[0099] FIGURES:

[0100] Figure 1. Protocol for the Artificial Marrow Organoid (AMO) formation and observation. AMO can be formed by concentrating MSCs and other cell types and depositing a drop of cell concentrate in a membrane placed over cell-culture medium. (A) CD34+ hematopoietic stem and progenitor cells isolated from BM or cord blood were differentiated into NK cells in AMO using an appropriate cocktail of cytokines. Direct optical microscopic observations (xlO) of AMO with CD34+ HSPCs after one and two weeks of culture are shown.

[0101] Figure 2. Myeloid differentiation compared to standard 2D MSC co-culture

[0102] Morphological evaluation of one representative myeloid cell differentiation at 21 days observed after MGG staining of AMO smears at x 100 magnification. One representative experiment out of three is presented. (A) Quantification of the CD45 intensity (n=15808 and 11237 cells, at day7 and day21 respectively) from 3 different experiments analysing, by fluorescent microscopy, organoids stained with Hoechst (nuclei) and CD45-AF647 (hematopoietic counterpart cell membrane) ; Mann Whitney test p-value <0,0001). Data are represented as mean ± SD. (B) Spatial cells' distance of CD45+ cells to organoid border at day 7 for 4 independent AMO numerated from 1 to 4 (N=1493, 268369, 28548, 4842 cells analyzed for independent organoid). Data are represented as mean ± SD. (C) Percentage of HSPCs (n=3, Wilcoxon test p-value: 0.03, 0.02 and 0.005 for day 7, day 14 and day 21 respectively) PMNs (n=3, Wilcoxon test p-value: 0.4, 0.07 and 0.007 for day 7, day 14 and day 21 respectively) and monocytes at each time point in the AMO and the 2D model (n=3, Wilcoxon test p-value: 0.03, 0.0001 and 0.002 for day 7, day 14 and day 21 respectively). Figure 3. NK cell differentiation from HSPC to mature effector cells. (A) Schematic representation of NK cell maturation stages used for manual gating. Image from Di Vito et al (3). (B) Gating strategy allowing to distinguish the different stages of NK cell maturation: HSC (CD34+ CD38-), CLP (CD34+ CD38+ CD 10+ CD45RA+), immature NK cells (stage 3: CD34- CD45RA+ CD56- CD16-), and mature NK cells (stage 4: CD34- CD45RA+ CD56bright CD16-, stage 5: CD34- CD45RA+ CD56dim CD16+ and stage 6: CD34- CD45RA+ CD56dim CD 16+ CD57+ KIR2D+ / -). (C) Tree representation of SPADE clustering colored by different lineage markers. The size of nodes is proportional to the number of cells in the given cluster. (D) Evolution of NK cell differentiation stages in percentage at two and four weeks of maturation inside the AMO (n=2 at 2 weeks and n=3 at 4 weeks). (E) Phenotype of a representative AMO at two weeks of differentiation; density plot of live NK cells gated on stage 5 cell subset as defined in (B). The positivity of NK cell receptors is assessed by comparison to unstained cells gated on forward scatter.

[0103] Figure 4. NK Cell Differentiation from CD34+ HSPCs in AMO. (A) Evaluation of NK cell’s polyfunctionality after 3 weeks of differentiation inside the AMO (n=4). After organoid dissociation, NK cells were cultured for 4 h at 37°C either in presence of the NK-sensitive K562 cell line or with PMA-Ionomycin. NK cell’s degranulation of cytolytic granules (surface expression of CD107a) and IFN-y and TNF-a intracellular richness were determined by flow cytometry. Proportion of NK-cells with monofunction, bifunction and trifunction significantly increased after stimulation by K562 cell line or PMA and ionomycin (p-value of 0.02 and <0.01 for K562 and PMA-Ionomycin versus control respectively, Fisher exact test). (B) Quantification of the cytotoxic activity measured by calcein-release (results expressed as percentage of specific lysis) after three weeks of NK cell differentiation inside the AMO system compared to mature NK sorted from PB.

[0104] Figure 5. Myeloid Cell Differentiation from CD34+ HSPCs in AMO. (A) Evolution of the main myeloid differentiation markers, quantified overtime by spectral flow-cytometry, after gating alive cells and eliminating doublets. Data were expressed as Median of Fluorescence in loglO scale. The smoothing was performed with Graph Pad Prism v8.0 software. (B) Quantitative evaluation of one representative myeloid cell differentiation at two timepoints (4 and 15 days) observed after MGG staining of AMO smears at xlOO magnification. One representative experiment out of two is presented. EXAMPLE 1: METHODS

[0105] Primary cells. Bone marrow (BM) samples were obtained from patients (ages 18- 80), free of hematological disease and coming to the Orthopedic and Trauma surgery Department of Lariboisiere Hospital (Paris, France) for a total hip replacement surgery. Cord blood (CB) and peripheral blood samples were provided from the blood biobank of Saint-Louis Hospital (Paris, France). All the patients provided written informed consent, in accordance with the Helsinki Declaration. Approval from the Institutional Review Board Paris-Nord was obtained before the use of the clinical materials for research purposes. White cells were purified from the BM or blood samples with a Ficoll gradient (Pancoll Human, PAN-BIOTECH). Human CD34+ cells were magnetically enriched with the EasySep™ Human CD34 Positive Selection Kit II (STEMCELL Technologies) from either normal BM or CB samples. In the same way, mature NK cells were isolated from peripheral blood mononucleated cells with the EasySep™ Human NK Cell Enrichment Kit (STEMCELL Technologies). The human mesenchymal stromal cells (h-MSC) were derived from BM samples and separated from other hematopoietic cells through their plastic adherence after 2-3 weeks of in vitro culture. H-MSC were then expanded in aMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM glutamine, 100 U / mL penicillin and 100 pg / mL streptomycin (Gibco). When the h-MSC reached the 80- 90% confluence, the cells were detached with Trypsin (Gibco) and cryopreserved before used.

[0106] Artificial Marrow Organoid (AMO) culture. The 3D culture was adapted from Sect at al. (1). In details, h-MSC were harvested by trypsinization and resuspended in serum-free culture medium, so-called AMO medium, composed of RPMI 1640 (Gibco), 4% B27 supplement (ThermoFisher Scientific), 30 pM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma) reconstituted in PBS, 100 U / mL penicillin and 100 pg / mL streptomycin (Gibco), 2 mM glutamine (ThermoFisher Scientific) and a precise cocktail of cytokines depending on the experiments and constituted as follows: myeloid differentiation cytokines’ cocktail: IL-3 7 ng / mL (Miltenyi), SCF 100 ng / mL (Miltenyi), TPO 10 ng / mL (Miltenyi), FLT3 200 ng / mL (Miltenyi); NK differentiation cytokines’ cocktail: IL-7 10 ng / mL (Miltenyi), SCF 10 ng / mL (Miltenyi), TPO 10 ng / mL (Miltenyi), FLT3-L 10 ng / mL (Miltenyi), IL- 15 20 ng / mL (Miltenyi), SR-1 2 pM (Miltenyi); NK immunomodulation cocktail: IL-2 100 UI / mL (Miltenyi). Depending on the experiment, different number of h-MSC cells, from 8* 104to 1.5 * 105, were combined with 6* 103-7,5x l03purified CD34+ cells or 4xlO4NK cells per AMO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min. Supernatants were carefully removed and the cell pellet was harvested. For each AMO, a 0.4 pm Millicell transwell insert (EMD Millipore) was placed in a 6-well plate containing 1 mL of AMO medium per well supplemented with the appropriate cytokines. Medium was changed completely every 3 days. The AMO were kept in culture for different time points depending on the experiments: up to two weeks for the myeloid maturation, three weeks for the NK ontogenesis and 1 to 7 days maximum for the NK immunomodulatory experiments. At the indicated time points, AMOs were harvested and dissociated in collagenase (Thermofisher) at a final concentration of 200 U / mL in IX PBS for 30 minutes at 37°C. If the analysis of the MSC is not required, a passage through a 50 pm nylon strainer is recommended.

[0107] 2D cell culture. MSCs were seeded overnight on 24 or 6 well plates in normal aMEM supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM glutamine, 100 U / mL penicillin and 100 pg / mL streptomycin (Gibco). The day after, the media was removed and the NK cells were added to the culture conditions (at the same ratio NK:MSC used in the AMO culture). The co-cultures were maintained in the same AMO medium and analyzed in parallel with the 3D systems.

[0108] H-MSC differentiation. To evaluate differentiation and clonogenic capacities of h-MSC after 3D culture, AMOs were dissociated and cells were counted. CFU-F (colony forming units- fibroblast), adipogenic and osteogenic differentiations were performed as described elsewhere (2). Briefly, for CFU-F, 200 h-MSCs were distributed in 25 cm2flask in 7mL of complete a- MEM FBS medium for three weeks with a medium change once a week. 5.000 h-MSCs were distributed in 24-well plates for osteogenic and adipogenic differentiation for 21 days. After 24h of adhesion, the osteogenic or adipogenic media were added onto the induced wells, whereas complete a-MEM FBS medium or adipogenic maintenance medium was added onto the control wells. At the end of the experiments, medium was removed, cells were fixed and stained following fabricant’s recommendations with 2% crystal violet (SIGMA), 2% red alizarin (SIGMA) or oil red (Lipid oil red staining kit, SIGMA) to assess CFU-F, osteogenic and adipogenic differentiation respectively.

[0109] Flow-cytometry analysis. After AMO dissociation, cells were washed in PBS and stained according to different panels presented in Table 1. Supervised analyses were performed on data extracted from FlowJo vl0.7 software and analyzed with Graph Pad Prism v8.0 software. Uniform manifold approximation and projection (UMAP) and trajectory analyses were performed with OMIC software from Dotmatics (www.omiq.ai, www.dotmatics.com).

[0110] Functional assays on NK cells. The cytotoxicity of NK cells was assessed by a calcein release assay. The NK-sensitive K562 cell line was labeled with 1 pg / mL calcein (ThermoFisher Scientific) in PBS for Ih at 37°C. NK cells were dissociated from the AMO, enumerated and co-cultured with calcein-labeled K562 cells in 96-well U-bottom plates in a medium supplemented with Probenecid at 1 : 1 effectortarget ratio. The killing was quantified after 4 h of incubation at 37 °C by measuring calcein-release into the supernatant. The specific killing was calculated as follows: (Measured fluorescence of K562 +NK cell well — Spontaneous Fluorescence) / (Maximum fluorescence — Spontaneous fluorescence)* 100. For the IFN-y and TNF-a quantification, enriched NK cells were cultured with PMA-Ionomycin (SIGMA, 50 and 500 ng / mL, respectively) or K562 cells (1 : 1 effectortarget ratio) for 5 h at 37 °C. Brefeldin A (Sigma) was added at a final concentration of 10 pg / ml after 1 h of incubation. The percentage of CD107a, IFN-y and TNF-a positive cells was estimated by flow cytometry in the CD3-CD56+ NK cells. Spontaneous release was detected in the absence of target cells.

[0111] Staining and microscopy. The AMO were fixed, without prior dissociation, with 4% paraformaldehyde (PF A, Sigma) for 15 minutes and permabilized with 0,3% Triton-X-100 (Sigma) in PBS for 15 minutes. Blocking solution (0,5% BSA in PBS) was then used for at least 1 hour. After the blocking step, the AlexaFluor647-labeled CD45-specific monoclonal antibody (Biolegend), FITC-labeled Phalloidin (Sigma) and Hoechst 33342 (ThermoFisher Scientific) were used in a solution with saponin 0.025% in PBS, and incubated with the AMO overnight at 4°C. Images were acquired by laser scanning microscopy on a LSM 800 AiryScan system mounted on an Axio Observer stand (Zeiss, Oberkochen, Germany) and with a Plan Apochromat 63X N.A. 1.4 oil-immersion objective using the Zen Blue edition software (Zeiss, v2.3). The pixel size was set at 0,71 pm and a z-step of 0.5 pm was used. The quantification of doxorubicin fluorescence was performed using QuPath software (vO.3.2). May Grunwald Giemsa (MGG) staining was performed on harvested and cytospined cells after AMO coculture with myeloid differentiation induction and dissociation. Different stages of the myeloid differentiation were morphologically identified by optical microscopy at x 100 magnification and counted over a total of 200 cells. In vitro cell treatment and functional assay. AMO were treated in AMO cell culture medium with 0.5 pM of AZA, DAC (Sigma). DMSO condition was used as control. The treatments were maintained for 3 days with a half change of medium after 48 h (IL-2 + drugs or DMSO). Then, cells were collected and NK cells were analyzed for their KIR expression and cytotoxic capacity.

[0112] References:

[0113] 1. Seet CS, He C, Bethune MT, Li S, Chick B, Gschweng EH, et al. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nat Methods. 2017 May;14(5):521-30.

[0114] 2. Bisio V, Espeli M, Balabanian K, Anginot A. Culture, Expansion and Differentiation of Human Bone Marrow Stromal Cells. Methods Mol Biol Clifton NJ. 2021;2308:3-20.

[0115] 3. Di Vito C, Mikulak J, Mavilio D. On the Way to Become a Natural Killer Cell. Front Immunol [Internet], 2019 [cited 2023 Sep 19]; 10. Available from: https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2019.01812

[0116] EXAMPLE 2: RESULTS

[0117] Here, we describe an easy-to-access, reliable and reproducible self-organizing in vitro 3D model of human BMM for studying NK progenitor and mature hematopoietic cells in the frame of their physical crosstalk with the human mesenchymal BM stroma.

[0118] The Artificial Marrow Organoid (AMO), is based on the Seet et al. model (8) in which we substituted the murine stromal cell line MS5-hDLLl with ex vivo expanded human primary MSCs (h-MSC). In the model, the MSC conserved mesenchymal -lineage markers (CD73+CD90+CD106+), formed compact clusters with fibrous connections, and retained lineage differentiation capacity (Figure 1). Importantly, this self-organizing model does not need any chemical or mineral support by contrast to other systems.

[0119] Taking advantage of the simplicity and high reproducibility of this model, we tested whether it could support myeloid and NK-cell development starting from freshly isolated human cord blood CD34+ HSPCs. H-MSCs, sorted by cell-to-plastic adhesion of BM samples from hip replacement surgery, were co-cultured with a small number of CD34+ HSPCs (n= 6000 / 7500) in presence of either myeloid or NK-specific cocktail of cytokines (Figure 1 and methods). Myeloid cell development within the AMO has been investigated with May Grunwald Giemsa- stained cytospins of cells after 3 weeks of culture. It showed characteristic morphology of the myeloid progenitor and monocytic and granulocytic cells covering all the stages of the myeloid differentiation (Data not shown). In line with this observation, confocal microscopy analysis showed an overall increase of the CD45 expression over time (p-value of paired t test <0.001, Figure 2A and data not shown). Moreover, we analyzed the spatial localization of the CD45+ cells in the AMO, observing random distribution throughout the organoid (Figure 2B). Next, we quantified more precisely the myeloid differentiation program by flow cytometry. Myeloid progenitors were distinguished from their erythroid counterparts, by expression of either CD71 or CD235a. After lymphocyte elimination mature myeloid cells were defined as CD 15+ for polymorphonuclear neutrophils (PMN) or CD33+ CD64+ for monocytes. Based on the expression of these specific cell surface markers, the myeloid differentiation was tracked from promyelocytes to myelocytes and beyond. Mature cells were identified in the AMOs from week 1 to week 3 (Data not shown). We observed a general decrease of the immature cell marker, CD34, together with an increase in all mature myeloid markers (CD33, CD13, CD45, CD64, CD 15, CD 1 lb) over time.

[0120] Interestingly the AMOs showed a persistence of CD34+ HSPCs for longer time compared to a classical 2D co-culture model composed by the same MSC and CD34+ cells (Mann Whitney test p-values: 0.03, 0.02 and 0.005 for Wl, W2 and W3 respectively, Figure 2C). The maintenance of immature progenitors was also reflected by the presence of myeloblasts and immature granulocytic cells visible by morphological examination (Data not shown). Secondly, compared to the 2D culture, the AMO model showed an increased percentage of PMNs at week 3 (Mann Whitney test p-value: 0.007) and a decreased percentage of monocytes at all time points (Mann Whitney test p-values: 0.03, <0.001 and 0.002 for Wl, W2 and W3 respectively), suggesting that the 3D organization of the model favored granulocytic over monocytic lineage differentiation, contrary to the 2D co-culture. Only few lymphoid cells (B, T or NK cells) were identified with this cocktail of cytokines, underlining the specificity of our protocol for the development of myeloid subsets (Figure 1A).

[0121] These findings indicate that the AMO model promotes maturation of monocytes and neutrophils providing a model to study the emergence of myeloid cells and in particular of granulopoiesis in a fully human context. NK-cell commitment was completed within 1 to 4 weeks and up to 2xl05total NK-cells were generated. Flow-cytometry analysis, using an appropriate gating strategy, based on previously described NK-cell maturation stages (9) (Figures 3 A and 3B) and pseudotime trajectories (Figure 3C), were used to visualize the progression of HSPCs from immature into various NK maturation stages. In details, the AMO supported efficient NK-cell lineage commitment from CD34+CD38-hematopoietic stem cells (HSCs), as shown by a predominance of CD3- CD56+CD16+ / - cells among the CD45RA+ hematopoietic cells (Figure 3D). The AMO model recapitulated the full NK-cell differentiation starting from HSCs (stage 1) to common lymphoid progenitor (CLP, stage 2), Lin-CD56- (stage 3), Lin-CD56+CD16- (stage 4), CD56+CD16+ (stage 5) and CD56+CD16+CD57+ (stage 6) NK-cells (Figure 3B and 3D) (9). Pseudotime analysis recapitulates this maturation trajectory from CD34+ HSC and CLP with a progressive acquisition of CD56+ and others NK markers such as CD94 and NKp46, acquired simultaneously during stage 3, and NKp80 acquired at stage 4 (Figure 3C). Very few immature T (CD7+CD56-) and mature B cells (CD 19+) originate from HSPC under this differentiating protocol. The supervised population gating showed that, by week 2, the majority of cells have engaged into NK-cell differentiation (stage 3: 47,90% and stage 4: 41,30%) while only few HSC (0,30%) and CLP (2,10%) were present (Figure 3D). These immature cells completely disappeared by week 4 where the majority of cells belong to stage 4 (77,20%) with an increase of cells in stage 5 (13,50%). As expected, the NK stage 5 was characterized by the expression of activating (NKp30 / DNAM-l) and inhibitory receptors (NKG2A / KIR molecules) crucial for NK-cell functionality (Figure 3E). By contrast to in vitro 2D co-culture (5,7), the AMO is not only able to support NK-cell differentiation but also to reproduce the natural NK-cell heterogeneity (according to the expression of NKG2A and KIR molecules), until the terminally differentiated NKG2A-KIR+CD57+ NK-cell subset (stage 6). Importantly, NK-cells produced after AMO-based differentiation were polyfunctional, as demonstrated by their capacity to degranulate cytolytic granules and produce cytokines (Figure 4A, Table 2). In fact, the proportion of NK-cells with monofunction, bifunction and trifunction significantly increased after stimulation by K562 cell line or PMA and ionomycin. Remarkably, the cytotoxic capacity against K562 cells, albeit variable, tended to be higher than that of mature NK-cells sorted directly from peripheral blood (n=3, Figure 4B), thus confirming that this model allowed the production of functional and fully differentiated NK-cells with a high efficiency.

[0122] In parallel, to verify the versatility of the system, we tested the ability of the AMO to recapitulate another cell fate process, the myeloid differentiation. The AMO, setup as described above, was cultured in a different cocktail of cytokines promoting myeloid differentiation. In this case, the CD34+Lin- HSPCs progressively differentiated losing early myeloid markers such as CD34, CD117 and HLA-DR, and acquiring CD45 and CD 15 overtime (Figure 5A). The typical biphasic evolution of CD13 and CD33 with a maximum expression at the promyelocyte stage (here achieved in around 7 days) was in accordance with the myeloid marker evolution already described (10). The trajectory -based analysis performed at one and two weeks individualized both monocytic and neutrophils branches containing CD64+CD14+ and CD 15+CD 13+ cells respectively (data not shown). All this was confirmed by cytological evaluation (Figure 5B). Altogether, these data demonstrated the versatility of the AMO to recapitulate innate hematopoiesis, as demonstrated by NK-cells or myeloid subsets generation, by varying only the cytokines cocktails within a primary human bone marrow environment.

[0123] Finally, to verify if the model may better reproduce the immunomodulating effect of the BM stroma over immune cells (11), mature NK-cells isolated from PBMC were co-cultured with h- MSC in the AMO model (data not shown) and compared to the standard 2D co-culture. Histological analysis showed the infiltration of isolated NK-cells, surrounded by MSC within the 3D structure (data not shown). After three days of co-culture of the same MSC and NK- cells in either 2D or AMO systems at the same MSC / NK ratio, the multidimensionality reduction performed on extracted MFIs (mean fluorescence intensity) on gated NK-cells showed dissimilarities in NK-cell phenotype according to the co-culture model (data not shown). Contrary to lineage markers that showed a conserved expression on NK-cells cocultured in 2D and AMO, we found that some adhesion markers, as integrin beta chains 1 and 4 (CD 18 and CD49d respectively), were downregulated in the AMO compared to 2D co-culture (data not shown). We hypothesized that this downregulation could be due to a higher engagement of these particular integrins as 3D co-cultures favor tighter contacts between cells. We also noticed significant different expression for the immune-checkpoint markers CD73 and Tim-3 and wondered how this could affect NK-cell functionality. Interestingly, NK-cells cocultured in AMO displayed reduced proliferation and cytotoxic activity compared to the 2D model (data not shown), prompting us to conclude that AMO co-cultured MSC displayed higher immunomodulatory properties, as already shown by enhanced pro-survival and antiinflammatory properties in other spheroid systems (12,13) and closer to the one described in BM NK-cells (14). These results suggested that the AMO model could more accurately reproduce the immunoregulations provided by the microenvironment within the BM. Finally, we explored whether the AMO may be suitable to test drug ex vivo. For this purpose, mature NK-cells isolated from PBMC were co-cultured with h-MSC in the AMO model, recapitulating by this mean the MSC-enriched BM microenvironment expected surrounding leukemic cell niches (Nauta et al., 2006; Poon et al., 2019; Woods and Guezguez, 2021). Histological analysis showed the infiltration of isolated NK-cells (in red), surrounded by MSC (in green) within the 3D structure (Data not shown). After one week of culture, the NK cells maintained the expression of lineage specific markers (CD 16 and CD56; Data not shown), the capacity to proliferate and to kill cancer cells (Data not shown).

[0124] Therefore, the AMO cultured in presence of the classical chemotherapy doxorubicin showed a good drug delivery and permeability in the whole structure as shown by fluorescence measure on confocal imaging compared to DMSO alone (data not shown). This result prompted us to test treatments with the hypomethylating agents azacytidine (AZA) and decitabine (5-aza-2’- deoxycytidine, DEC), commonly used to treat high-risk MDS and AML patients, and known to increase the NK cytotoxic functionality (7,15). Indeed, we noticed the expected increase of NK cytotoxic activity in particular after DEC treatment, more markedly in AMO system than in 2D (data not shown), confirming the possible use of the AMO for drug testing.

[0125] Herein, we illustrated the robustness and simplicity of the AMO model to study cell ontogenesis and the immunomodulatory microenvironment allowing better evaluations of the immunological response and drug targeting. The advantages of this system over the others are (i) the presence of primary human BM stromal cells, which create an in vzvo-mimicking environment for the hematological cells, (ii) the possibility to study different hematological cells changing only the cocktail of cytokines, (iii) the 3D context granting better prescreening data. Further studies complexifying the AMO with other cell types (as immune CD8+ T or leukemic cells) or in autologous setting could further increase the reliability of the model. Meanwhile, the AMO is a straightforward, versatile, reproducible and scalable platform meeting basic and clinical exigencies.

[0126] TABLES:

[0127] Table 1. List of antibodies used for flow cytometry

[0128]

[0129] Table 2: Polyfunctional Assay after NK differentiation from cord blood CD34+ cells.

[0130] Expressed as percentage of positive cells among NK cells (n=4)

[0131] TNF-a+

[0132] Resting TNF-«+ TNF-«+

[0133] CD107a+ IFN-V+ TNF-«+ IFN-y+ IFN-y+ cells IFN-V+ CD107a+

[0134] CD107a+

[0135] 94.1 1.1 0.5 2.3 0.2 0.9 0.2 0.7

[0136] T^ftsclmc (91.8-95.5) (0.6-1.7) (0.1-1.1) (1.3-2.8) (0.1-0.6) (0.3-1.7) (0.1-0.4) (0.1-1.1)

[0137] 83.4 9.2 1.9 1.4 1.4 0.7 0.7 1.3

[0138] K562 (79.6-86.4) (6.2-13.2) (0.4-4.9) (0.5-2.1) (0.4-2.1) (0.3-1.1) (0.5-1.4) (0.5-3.0)

[0139] PMA + 37.3 11.0 26.3 5.4 9.7 4.5 0.6 5.2 ionomycin (29.2-42.3) (9.3-14.4) (24.2-29.) (4.1-6.8) (5.3-17.2) (2.8-5.9) (0.5-0.9) (2.9-6.8)

[0140] REFERENCES:

[0141] 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.

[0142] 1. Lang Y, Lyu Y, Tan Y, Hu Z. Progress in construction of mouse models to investigate the pathogenesis and immune therapy of human hematological malignancy. Front Immunol [Internet], 2023 [cited 2023 Sep 19]; 14. Available from: https: / / www.frontiersin.Org / articles / 10.3389 / fimmu.2023.l 195194 2. Pievani A, Savoldelli R, Poelchen J, Matti oli E, Anselmi G, Girardot A, et al.

[0143] Harnessing Mesenchymal Stromal Cells for the Engineering of Human Hematopoietic Niches. Front Immunol [Internet], 2021 [cited 2023 Sep 19]; 12. Available from: https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2021.631279

[0144] 3. Three-Dimensional Human Bone Marrow Organoids for the Study and Application of Normal and Abnormal Hematoimmunopoiesis - PubMed [Internet], [cited 2023 Sep 19],

[0145] Available from: https: / / pubmed.ncbi.nlm.nih.gov / 36947817 /

[0146] 4. Kyrysyuk O, Wucherpfennig KW. Designing Cancer Immunotherapies That Engage T Cells and NK Cells. Annu Rev Immunol. 2023 Apr 26;41 : 17-38. 5. Roeven MWH, Thordardottir S, Kohela A, Maas F, Preijers F, Jansen JH, et al. The Aryl Hydrocarbon Receptor Antagonist StemRegeninl Improves / / ? Vitro Generation of Highly Functional Natural Killer Cells from CD34+ Hematopoietic Stem and Progenitor Cells. Stem Cells Dev. 2015 Dec 15;24(24):2886-98.

[0147] 6. Granzin M, Wagner J, Kohl U, Cerwenka A, Huppert V, Ullrich E. Shaping of Natural

[0148] Killer Cell Antitumor Activity by Ex Vivo Cultivation. Front Immunol [Internet], 2017 [cited 2023 Sep 19];8. Available from: https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2017.00458

[0149] 7. Cany J, van der Waart AB, Spanholtz J, Tordoir M, Jansen JH, van der Voort R, et al. Combined IL-15 and IL-12 drives the generation of CD34+-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer. Oncolmmunology. 2015 Jul 3;4(7):el017701.

[0150] 8. Seet CS, He C, Bethune MT, Li S, Chick B, Gschweng EH, et al. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nat Methods. 2017 May;14(5):521-30.

[0151] 9. Di Vito C, Mikulak J, Mavilio D. On the Way to Become a Natural Killer Cell. Front Immunol [Internet], 2019 Aug 2 [cited 2020 Mar 15];10. Available from: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6688484 /

[0152] 10. Loken MR, van de Loosdrecht A, Ogata K, Orfao A, Wells DA. Flow cytometry in myelodysplastic syndromes: Report from a working conference. Leuk Res. 2008 Jan 1 ;32(1): 5— 17.

[0153] 11. Sarsenova M, Kim Y, Raziyeva K, Kazybay B, Ogay V, Saparov A. Recent advances to enhance the immunomodulatory potential of mesenchymal stem cells. Front Immunol [Internet], 2022 [cited 2023 Jun 29]; 13. Available from: https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2022.1010399

[0154] 12. Cesarz Z, Tamama K. Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int. 2016;2016:9176357.

[0155] 13. Ferreira JR, Teixeira GQ, Santos SG, Barbosa MA, Almeida-Porada G, Goncalves RM. Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Preconditioning. Front Immunol [Internet], 2018 [cited 2022 Mar 7];9. Available from: https: / / www.firontiersin.org / article / 10.3389 / fimmu.2018.02837

[0156] 14. Yang C, Siebert JR, Burns R, Gerbec ZJ, Bonacci B, Rymaszewski A, et al. Heterogeneity of human bone marrow and blood natural killer cells defined by single-cell transcriptome. Nat Commun. 2019 Sep 2; 10(l):3931. 15. Sohlberg E, Pfefferle A, Andersson S, Baumann BC, Hellstrom-Lindberg E, Malmberg KJ. Imprint of 5-azacytidine on the natural killer cell repertoire during systemic treatment for high-risk myelodysplastic syndrome. Oncotarget. 2015 Oct 21;6(33):34178-90.

Claims

CLAIMS:

1. An in vitro method for generating NK cells comprising the steps of i) aggregating hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs), ii) seeding and culturing the cell pellet obtained at step i) on a culture support at the air-fluid interface in a medium supplemented with appropriate cytokines for allowing NK differentiation and iii) harvesting the generated NK cells.

2. An in vitro method for generating myeloid cells comprising the steps of i) aggregating hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs), ii) seeding and culturing the cell pellet obtained at step i) on a culture support at the air-fluid interface in a medium supplemented with appropriate cytokines for allowing myeloid differentiation and iii) harvesting the generated myeloid cells.

3. The method of claim 1 or 2 wherein the step i) is performed by centrifugation.

4. The method of claim 1 wherein the culture medium used at step ii) for the NK differentiation comprises at least 1, 2, 3, 4 or 5 cytokines selected from the group comprising or consisting of IL-7 (interleukin-7), SCF (stem cell factor), TPO (thrombopoietin), Flt3-L (Flt3 ligand), and IL-15 (interleukin-15).

5. The method of claim 4 wherein the culture medium used at step ii) comprises at least at least 1, 2, 3, 4 or 5 cytokines selected from the group comprising or consisting of human IL-7, human SCF, human TPO, human Flt3-L, and human IL-15.

6. The method of claim 4 or 5 wherein the culture medium comprises IL-7 (preferably hlL- 7), SCF (preferably hSCF), Flt3 -L (preferably hFlt3-L), TPO (preferably hTPO) and IL-15 (preferably hIL-15).

7. The method according to claim 1, 4, 5, or 6 wherein the culture medium used at step ii) comprises an antagonist of aryl hydrocarbon receptor (AHR).

8. The method of claim 7 wherein the antagonist is selected from the group consisting of StemRegeninl (SR-1), PDM2, GNF351, CH-223191, BAY 2416964, PDM-11, and BAY-218.

9. The method of claim 2 wherein the culture medium used at step ii) for the myeloid differentiation comprises at least 1, 2, 3, or 4 cytokines selected from the group comprising or consisting of IL-3 (interleukin-3), SCF (stem cell factor), TPO (thrombopoietin), and Flt3-L (Flt3 ligand).

10. The method of claim 9 wherein the culture medium used at step ii) comprises at least at least 1, 2, 3, or 4 cytokines selected from the group comprising or consisting of human IL-3, human SCF, human TPO, and human Flt3-L.

11. The method of claim 9 or 10 wherein the culture medium comprises IL-3 (preferably hIL-7), SCF (preferably hSCF), Flt3 -L (preferably hFlt3-L), and TPO (preferably hTPO).

12. The method of claim 1 wherein the pellet is cultured for at least 2 weeks, preferably 3 weeks for generating NK cells.

13. The method of claim 2 wherein the pellet is cultured for at least 1 week, preferably 2 weeks for generating myeloid cells.

14. The method according to any one of claim 1 to 13 that further comprises a genetic modification step that consists to a gene disruption step, a gene correction step or a gene addition step, preferably a gene addition step.

15. The method of claim 14 wherein an exogenous nucleic acid sequence expressing a gene of interest is introduced into the HSPCs, preferably before step i) of the methods.

16. The method of claim 15 wherein the exogenous nucleic acid sequence to be introduced into the cells encodes a Chimeric Antigenic Receptor (CAR).

17. An isolated population of NK or myeloid cells susceptible to be obtained, or obtained, by the in vitro method according to any one of claims 1 to 16.

18. A pharmaceutical composition that comprises the isolated population of cells according to claim 17.

19. A method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of cells according to claim 17.

20. The method of claim 19 wherein the subject suffers from a cancer or an infectious disease.

21. In some embodiments, the myeloid cells of the invention are CD45+ CD 15+ myeloid cells.

Images