Bone marrow organoids produced from induced pluripotent stem cells and uses of these organoids

Abstract

The present invention relates to a method for producing mammalian vascular network or mature mammalian bone marrow organoid.Furthermore, the use of produced vascular network or mammalian bone marrow organoid is provided for in-vitro production of BMO or mammalian blood cell for use in the treatment of bone marrow-related disease, as a model system for the pathogenesis of bone marrow-related disease, and as a system for identifying and / or testing pharmacologic effective compounds for the treatment or prevention of bone marrow-related disease.

Description

[Technical field]

[0001] The present invention relates to a method for producing mammalian vascular network or mature mammalian bone marrow organoid.Furthermore, the use of produced vascular network or mammalian bone marrow organoid is provided for in-vitro production of BMO or mammalian blood cell for use in the treatment of bone marrow-related disease, as a model system for the pathogenesis of bone marrow-related disease, and as a system for identifying and / or testing pharmacologic effective compounds for the treatment or prevention of bone marrow-related disease. [Background technology]

[0002] Postnatal hematopoiesis in humans takes place in the bone marrow and involves a tightly controlled process of constant differentiation of hematopoietic stem cells into mature blood cells while maintaining the hematopoietic stem cell (HSC) pool by self-renewal. The surrounding microenvironment is called the bone marrow niche and consists of a heterogeneous cell population including mesenchymal cells (e.g., pericytes, adipocytes) and endothelial cells. The bone marrow niche plays a crucial role in the regulation and maintenance of hematopoiesis throughout life (1).

[0003] The dense vascular network within the bone marrow is essential because it provides nutrients, growth factors, and important cell-cell interactions to other niche cells. Furthermore, endothelial cells, which are surrounded by perivascular PDGFRβ+ mesenchymal cells (pericytes), directly promote hematopoietic homeostasis by secreting a variety of factors, and thus HSCs are often located in close proximity to blood vessels (2).

[0004] Hematopoietic disorders can be caused by specific genetic mutations, which in the case of germline mutations manifest themselves in the form of congenital bone marrow failure (IBMFS), e.g., severe congenital neutropenia (CNF), or in the case of somatic mutations manifest themselves in the form of diseases such as myelodysplasia and leukemia. Interestingly, it has been shown that disruption of the bone marrow microenvironment leads to myelodysplasia (3).

[0005] Human organoid models have been established in recent years as a model system for studying the development of diseases in various tissues. Organoids are self-organizing 3D structures that mimic the most important functional and structural organization of organs and can be differentiated from induced pluripotent stem cells (iPSCs). Organoids are superior to traditional 2D cultures in part because they mimic the natural environment of certain cells through cell-cell and cell-matrix interactions (7, Non-Patent Document 1).

[0006] Organoid formation and maturation are preceded by proliferation and reorganization of single cells or small cell clusters. Based on the choice of stem cells, there are two main types of organoids. The first type is derived from PSCs, including both embryonic stem cells (ESCs) and iPSCs, and the second type is derived from organ-specific adult stem cells (ASCs). Various workflows have been developed to generate organoids, but specialized organoid types require specific culture methods, and not all common workflows are suitable. The choice of cell culture conditions and 3D matrix is ​​critical for this complex tissue.

[0007] US Patent No. 5,999,943 discloses co-cultures of organoids and immune cells and methods of using them to identify drugs for treating diseases.

[0008] Breunig et al. (Non-Patent Document 2) describe a scalable in vitro differentiation protocol for stepwise induction of human pluripotent stem cells into pancreatic duct-like organoids. This protocol mimics pancreatic duct development and has been successfully used to model the onset and progression of pancreatic ductal adenocarcinoma, making this technique suitable for multiple downstream applications. However, this protocol is costly and time-consuming.

[0009] Vallmajo-Martin, Q. et al. (in Non-Patent Document 3) disclose that bone marrow (BM) organoids provide a powerful tool to study the important interactions between the BM microenvironment and resident cells. A transglutaminase (TG) crosslinking system is shown to seamlessly incorporate poly(ethylene glycol) (PEG) and hyaluronic acid (HA) into hybrid hydrogels to form BM analogs. The utility of TG-PEG / HA hybrid hydrogels to maintain, expand, or differentiate human bone marrow-derived stromal cells and human hematopoietic stem and progenitor cells in vitro is demonstrated. It is described that TG-PEG / HA hybrid hydrogels are superior to currently used natural biomaterials in the formation of humanized BM organoids in xenograft models. Engineered humanized BM organoids as shown can be an effective tool for the study of this complex organ.

[0010] Isem J et al. (in Non-Patent Document 4) discuss strategies for expanding hematopoietic stem cells (HSCs), including co-culture with cells that recapitulate the native microenvironment, such as bone marrow stromal stem / progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to maintain primitive HSCs. They describe a method to isolate and culture human BMSCs as non-adherent mesenchymal spheres. Human mesenspheres were derived from CD45-CD31- CD71- CD146+ CD105+ nestin+ cells, but could also simply be grown from fetal and adult BM CD45--enriched cells. Human mesenchymal spheres were strongly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, reduced adhesion to plastic, and increased self-renewal, they promoted enhanced proliferation of cord blood CD34+ cells by secreted soluble factors. The expanded HSCs could be serially transplanted into immunodeficient mice, greatly increasing long-term human hematopoietic engraftment. They discuss how culture techniques maintain the self-renewal of human BMSCs and their ability to support functional HSCs.

[0011] Sun et al. (in Non-Patent Document 5) induced vascular and cerebral organoids, respectively, and then fused the two types of organoids together to obtain vascularized cerebral organoids.

[0012] Janagama and Hui (in Non-Patent Document 6) review the state of the art in bone marrow tissue engineering (BMTE) and hematological cancer tissue engineering (HCTE) in light of the recent interest in the bone marrow environment and the pathophysiology of hematological cancers. They focus on engineered BM tissues and organoids as in vitro models of hematological cancer therapy, along with the identification of BM components and their integration as synthetically engineered BM-mimicking scaffolds. Furthermore, the review details the interaction dynamics of various BM and hematological cancer (HC) cell types in engineered BM tissue / phantom co-culture systems and their relationship to drug resistance and cytotoxicity. Differences in the interaction of hematological cancer cells with their niches and with respect to the healthy niche microenvironment are described. Future perspectives of BMTE for in vitro disease models, BM regeneration, and large-scale ex vivo expansion of hematopoietic and mesenchymal stem cells for transplantation and therapy are discussed. They conclude by outlining the clinical applications of biomaterials in BM and HC pathophysiology as well as their challenges and opportunities.

[0013] Bessy T (in Non-Patent Document 7) provides another comprehensive review focused on the engineering of vascularized BM niche models and summarizes current approaches including bioengineered microfluidic chips.

[0014] Cornelia Lee-Thedieck et al. (in Non-Patent Document 8) review that hematopoietic stem cells (HSCs) are a lifelong source of all types of blood cells. Their function is controlled by the HSC niche in their immediate microenvironment, the bone marrow. The importance of the extracellular matrix (ECM) in the niche by orchestrating niche structure and cell function is widely recognized but remains underexplored. In this review, they give a comprehensive overview of the ECM in the HSC niche. To this end, they briefly outline HSC niche biology and then review the role of each of the different classes of ECM molecules in the niche and how they are recognized by cells. The emerging importance of matrix remodeling and biophysics in HSC niche function is discussed. Finally, the application of the current knowledge of the ECM in the niche in the form of artificial HSC niches for HSC proliferation or targeted differentiation as well as drug testing is reviewed.

[0015] Because mouse models often do not fully recapitulate the human phenotype due to differences in hematopoiesis between mice and humans, alternative approaches are needed to investigate the genetic causes and mechanisms leading to bone marrow disorders. Previous studies on in vitro modeling of the niche in human bone marrow have relied on the use of primary endothelial and mesenchymal cells, which do not reflect the multicellular complexity of the native niche system, and broader applications are limited by the supply and limited lifespan of these cells (4-6).

[0016] In view of the above, there is an unmet need for novel in vitro approaches to recapitulate hematopoiesis in a complex myeloid niche system to study hematopoietic disorders and develop novel therapies. It is therefore an object of the present invention to provide respective approaches and methods that can be used to establish suitable systems to study hematopoietic disorders and develop novel therapies. Other objects and advantages will become apparent to those skilled in the art upon study of the instant invention. [Prior art documents] [Chartered documents]

[0017]

Patent Document 1

Non-licensed literature

[0018]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

[0019] In a first aspect of the present invention, the present invention provides a method for producing mature mammalian bone marrow organoids, comprising the steps of: a) generating embryoid bodies from a substantially single induced pluripotent stem cell (iPSC) obtained from at least one mammalian species, comprising culturing the single iPSC in an aggregation medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day, followed by culturing the embryoid body in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells without a ROCK inhibitor for about 48 hours; and b) inducing mesoderm in said embryoid bodies formed in step a), comprising culturing said embryoid bodies in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells for about 48 hours, with resuspension of the mesoderm-induced embryoid bodies every about 24 hours, the medium comprising 80 ng / ml bone morphogenetic protein 4 (BMP4), 4 μM glycogen synthase kinase (GSK) 3 inhibitor, and 80 μM erythrocyte-derived blastocyst (EC) ... c) replacing the medium of the culture of b) with Essential 6 medium supplemented with 80 ng / ml VEGF, 25 ng / ml fibroblast growth factor (FGF)-2, 50 ng / ml stem cell factor (SCF), and 2 μM SB431542 for about 48 hours with gentle rocking while resuspending the embryoid bodies about every 24 hours; and d) embedding the mesoderm-derived embryoid bodies of step c) in a suitable polymerized 3D collagen I / Matrigel™ matrix, followed by covering the matrix with StemPro™-34 medium supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF, and 2 μM SB431542 for about 48 hours, followed by 50 ng / ml VEGF, 50 ng / ml SCF, 50 ng / ml Cytokine replacement with IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours, followed by a cytokine boost to 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours; e) harvesting the individual vascular networks generated in step d), followed by a cytokine boost of 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours;The above problem is solved by providing a method comprising the steps of: culturing in StemPro™-34 medium supplemented with SCF, 50 ng / ml IL-3, 50 ng / ml Flt-3L and 5 ng / ml TPO for about 7-11 days, refreshing the medium every 3-4 days, thereby producing mature mammalian bone marrow organoids, preferably no lineage-targeting cytokines such as EPO, IL-6 and / or G-CSF are used in the method. Preferably, the mammalian bone marrow organoids are mature organoids, i.e., they exhibit the main physiological characteristics of the respective tissue(s) in vivo.

[0020] In the second aspect of the present invention, the present invention solves the above-mentioned problem by providing a vascular network or mammalian bone marrow organoid, preferably mature organoid, produced according to the method of the present invention, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid of the present invention.Furthermore, a pharmaceutical composition comprising the vascular network or mammalian bone marrow organoid, preferably mature organoid, produced according to the method of the present invention, or the vascular network and / or mature mammalian bone marrow organoid of the present invention, for use in the treatment of disease, is provided.

[0021] In a third aspect of the present invention, the present invention solves the above mentioned problem by providing the use of the vascular network or the mammalian bone marrow organoid or the pharmaceutical composition according to the present invention as a model system in the pathogenesis of bone marrow related diseases such as, for example, hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV associated conditions, sickle cell disease and chemotherapy or transfusion complications.

[0022] In a fourth aspect of the present invention, the present invention solves the above problem by providing the use of the vascular network or mammalian bone marrow organoid or pharmaceutical composition according to the present invention as a model system for identifying and / or testing pharmacologic compounds for the treatment or prevention of bone marrow-related diseases, such as hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and complications from chemotherapy or transfusion. Another aspect of this embodiment is a method for screening pharmacologic compounds for the treatment or prevention of bone marrow-related diseases, such as hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and complications from chemotherapy or transfusion, comprising the use of the vascular network or mammalian bone marrow organoid or pharmaceutical composition according to the present invention as a model system.

[0023] In a fifth aspect of the present invention, the present invention solves the above problem by providing an assembloid comprising a vascular network or a mature mammalian bone marrow organoid according to the present invention, having at least one additional iPSC-derived organoid.

[0024] In a sixth aspect of the invention, the invention solves the above mentioned problem by providing the use of a vascular network or a mammalian bone marrow organoid or a pharmaceutical composition according to the invention for the in-vitro production of BMOs or mammalian blood cells, in particular autologous BMOs or mammalian blood cells, in particular for transplantation purposes.

[0025] In a seventh aspect of the present invention, the present invention solves the above problem by providing a vascular network or mature mammalian bone marrow organoid or assembloid or pharmaceutical composition according to the present invention in a pharmacologic effective amount, preferably for transplantation, for use in the treatment of bone marrow-related diseases, such as hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, conditions associated with HIV, sickle cell disease, and complications from chemotherapy or transfusion. Another aspect of this embodiment is a method for treating bone marrow-related diseases, such as hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, conditions associated with HIV, sickle cell disease, and complications from chemotherapy or transfusion, comprising administering to a subject in need thereof a pharmacologic effective amount of the vascular network or mammalian bone marrow organoid or pharmaceutical composition according to the present invention. Preferably, the vascular network or mammalian bone marrow organoid or assembloid or pharmaceutical composition according to the present invention is administered as a transplant, such as an autologous transplant. Another aspect of this embodiment is a method of transplanting the vascular network or mammalian bone marrow organoid or pharmaceutical composition according to the present invention into a mammalian subject in need thereof, preferably for treating bone marrow-related diseases such as, for example, hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and / or complications from chemotherapy or transfusion.

[0026] The present inventors present a novel approach to generate bone marrow organoids (BMOs) (wherever possible, this term shall include both bone marrow organoids and manufactured mature bone marrow organoids) that mimic key structural and cellular features of the human bone marrow niche. These BMOs are generated exclusively from human induced pluripotent stem cells (hiPSCs) and have hematopoietic, stromal (mesenchymal) and vascular compartments. The present inventors show that this system induces the formation of mature blood cells of the myeloid lineage, such as neutrophils, eosinophils and basophilic granulocytes and monocytes, as well as megakaryocytic lineage, mast cells, dendritic cells, lymphoid progenitors and early erythroid progenitors. Furthermore, the present inventors can show that the stromal compartment is composed of mesenchymal stem cells and progenitors, such as CXCL12-enriched reticular (CAR) cells and perivascular cells, while the vascular compartment is composed of endothelial cells that self-assemble, connect and form lumens.

[0027] Khan et al. (in Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discov. 2023 Feb 6;13(2):364-385. doi: 10.1158 / 2159-8290.CD-22-0199. PMID: 36351055; PMCID: PMC9900323) describe a stepwise directed differentiation protocol in which organoids are generated from iPSCs committed to mesenchymal, endothelial, and hematopoietic lineages. These three-dimensional structures were reported to capture bone marrow cells, including stroma, lumen-forming sinusoidal vessels, and proplatelet-forming megakaryocytes, which are key features of human bone marrow. Organoids were reported to support the engraftment and survival of cells from patients with hematological malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of organoids occurred after TGFβ stimulation and engraftment with myelofibrosis, but not with cells from healthy donors. Khan et al. did not mention the exact concentrations used in their protocol.

[0028] Importantly, no Wnt activators (e.g., CHIR99021) or Nodal inhibitors (e.g., SB431542), which have been shown to be important for mesoderm patterning leading to the induction of definitive hematopoiesis, were used. + CD235a + ) generation depends on stage-specific inhibition of activin-nodal signaling and the Wnt-β-catenin pathway, whereas definitive progenitor (KDR + CD235a -) requires Wnt-β-catenin signaling during this same time frame (Sturgeon, CM, Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat. Biotechnol. 32, 554-561 (2014)).

[0029] Also, in Khan et al., lineage-targeting cytokines such as EPO, IL-6 and G-CSF were used, which precludes the possibility to study endogenous cytokine signaling within bone marrow organoids and likely represents an obstacle to modeling inherited bone marrow failure syndromes due to some possible rescue effects of these cytokines.

[0030] In the present invention, maturation of neutrophil granulocytes in BMOs proceeds without the addition of recombinant cytokines, reminiscent of the in vivo situation (see below).

[0031] Thus, structurally, the present invention differs from Khan et al. in that, for example, blood vessels consist of arterial-type endothelial cells rather than sinusoids. This is very important because it has been shown that long-term HSCs arise from arterial-type hemogenic endothelium (Calvanese et al., Nature 2022). For the mesenchymal cell compartment, a desirable heterogeneous mesenchymal cell type composition was found, including mesenchymal stem and progenitor cells that give rise to osteogenic, chondrogenic and adipogenic precursor cells. For hematopoietic cells, clear evidence by scRNA sequencing of potential differentiation into all hematopoietic lineages, not just erythromyeloids as in Khan et al. Finally, whereas Khan et al. did not perform transplantation, the present organoids were transplantable in NOD-SCID mice in vivo and showed engraftment of human CD45+ in mouse bone marrow, indicating the HSC characteristics of BMO-derived cells.

[0032] Our organoids displayed a consistent spherical morphology throughout differentiation (see FIG. 1), which was reproducible in different iPS cell lines derived from renal epithelial cells.

[0033] Therefore, the inventors have developed an improved protocol that allows the reliable production of mammalian bone marrow organoids, in particular mature mammalian bone marrow organoids derived exclusively from iPSCs and composed of blood cells and various niche cells such as endothelial and mesenchymal cells. The method according to the present invention comprises the steps of: a) generating embryoid bodies from substantially single induced pluripotent stem cells (iPSCs) obtained from at least one mammalian species, comprising culturing the single iPSCs in aggregation medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day, followed by culturing the embryoid bodies in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells without a ROCK inhibitor for about 48 hours; and b) inducing mesoderm in the embryoid bodies formed in step a), comprising culturing the embryoid bodies in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells for about 48 hours, with resuspending and gently rocking the mesoderm-induced embryoid bodies every about 24 hours, the medium comprising 80 ng / ml bone morphogenetic protein 4 (BMP4), 4 μM glycogen synthase kinase (GSK) 3 inhibitor, and 80 ng / ml erythrocyte spheroids. c) replacing the medium of the culture of b) with Essential 6 medium supplemented with 80 ng / ml VEGF, 25 ng / ml fibroblast growth factor (FGF)-2, 50 ng / ml stem cell factor (SCF), and 2 μM SB431542 for about 48 hours, with resuspension of the embryoid bodies about every 24 hours; d) embedding the mesoderm-derived embryoid bodies of step c) in a suitable polymerized 3D collagen I / Matrigel™ matrix, followed by covering the matrix with StemPro™-34 medium supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF, and 2 μM SB431542 for about 48 hours, followed by resuspension of the embryoid bodies about every 24 hours. Cytokine replacement was performed with 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for approximately 48 hours, and then 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours; and e) extracting the individual vascular networks generated in step d), followed by culturing in StemPro™-34 medium supplemented with 25 ng / ml VEGF, 50 ng / ml SCF, 50 ng / ml IL-3, 50 ng / ml Flt-3L and 5 ng / ml TPO for about 7-11 days, refreshing the medium every 3-4 days, thereby producing mammalian bone marrow organoids, preferably without lineage-targeting cytokines such as EPO, IL-6 and / or G-CSF. The method for producing mammalian bone marrow organoids according to the present invention preferably further comprises culturing the mature organoids for up to about 47 days, preferably about 60 days.

[0034] The present invention provides a vascularized BMO preparation or system derived exclusively from iPSCs (especially hiPSCs) that mimics hematopoiesis in the multicellular context of native bone marrow, including blood cells and various bone marrow "niche cells" such as endothelial and mesenchymal cells. This system allows modeling of bone marrow-related diseases, i.e., allows the study of the interactions between niche cells and hematopoietic cells in the pathogenesis of hematological diseases. This can further be achieved in the context of drug testing, i.e., studying the effect of drugs on the system, which allows the testing of novel therapies in the complex mammalian, e.g., human, in-vitro model system of the present invention. It is also possible to carry out in a high-throughput format, e.g., in a 96-well plate format. Furthermore, the generated "product" can be used for therapeutic applications, i.e., bone marrow-related diseases such as hematological diseases. This method offers the possibility of transplantation of the produced human BMO, or improved in vitro generation of human blood cells from iPSCs, e.g., autologous patient-specific production, and subsequent infusion or transplantation.

[0035] So far, ex-vivo expansion of HSCs for stem cell transplantation has not been possible. Advantageously, the vascular network or mature mammalian bone marrow organoids according to the present invention maintain the stemness of hematopoietic stem cells (HSCs), allow to keep HSCs in vitro for further quality control studies, and to select for therapeutic use only those cells that show the desired molecular changes without any undesirable side effects.

[0036] The method for producing mammalian bone marrow organoids according to the invention preferably further comprises in step e) fixing the vascular network and carrying out immunofluorescence analysis and / or dissociating the vascular network into individual cells and carrying out flow cytometric analysis and / or live cell imaging. This can be carried out to characterize the components of the developed vascular network and / or to study any effects of the drugs being tested (see also below).

[0037] The method for producing mammalian bone marrow organoids according to the present invention preferably further comprises the steps of fixing the produced (mature) organoids and carrying out immunofluorescence analysis and / or dissociating mature organoids into individual cells and carrying out flow cytometric analysis and / or live cell imaging.This can be carried out to characterize the components of the developed organoids (e.g. mature) and / or to study any effects of the drugs being tested (see also below).

[0038] In the first step of the method according to the invention, embryoid bodies are generated from substantially single / individual induced pluripotent stem cells (iPSCs) obtained from at least one mammalian species. Individual cells can be obtained by several methods, for example by mechanical dissociation (for example by using a pipette, etc.). Nevertheless, the method for producing mammalian bone marrow organoids according to the invention is preferred, in which single / individual iPSCs are provided by dissociating iPSC cells into single cells by Accutase™ digestion, which is a gentler procedure. Accutase™ is gentle to cells and self-inhibits at 37°C without the need for a neutralizing solution like trypsin. Accutase™ is commercially available from Sigma and acts on all mammalian iPSCs. The method further comprises culturing individual / single iPSCs in aggregation medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day.

[0039] In the method for producing mammalian bone marrow organoids according to the present invention, it is preferable that the aggregation medium is KnockOut DMEM / F12 (Thermo Fisher) containing 20% ​​serum replacement, 1% L-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin, and 100 μM β-mercaptoethanol.

[0040] In the method for producing mammalian bone marrow organoid according to the present invention, in the first step, cell is further cultured in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor.ROCK inhibition allows maintaining stem cell phenotype, and its metabolic impact is unknown.

[0041] In the method for producing mammalian bone marrow organoids according to the present invention, the ROCK inhibitor is preferably selected from the group consisting of Y-27632 ((lR,4r)-4-((R)-l-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide), fasudil, and TS-f ROCK inhibitors as disclosed in Shen et al. (Shen, M., Tian, ​​S., Pan, P. et al. Discovery of Novel R0CK1 Inhibitors via Integrated Virtual Screening Strategy and Bioassays. Sci Rep 5, 16749 (2015). https: / / doi.org / 10.1038 / srepl6749, incorporated herein by reference), in particular TS-f5 or TS-f22. Other suitable ROCK inhibitors are known to those skilled in the art and are disclosed in the respective literature.

[0042] Typically, step a) is preferably carried out for 60 to 84 hours, preferably about 72 hours as described above, i.e., for about 3 days, and the medium is changed to a suitable medium (e.g., TeSRplus) that does not contain a Rock inhibitor after about 24 hours.

[0043] In the context of the present invention, the term "about" is intended to mean a deviation of + / - 10% from a given value, unless otherwise indicated.

[0044] In the context of the present invention, incubation is generally carried out at about 37° C., unless otherwise indicated.

[0045] In a second step of the method according to the invention, mesoderm is induced in said embryoid bodies formed in step a), which comprises culturing the embryoid bodies in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells and human iPSCs.

[0046] Preferably, the serum-free stable cell culture medium suitable for feeder-free maintenance and growth of human embryonic stem cells is mTeSR™ Plus medium (STEMCELL Technologies, Fisher Scientific). The medium is supplemented with 80 ng / ml bone morphogenetic protein 4 (BMP4), 4 μM glycogen synthase kinase (GSK) 3 inhibitor, and 80 ng / ml vascular endothelial growth factor (VEGF).

[0047] For example, inhibition of GSK3 by CHIR99021 promotes mESC self-renewal by stabilizing cytoplasmic β-catenin, an essential component of the classical Wnt signaling pathway, and suppressing TCF3-mediated transcriptional repression of pluripotency-related genes, including Oct4, Nanog, Tfcp2l1, and Esrrb. Therefore, in the method for producing mammalian bone marrow organoids according to the present invention, the glycogen synthase kinase (GSK) 3 inhibitor is preferably CHIR99021 or an aurone derivative, NPD13432 (Hiroki Kobayashi, et al. A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells, Bioscience, Biotechnology, and Biochemistry, Volume 84, Issue 10, 2 October 2020, Pages 2113-2120, https: / / doi.org / 10.1080 / 09168451.2020.1789445). Glycogen synthase kinase (GSK) 3 inhibitors can be combined with MEK inhibitors (e.g., PD0325901, referred to as "2i") to support long-term self-renewal of mouse embryonic stem cells (mESCs), while blockade of the MEK / ERK pathway with PD0325901 increases the expression of Nanog, Tfcp2l1, and Klf4 in mESCs, thereby promoting self-renewal.

[0048] Typically, step b) is preferably carried out for 36 to 60 hours, preferably about 48 hours, ie, for about 2 days, with gentle mixing or resuspension of the mesodermally-derived embryoid bodies about every 24 hours.

[0049] In the third step of the method according to the invention, the medium of the culture of step b) is replaced with Essential 6 medium supplemented with 80 ng / ml VEGF, 25 ng / ml fibroblast growth factor (FGF)-2, 50 ng / ml stem cell factor (SCF) and a suitable inhibitor of the TGF-β / activin / NODAL pathway, preferably 2 μM SB431542 (STEMCELL Technologies). SB431542 inhibits ALK5 (IC) by competing for the ATP binding site. 50 = 94 nM), ALK4 (IC 50 =140 nM), and ALK7. SB431542 does not inhibit the BMP type I receptors ALK2, ALK3, and ALK6. Essential 6 Medium (Thermo Fisher) is a feeder-free and xeno-free medium that supports somatic cell reprogramming and spontaneous or directed differentiation of human pluripotent stem cells (PSCs).

[0050] Typically, step c) is preferably carried out for 36 to 60 hours, preferably about 48 hours, i.e., about 2 days, by gently mixing or resuspending the embryoid bodies about every 24 hours and placing the embryoid bodies on a pulsating shaker.

[0051] The fourth step of the method according to the present invention comprises embedding the mesoderm-derived embryoid bodies generated in step c). Embedding the embryoid bodies in a suitable polymerized 3D matrix prevents the embryoid bodies from sinking to the bottom of the dish or well, thereby preventing or imparting further development into mature embryoid bodies and vascular structures. The extracellular matrix (ECM) further provides biochemical cues and structural support, such as porosity and rigidity, which mediate signaling for cell migration, cell behavior and polarization in the organoid structure. In general, any suitable polymerized 3D matrix can be used. The present invention preferably uses a collagen I / Matrigel™ matrix bottom layer in the culture vessel (e.g., vial, dish or well). For example, 500 μl / well of collagen I-Matrigel™ mixture is first prepared and polymerized at 37° C. for about 1 hour as the bottom layer. Then, the embryoid bodies are resuspended in 500 μl / well of the same collagen-I-Matrigel mixture to prepare a second layer, which is also polymerized at 37° C. for about 1 hour. The two layers are then overlayed with a suitable medium, such as StemPro™-34 medium supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and a suitable inhibitor of the TGF-β / activin / NODAL pathway as described above, preferably 2 μM SB431542 (STEMCELL Technologies). StemPro™-34 SFM (e.g., from Thermo Fisher) is a serum-free medium specifically formulated to support the growth of human hematopoietic cells in culture (Burridge PW, et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One. 2011 Apr 8;6(4):el8293. doi: 10.1371 / journal.pone.0018293. PMID: 21494607; PMCID: PMC3 072973).

[0052] Usually, preferably, the first part of step d) is carried out for 36 hours to 60 hours, preferably about 48 hours, i.e., for about 2 days. Then, cytokine replacement followed in the same medium with 50 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO. Usually, preferably, the second part of step d) is carried out for 36 hours to 60 hours, preferably about 48 hours, i.e., for about 2 days. Finally, cytokine boost followed in the same medium up to 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO, usually, preferably, the third part of step d) is carried out for 36 hours to 60 hours, preferably about 48 hours, i.e., for about 2 days. At the end of the fourth step of the method according to the invention, a vascular network is generated. Because these already contain hematopoietic cells, they are called "germinated embryoid bodies."

[0053] In a fifth step of the method according to the invention, the individual vascular networks generated in step d) are excised / isolated and transferred, for example, to one well of a low-attachment 96-well plate, followed by culturing the vascular networks for about 6-8 days, preferably about 7 days, in StemPro™-34 medium supplemented with 25 ng / ml VEGF, 50 ng / ml SCF, 50 ng / ml IL-3, 50 ng / ml Flt-3L and 5 ng / ml TPO, the medium being refreshed about every 3-4 days.

[0054] Then, the mature mammalian bone marrow organoid according to the present invention is produced.The method advantageously uses Wnt activators (e.g., CHIR99021) and Nodal inhibitors (e.g., SB431542), which are important for mesoderm patterning leading to definitive hematopoiesis induction, and lineage-directed cytokines such as EPO, IL-6 and G-CSF are avoided.Nevertheless, EPO can be used at a later stage to generate more mature erythroid cells.

[0055] The method for producing mammalian bone marrow organoids according to the invention is preferably such that the ratio of 3D collagen I to Matrigel™ in the matrix used is about 1:1 to 4:1, preferably about 3:1 or 1.2:1. Matrigel™, in which the organoids are embedded, is derived from the mouse Engelbreth-Holm-Swarm sarcoma cell line, which currently prevents the production of BMO in a cGMP-compliant manner. Synthetic scaffold materials free of xenogeneic components have been developed and tested for organoid differentiation (reviewed in Aisenbrey EA, Murphy WL. Synthetic alternatives to Matrigel. Nat Rev Mater. 2020 Jul;5(7):539-51) and can be advantageously included in the method according to the invention.

[0056] It is further preferred that the method for producing mammalian bone marrow organoids according to the present invention comprises resuspending the mesodermally derived embryoid bodies using a pipette.

[0057] The method for producing mammalian bone marrow organoids according to the present invention preferably involves mechanical dissociation and / or extraction and separation of individual vascular networks, preferably using sterile dissection tools.

[0058] In the method for producing mammalian bone marrow organoid according to the present invention, mammalian animal is preferably selected from the group consisting of human, mouse, monkey, rat, pig, dog, cat, rabbit, sheep, cow, horse and goat.Most preferred is human, thus providing hiPSC.

[0059] It is also possible to carry out the methods according to the invention in a high throughput format, for example in a 96 well plate format.

[0060] The method according to the invention may further comprise the step of testing and / or analyzing the produced BMO for its suitability as a pharmaceutical preparation for transplantation and other therapeutic purposes.

[0061] The method according to the invention may further comprise the step of separating cells and cell types from the produced BMO. In this context, BMO can be used as a source of functional endothelial cells, pericytes, hematopoietic stem cells, and mesenchymal stem cells. The method according to the invention may be used to separate mesenchymal stem cells (CD45 - CD31 - CD34 - CD90 + CD105 + CD271 + CD73 + ) or HSC isolation. This can preferably be done by cell sorting, e.g. FACS. In the context of the present invention, FACS-selected MSCs were found to have the capacity to differentiate into osteogenic, chondrogenic and adipogenic cells in a triple lineage differentiation assay, visualized by staining with Alizarin Red S, Alcian Blue and Oil Red O, respectively. Sorted BMO-derived MSCs expanded in culture and showed continuous reseeding capacity.

[0062] A further aspect of the invention then relates to preparations of the produced HSCs or MSCs.

[0063] A further aspect of the present invention then relates to a vascular network or mature mammalian bone marrow organoid produced according to the method of the present invention, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid.

[0064] Another aspect of the present invention then relates to the assembloid of the present invention, comprising vascular network or mature mammalian bone marrow organoid, with at least one additional iPSC-derived organoid.Preferably, the assembloid of the present invention comprises immune cells, blood vessels and pericytes.

[0065] Since the lack of immune cells and vasculature represents a general drawback of organoid models, the present invention includes the combination of the present BMOs with other iPSC-derived organoids to form so-called assembloids that integrate immune cells, blood vessels and pericytes (see, e.g., Sharma, A., Sances, S., Workman, MJ & Svendsen, CN Multi-lineage Human iPSC-Derived Platforms for Disease Modeling and Drug Discovery. Cell Stem Cell 26, 309-329 (2020).; Kanton, S. & Pasca, SP Human assembloids. Development 149, dev201120 (2022)). While previous approaches required mixing these cell types (see, for example, Wang, L. et al. A human three-dimensional neural-perivascular 'assembloid' promotes astrocytic development and enables modeling of SARS-CoV-2 neuropathology. Nat. Med. 27, 1600-1606 (2021)), the iPSC-derived BMO system better models developmental and functional interactions in a spatial context. In addition, the combination of bone marrow organoids with other organoids has the advantage of including hematopoietic cells as well as vascular cells.

[0066] Further provided is a vascular network or a mammalian bone marrow organoid produced according to the method according to the invention, preferably a mature organoid, an assembloid according to the invention, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid and / or assembloid according to the invention, for use in the treatment of a disease.

[0067] The pharmaceutical composition used may optionally include a pharma- ceutically acceptable carrier. A person skilled in the art knows suitable formulations for cells and cell products and can easily select a suitable pharma- ceutically acceptable carrier or excipient depending, for example, on the formulation and administration route of the pharmaceutical composition.

[0068] Pharmaceutically acceptable carriers or excipients include diluents (fillers, bulking agents, e.g., lactose, microcrystalline cellulose), disintegrants (e.g., sodium starch glycolate, croscarmellose sodium), binders (e.g., PVP, HPMC), lubricants (e.g., magnesium stearate), glidants (e.g., colloidal SiO2), solvents / co-solvents (e.g., aqueous vehicles, propylene glycol, glycerol), buffers (e.g., citrate, gluconate, lactate), preservatives (e.g., Examples of suitable pharma- ceutically acceptable excipients include sodium benzoate, parabens (Me, Pr, and Bu), BKC, antioxidants (e.g., BHT, BHA, ascorbic acid), humectants (e.g., polysorbates, sorbitan esters), thickeners (e.g., methylcellulose or hydroxyethylcellulose), sweeteners (e.g., sorbitol, saccharin, aspartame, acesulfame), flavoring agents (e.g., peppermint, lemon oil, butterscotch, etc.), and humectants (e.g., propylene, glycol, glycerol, sorbitol). Other suitable pharma- ceutically acceptable excipients include those described in, among others, Remington's Pharmaceutical Sciences, 15 th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5 th Ed., Govi-Verlag Frankfurt (1997).

[0069] The pharmaceutical compositions can be administered in any suitable manner, for example in the form of a solution, syrup, emulsion or suspension. Administration is preferably carried out by transfusion, for example in the form of an injection or infusion solution.

[0070] In addition to the above-mentioned products of the present invention, pharmaceutical compositions can further contain customary, usually inert, carrier substances or excipients. Thus, pharmaceutical preparations can also contain, for example, fillers, spreaders, disintegrants, binders, glidants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavors or fragrances, buffer substances, and also additives such as solvents or solubilizers or agents for achieving a depot effect, and salts for changing osmotic pressure, coating agents or antioxidants. Pharmaceutical preparations can also contain other therapeutically active substances, as also described herein.

[0071] The present invention provides vascularized BMO preparations or systems (e.g., assembloids according to the present invention) derived exclusively from iPSCs (particularly hiPSCs) that mimic hematopoiesis in the multicellular context of native bone marrow, including blood cells and various bone marrow "niche cells" such as endothelial and mesenchymal cells.

[0072] The produced preparations can generally be used in two strategies: one can be used as a research tool, for example to study the interactions between niche cells and hematopoietic cells in the pathogenesis of hematological disorders. The tool can also be used to study the effects of drugs on the system and use the identified effects to develop and test new therapies. The tool can further be used to test cell therapies, such as CAR-T cells or antibodies, or combinations of T cells and antibodies (BiTe), for example, for their effects on leukemia stem cells (LSCs), or HSCs. Furthermore, the system can be used as a tool to screen and identify new drugs for hematological disorders and other diseases disclosed herein. In the second strategy, the produced "products" and compositions can be used as therapeutics themselves, i.e., to prevent and / or treat bone marrow-related disorders, such as hematological disorders and other diseases disclosed herein. This strategy includes the transplantation of produced human BMOs, or improved in vitro generation of human blood cells from iPSCs, for example, autologous patient-specific production, and the possibility of subsequent infusion or transplantation. The present invention has the advantage of maintaining the stemness of hematopoietic stem cells, which is normally lost when cultured in vitro with cytokines. When stemness is maintained, single cell studies can be performed to select stem cells with the desired genetic modifications.

[0073] The vascular network or mature mammalian bone marrow organoid or assembloid produced according to the present invention is preferably a model of bone marrow-related disease, such as blood disease, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormality of hematopoiesis and immunity, HIV-related condition, sickle cell disease, and chemotherapy or transfusion complications.In these cases, the model is generated in iPSCs that are mutated or modified to reflect the origin of bone marrow-related disease, and then this cell is used to generate organoid.Nevertheless, organoid or assembloid or vascular network or certain cellular components thereof may be modified to reflect the origin of bone marrow-related disease to be analyzed.

[0074] Therefore, another aspect of the present invention relates to a method for identifying pharmacologic active compounds for bone marrow-related diseases, comprising: a) providing vascular network or mature mammalian bone marrow organoid or assembloid produced according to the present invention, which is a model of at least one bone marrow-related disease; b) contacting said vascular network or mature mammalian bone marrow organoid or assembloid according to step a) with at least one potentially pharmacologic active compound; and c) identifying physiological effects that reflect or indicate the treatment or improvement of said bone marrow-related disease in the presence of said at least one potentially pharmacologic active compound, when compared with the absence of said at least one potentially pharmacologic active compound or control, and said effects identify pharmacologic active compounds for bone marrow-related diseases.Bone marrow-related diseases can be, for example, blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications from chemotherapy or blood transfusion.

[0075] Preferably, the method or use according to the invention comprises the candidate compound in vivo or in vitro, in solution or bound or coupled to a solid support, each format also being described in the art and known to the skilled person.

[0076] In the context of the present invention, anti-aging candidate compounds can be selected from any suitable molecule, such as chemical organic molecules, molecules selected from libraries of small organic molecules (molecular weight less than 500 Da), molecules selected from combinatorial libraries, cell extracts, in particular plant cell extracts, small molecule drugs, proteins, protein fragments, molecules selected from peptide libraries, antibodies or fragments thereof, etc.

[0077] These candidate molecules can also be used as standards for screening improved compounds (see below).

[0078] In the context of the present invention, any method suitable for detecting the effect of a compound can be used. The respective methods are known to those skilled in the art and are disclosed in the art. The components of the assays disclosed herein can be labeled, for example, with a radioactive or fluorescent label, or with an antigenic label.

[0079] The present invention further relates to the identification of improved compounds identified in the first round of screening / identification. Following the provision of the identified compounds, the compounds can be modified. In general, many methods of modifying the compounds of the present invention are known to those skilled in the art and are disclosed in the literature. Modification of compounds is usually classified into several categories, such as a) chemical modification, e.g., by adding additional chemical groups, b) changing the size, length and / or charge of the compound, and c) attaching additional groups to the molecule, including marker groups, labels, linkers or carriers, e.g., chelators. All of these modifications, any one of them or a combination thereof, ultimately present a novel strategy that leads to the "rational design" of improved molecules to be used in the context of the present invention. The present invention also includes a strategy for further improving compounds that have only partially undergone "directed evolution" or "directed mutagenesis", i.e., the compounds can undergo several successive rounds of the above methods.

[0080] In a next step, the modified compound is tested in the presence of the at least one potentially pharma- ceutical active compound for a change in physiological effect that reflects or is indicative of treatment or amelioration of the bone marrow-related disease when compared to the absence of the at least one potentially pharma- ceutical active compound, when compared to the unmodified pharma- ceutical active compound or a control.

[0081] Thus, yet another aspect of the present invention relates to a pharma- ceutical composition comprising a pharma- ceutical active compound against bone marrow-related diseases identified according to the present invention. This aspect also includes a method for producing a pharmaceutical composition comprising a pharma- ceutical active compound against bone marrow-related diseases identified according to the present invention, comprising formulating the compound with a suitable diluent and / or carrier. In general, the same conditions as above apply to this pharmaceutical composition.

[0082] Then, another aspect of the present invention relates to the use of vascular network or mature mammalian bone marrow organoid or assembloid or pharmaceutical composition (comprising identified pharmacoactive compound) according to the present invention as a model system for identifying and / or testing pharmacologic active compounds for treating or preventing bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications from chemotherapy or transfusion.Identification and testing are generally outlined above.

[0083] Yet another aspect of the present invention then relates to the use of the vascular network or mature mammalian bone marrow organoid or assembloid or pharmaceutical composition (containing the identified pharmacologic active compound) according to the present invention as a model system in the pathogenesis of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications from chemotherapy or blood transfusion. As mentioned above, in these cases, the model used can be generated in iPSCs that are mutated or modified to reflect the origin of the bone marrow-related disease, and then this cell is used to generate the organoid. Nevertheless, the organoid or assembloid or vascular network or certain cellular components thereof may be modified to reflect the origin of the bone marrow-related disease to be analyzed. The model system can also be used to test the suitability of BMO as a pharmaceutical preparation for transplantation and other therapeutic purposes.

[0084] A further aspect of the present invention then relates to the use of the vascular network or mature mammalian bone marrow organoids or assembloids or pharmaceutical compositions according to the present invention for the in vitro production of BMOs or mammalian blood cells, in particular autologous BMOs or mammalian blood cells, in particular for transplantation.

[0085] Then, another aspect of the present invention relates to the use of vascular network or mature mammalian bone marrow organoid or assembloid according to the present invention to maintain stemness of hematopoietic stem cells (HSC). It is advantageous to maintain HSC in vitro for further quality control studies and select only those cells that show desired molecular changes without any undesired side effects (e.g. off-target editing, or activation of oncogenes, etc.) for therapeutic use. The present invention has the advantage of maintaining stemness of hematopoietic stem cells, which is usually lost when cultured in vitro with cytokines. When stemness is maintained, single cell studies can be performed to select stem cells with desired genetic modifications.

[0086] Yet another aspect of the present invention then relates to a pharma- ceutically effective amount of the vascular network or mature mammalian bone marrow organoids or assembloids or pharmaceutical compositions according to the present invention (comprising the identified pharma- ceutical active compound) for use in the treatment or prevention of bone marrow-related diseases, such as, for example, blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and complications due to chemotherapy or transfusion, preferably for transplantation.

[0087] Then, another aspect of the present invention relates to a method for preventing or treating bone marrow-related diseases in a subject, such as blood disease, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications of chemotherapy or transfusion, comprising administering an effective amount of vascular network or mature mammalian bone marrow organoid or assembloid according to the present invention or pharmaceutical composition (comprising identified pharmacologic active compound) to said subject.Preferably, said method comprises transplantation of an effective amount of vascular network or mature mammalian bone marrow organoid or assembloid according to the present invention.

[0088] It is to be understood that the products, compounds and / or pharmaceutical compositions are used for administration to human patients. The term "administration" refers to administration of a single therapeutic agent or administration in combination with another therapeutic agent. It is therefore envisaged that the pharmaceutical compositions of the present invention are used in a combination therapy approach, i.e., simultaneous administration with other medicaments or drugs and / or any other therapeutic agents that may be beneficial in the context of the method of the present invention. Nevertheless, the other medicaments or drugs and / or any other therapeutic agents may be administered separately from the compound used, if necessary, as long as they act in combination (i.e., directly and / or indirectly, preferably synergistically) with the compound used.

[0089] The compounds of the invention can therefore be used alone or in combination with other active compounds (e.g. drugs and therapies already known for the treatment of the abovementioned diseases), whereby, in the latter case, a favorable additive enhancing effect, or preferably a synergistic effect, is observed.

[0090] As stated herein, the product or compound is administered to the subject in an effective dosage. This dosage can vary within a wide range and is appropriate for the individual conditions of each individual case. For the above uses, the appropriate dosage will vary depending on the mode of administration, the particular condition to be treated and the desired effect. In general, however, satisfactory results are achieved with dosages as stated above, for example about 1 mg / kg to 100 mg / kg animal body weight, in particular 1 mg / kg to 50 mg / kg. Suitable dosages for large mammals, for example humans, are on the order of about 10 mg / day to 3 g / day, conveniently administered in single or divided doses, for example 2 to 4 times a day, or in sustained release form. In general, for oral administration, a daily dosage of approximately 10 mg to 100 mg, in particular 10 mg to 50 mg per human individual is appropriate. The effective concentration reached at the cellular level can be set at 50 μM to 200 μM, preferably about 100 μM.

[0091] Another aspect of the present invention then relates to a kit, for example a diagnostic kit, comprising materials for carrying out the method according to the present invention, such as a set of media, such as the aggregation medium containing a Rho-associated protein kinase (ROCK) inhibitor according to step a) herein, a serum-free stabilized cell culture medium suitable for feeder-free maintenance and growth of human embryonic stem cells supplemented according to step b) herein, Essential 6 medium supplemented according to step c) herein, and materials for producing polymerized 3D collagen I / Matrigel™ matrix and StemPro™-34 medium supplemented according to step d) herein.

[0092] The kits may further include relevant antibodies that bind to cell markers of BMO, dyes and other labels, as well as buffers and matrices for carrying out the above methods.

[0093] The kits can be used in accordance with the methods of the invention, i.e., for the identification and / or testing of pharma- ceutically active compounds for the treatment or prevention of bone marrow-related diseases, for use in model systems in the pathogenesis of bone marrow-related diseases, and / or for the in vitro production of BMOs or mammalian blood cells, in particular autologous BMOs or mammalian blood cells, in particular for transplantation.

[0094] In summary, the present invention provides a vascularized BMO system derived exclusively from iPSCs and mimicking hematopoiesis in the multicellular context of native bone marrow.

[0095] Furthermore, a method is provided for generating complex self-organized bone marrow organoids, including de novo vascular networks, hematopoietic cells and stromal niche cells, by simultaneous differentiation from human iPSCs. BMOs exhibit key cellular, structural and molecular features of the native human bone marrow niche. They include not only multipotent hematopoietic stem and progenitor cells (HSPCs) but also mesenchymal stem and progenitor cells (MSPCs), modeling the developmental context of fetal bone marrow. This novel organoid system may be useful for studying hematopoietic development and disease progression. As mentioned above, further, a method is provided for generating complex assembloids according to the present invention, including (further) iPSC-derived immune cells, blood vessels and pericytes.

[0096] The present invention relates to the following items.

[0097] Item 1. A method for producing mammalian bone marrow organoids, comprising: a) generating embryoid bodies from a substantially single induced pluripotent stem cell (iPSC) obtained from at least one mammalian species, comprising culturing the single iPSC in an aggregation medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day, followed by culturing the embryoid body in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells without a ROCK inhibitor for about 48 hours; and b) inducing mesoderm in the embryoid body formed in step a), comprising culturing the embryoid body in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells for about 48 hours, with resuspending the mesoderm-induced embryoid body every about 24 hours, the medium containing 80 ng / ml bone morphogenetic protein 4 (BMP4), 4 μM glycogen synthase kinase (GSK) 3 inhibitor, and 80 μM glycerol. c) replacing the medium of the culture of b) with Essential 6 medium supplemented with 80 ng / ml VEGF, 25 ng / ml fibroblast growth factor (FGF)-2, 50 ng / ml stem cell factor (SCF), and 2 μM SB431542 for about 48 hours with resuspending the embryoid bodies about every 24 hours; d) embedding the mesoderm-derived embryoid bodies of step c) in a suitable polymerized 3D collagen I / Matrigel™ matrix, followed by covering the matrix with StemPro™-34 medium supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF, and 2 μM SB431542 for about 48 hours, followed by resuspending the embryoid bodies about every 24 hours. and cytokine replacement with 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours, followed by a cytokine boost to 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours; and e) isolating the individual vascular networks generated in step d) and subsequently replacing the individual vascular networks with 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours.and culturing in StemPro™-34 medium supplemented with Flt-3L and 5 ng / ml TPO for about 7-11 days, refreshing the medium every 3-4 days, thereby producing mammalian bone marrow organoids, preferably without the use of lineage-targeting cytokines such as EPO, IL-6 and / or G-CSF.

[0098] Item 2. The method for producing mammalian bone marrow organoids according to item 1, further comprising the steps of fixing the vascular network in step e) and performing immunofluorescence analysis, and / or dissociating the vascular network into individual cells and performing flow cytometry analysis and / or live cell imaging.

[0099] Item 3. The method for producing mammalian bone marrow organoids according to item 1 or 2, further comprising the steps of fixing the produced organoids, performing immunofluorescence analysis, and / or dissociating the organoids into individual cells, performing flow cytometry analysis and / or live cell imaging.

[0100] Item 4. The method for producing a mammalian bone marrow organoid according to any one of items 1 to 3, further comprising culturing the organoid for up to about 47 days, preferably about 60 days.

[0101] Item 5. The method for producing a mammalian bone marrow organoid according to any one of items 1 to 4, wherein the single iPSC is provided by dissociating iPSC cells into single cells by Accutase™ digestion.

[0102] Item 6. The method for producing a mammalian bone marrow organoid according to any one of items 1 to 5, wherein the ROCK inhibitor is selected from the group consisting of Y-27632 ((lR,4r)-4-((R)-l-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide) and fasudil.

[0103] Item 7. The method for producing mammalian bone marrow organoids according to any one of items 1 to 6, wherein the aggregation medium is KnockOut DMEM / F12 containing 20% ​​serum replacement, 1% L-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin, and 100 μM β-mercaptoethanol.

[0104] Item 8. The method for producing mammalian bone marrow organoids according to any one of items 1 to 7, wherein the serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells is mTeSR™ Plus medium.

[0105] Item 9. The method for producing a mammalian bone marrow organoid according to any one of items 1 to 8, wherein the glycogen synthase kinase (GSK) 3 inhibitor is CHIR99021.

[0106] Item 10. The method for producing mammalian bone marrow organoids according to any one of items 1 to 9, wherein the ratio of 3D collagen I / Matrigel™ matrix is ​​about 1:1 to 4:1, preferably about 3:1 or 1.2:1.

[0107] Item 11. The method for producing a mammalian bone marrow organoid according to any one of items 1 to 10, wherein resuspension of the embryoid body / mesoderm-derived embryoid body comprises use of a pipette.

[0108] Item 12. The method for producing mammalian bone marrow organoids according to any one of items 1 to 11, wherein the dissociation or removal of individual vascular networks comprises removal by using a sterile dissection tool.

[0109] Item 13. The method for producing a mammalian bone marrow organoid according to any one of Items 1 to 12, wherein the mammal is selected from the group consisting of humans, mice, monkeys, rats, pigs, dogs, cats, rabbits, sheep, cows, horses, and goats.

[0110] Item 14. A method for producing mesenchymal stem cells (MSCs) and / or hematopoietic stem cells (HSCs), comprising carrying out the method according to any one of items 1 to 13 and appropriately isolating mesenchymal stem cells (MSCs) or hematopoietic stem cells (HSCs) from a vascular network or a mammalian bone marrow organoid.

[0111] Item 15. A vascular network or mature mammalian bone marrow organoid produced according to the method according to any one of items 1 to 13, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid.

[0112] Item 16. An assemblen bloid comprising the vascular network or mature mammalian bone marrow organoid of item 15, having at least one additional iPSC-derived organoid.

[0113] Item 17. The assembloid according to item 16, comprising immune cells, blood vessels, and pericytes.

[0114] Item 18. Use of the vascular network or mature mammalian bone marrow organoid according to item 15 or the assembloid according to item 16 or 17 as a model system for bone marrow-related diseases, such as hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

[0115] Item 19. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition according to item 15 or the assembloid according to item 16 or 17 as a model system for the pathogenesis of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

[0116] Item 20. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition according to item 15 or the assembloid according to item 16 or 17 as a model system for identifying and / or testing pharmacologic active compounds for the treatment or prevention of bone marrow-related diseases, such as hematological diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

[0117] Item 21. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition according to item 15 for the in vitro production of BMO or mammalian blood cells, in particular autologous BMO or mammalian blood cells, in particular for transplantation.

[0118] Item 22. A vascular network or mature mammalian bone marrow organoid or pharmaceutical composition according to item 15 or assembloid according to item 16 or 17, preferably for transplantation, for use in the treatment of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or transfusion.

[0119] Item 23. MSCs or HSCs produced according to item 14, or a pharmaceutical composition comprising MSCs or HSCs.

[0120] Item 24. A pharma- ceutical effective amount of MSCs or HSCs or pharmaceutical composition according to item 23, for use in the treatment of bone marrow-related diseases, such as, for example, blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-associated conditions, sickle cell disease, and complications from chemotherapy or transfusion, preferably for transplantation.

[0121] The present invention will now be further described, but not limited, in the following examples and with reference to the accompanying figures and sequence listing. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief description of the drawings]

[0122] [Figure 1-1] Generation of human iPSC-derived bone marrow organoids and analysis of cellular composition by flow cytometry. a) Schematic of the workflow for BMO generation. b) Representative brightfield images of fibroblast-derived iPSC (FiPSC) embryoid bodies at day 0 and day 4, germinated embryoid bodies at day 6, and differentiated BMOs at day 17. c) Joint tSNE visualization of flow cytometry data of three independent differentiations, resulting in 105000 cells stained by protein expression. d) Overlay of manually gated populations on tSNE projection. Subsets were defined as CD45-CD31+ endothelial cells, CD45+ hematopoietic cells, CD45+CD11b+ myeloid cells, and CD45-CD31-CD271+ mesenchymal cells. CD45+CD11b-CD34+ as HSPCs and CD45-CD31-CD271+CD90+CD105+CD73+ as MSPCs. e) Frequencies of manually gated cell types, n=5 independent differentiations. f) Brightfield microscopy of sorted and May-Grünwald-Giemsa stained HSPCs and CD45+CD11b+ bone marrow cells. High nuclear to cytoplasmic ratio typical of HSPCs. Morphology of sorted MSPCs in culture in phase contrast. Representative images of n=6 experiments with two iPSC cell lines. iPSC, induced pluripotent stem cells; EB, embryoid bodies. Scale bars b) 500 μm, inset at day 6 100 μm, f) 20 μm. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 2-1]FIG. 1: Spatial organization of BMO recapitulates key features of the human bone marrow niche. a) Confocal imaging of whole mounted organoids reveals a dense interconnected meshwork of CD31-expressing vascular cells and CD271-expressing stromal cells, including hematopoietic cells marked with CD45. b) Zoomed-in view of a) displaying CD45+ cells within CD31+ and CD271+ niche cells. c) Surface rendering of 3D z-reconstruction of organoids stained for endothelial cells (CD31) and pericytes (PDGFRβ) imaged by confocal microscopy to visualize vascular networks and PDGFRβ+ pericytes. d) Pericyte-endothelial cell associations at days 10 and 21 of differentiation. Top: finger-like extensions of PDGFRβ+ pericytes at day 10 of differentiation. Bottom: close association of PDGFRβ+ pericytes with endothelial cells at day 21 of differentiation. e) The niche marker CXCL12 is expressed in specific cell subsets throughout the organoid visualized by confocal microscopy. f) CXCL12 expressing pericytes in close association with endothelial cells. g) Nestin+ cells lining CD31+ blood vessels and CD45+ hematopoietic cells. Note the CD45high expressing cells with band-like nuclei. h) Two-photon microscopy shows spatial organization throughout the organoid. Expression of the key niche marker Nestin in spatial association with CD31+ blood vessels and CD45+ hematopoietic cells. Z dimension 515 μm. Scale bars: a) 100 μm, b) 20 μm, c) 100 μm, d), e), f), g) 50 μm, h) 200 μm. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 2-4] Same as above [Figure 3-1]Figure 2. Vascular structures showing key features of blood vessels and surrounding hematopoietic cells. a) Endothelial cells are covered by a collagen IV+ (Col IV) basement membrane. b) TEM image of pericytes and endothelial cells connected by tight junctions (TJs). c) Orthogonal 2D z-projection of the vascular structures of the bottom image of Fig. 2d) in xz and yz directions. Note the lumen formation indicated by the arrows. d) TEM image of an organoid section showing capillary-like structures with endothelial cells (E) forming a lumen (L) and surrounded by pericytes (P). Asterisks indicate Weibel-Palade bodies. e) Confocal imaging of CD31+ blood vessels lined by nestin expressing cells and surrounding CD45+ hematopoietic cells. f) Surface rendering of the fluorescent image of e) to show the vascular structures. Transparent surfaces of CD31 and nestin reveal CD45+ cells within the vessels. g) Histological sections stained with hematoxylin and eosin (HE) morphologically reveal hematopoietic cells within the lumen of the vessel-like structures. h) TEM images of organoid sections showing endothelial cells (E) enveloping round cells similar to myeloid cells (M). Scale bars: a) 10 μm, b) 1 μm, c) 20 μm, d) 2 μm, e) 10 μm, f) 20 μm, h) 2 μm. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4-1]Figure 2: Bone marrow organoids are capable of granulopoiesis. Expression of a) S100A8 / A9 and b) myeloperoxidase (MPO) in cells with banded / lobulated nuclei. Enlarged image of rectangular area shown in inset. c) MPO expression was confirmed by immunohistochemistry of organoid sections. Enlarged image of rectangular area shown in inset. d) Representative TEM image of typical morphology of cells resembling neutrophilic granulocytes in organoids. e) Gating scheme for neutrophil differentiation analysis of dissociated BMOs at day 21 of differentiation by flow cytometry. f) Histogram of surface marker expression of different neutrophil progenitor stages up to mature neutrophil-like state of BMO-derived neutrophils. g) Frequency of neutrophil progenitor subpopulations within CD45+ population, n=6 independent experiments. h) May-Grünwald-Giemsa staining of sorted neutrophil progenitor cells from BMOs shows high morphological similarity to human neutrophil progenitor cells in vivo. Scale bars: a), b), 10 μm, c) 50 μm, insets 10 μm, d) 2 μm, h) 20 μm. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 4-4] Same as above [Figure 5-1]Figure 1: Single-cell transcriptome analysis of BMO identifies diverse cell populations. a) Coarse-grained clustering of scRNA-sequencing data reveals three major populations including endothelial, hematopoietic and mesenchymal cells. b) Expression of characteristic markers indicative of the three major populations. c) UMAP projection of a total of 31040 cells colored according to detailed cell type annotation. d) Expression of marker genes for hematopoietic lineage annotation. e) Expression of marker genes for endothelial subtypes. f) UMAP projection of arterial endothelial and pre-HE cells co-expressing the indicated marker genes. g) Expression of marker genes for mesenchymal cell clusters. h) UMAP projection of different mesenchymal subsets co-expressing marker genes. DC, dendritic cell; ELP, early lymphoid progenitor; eo / baso, eosinophil / basophil; GMP, granulocytic / monocytic progenitor; HE, hemogenic endothelium; HSC / MPP, hematopoietic stem cell / multipotent progenitor; LMPP, lymphoid multipotent progenitor; MEP, megakaryocyte / erythroid progenitor; MK, megakaryocyte; mono. mac., monocyte-like macrophage; PS, primitive streak-like. [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 5-4] Same as above [Figure 5-5] Same as above [Figure 5-6] Same as above [Figure 6-1]Figure 3. Functional characterization of BMO in vitro and in vivo and modeling of monogenic bone marrow failure syndromes. a) Trilineage differentiation assay of FACS-sorted BMO-derived MSPCs. MSPCs were cultured in osteogenic, adipogenic or chondrogenic differentiation medium for at least 21 days. Osteogenic, adipogenic and chondrogenic differentiation were visualized by staining with Alizarin Red S, Oil Red O or Alcian Blue, respectively. n=4 independent experiments with two iPS cell lines. b) Colony forming unit assay of FACS-sorted BMO-derived CD45+CD11b-CD34+ HSPCs. CFU-GM, granulocyte-macrophage colony forming unit; BFU-E, erythroid burst forming unit; CFU-GEMM, granulocyte, erythroid, monocyte and megakaryocyte colony forming unit; representative phase contrast microscopy of colony morphology (left) and May-Grünwald-Giemsa staining of harvested colonies (right). c) Frequency of BMO hPSC-derived colonies, n=4 independent experiments with two iPS cell lines, showing the number derived from FiPSCs. d) Xenografting of BMO under the kidney capsule of NSG mice. e) Gross morphology after 3.5 months showing further growth in vivo. f) Hematoxylin-eosin stained section of paraffin-embedded organoid 3.5 months after transplantation showing blood vessels filled with red blood cells within BMO. g) Representative plot of flow cytometry analysis reveals human CD45+ in mouse bone marrow of transplanted mice but not in control mice (n=2 mice). h) Modeling VPS45 deficiency in bone marrow organoids. Schematic of gene editing and experimental setup to generate VPS45 mutant iPS cell lines with isogenic background. i) Histological comparison of control and VPS45 mutant BMO by HE and Gomori staining reveals reticulin fibrosis in VPS45 mutant BMO. n=8 organoids for each condition in two batches. j) Alpha-smooth muscle actin (SMA) expression in control and VPS45 mutant BMO analyzed by immunofluorescence. k) Quantification of the mean fluorescence intensity (MFI) of SMA expression, four different regions of n=5 organoids per condition in two batches.l) Flow cytometry shows increased expression of Annexin V in mature Neu in VPS45 mutant BMO (n=3). m) Quantification of Annexin MFI in mature Neu, n=3, *p<0.05, ****p<0.0001, unpaired two-tailed t-test. Scale bars: a) 100 μm, b) 200 μm (left) and 50 μm (right), f) 20 μm, i), j) 50 μm. [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 6-4] Same as above [Figure 6-5] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0123] background A 2D system for hematopoietic differentiation was described in 2011, where blood precursor cells were generated from human induced pluripotent stem cells (hiPSCs) by sequential induction of cytokines (8). At that time, serum-free monolayer cultures were established that allowed tracing the in vivo hematopoietic pathway from ES / iPS cells through mesodermal progenitors to functional definitive blood cells, and hematopoietic mesodermal progenitors were induced via primitive streak cells by stepwise adjustment of exogenous cytokine cocktails. These precursor cells were then differentiated into various cell lineages depending on the hematopoietic cytokines present.

[0124] The protocol of (8) was extensively modified to differentiate hematopoietic progenitors into functional neutrophils and macrophages. For the generation of vascularized bone marrow organoids, we carried out a part of the protocol for the generation of vascular organoids recently published by Wimmer et al. (9). These hiPSC-derived vascular organoids were not only composed of functional endothelial cells and pericytes, but also contained very small numbers of hematopoietic cells and mesenchymal stromal cells (10).

[0125] The inventors concluded, and surprisingly found to be correct, that the association of the above hematopoietic differentiation protocol with a three-dimensional vascularized culture system would promote the formation of complex cellular interactions and thus lead to the formation of a structurally organized microenvironment similar to the native bone marrow niche.

[0126] Summary of the Preferred Method According to the Invention Bone marrow organoids were generated from iPSCs by mesoderm induction and subsequent hematopoietic induction in a 3D matrix cytokine induction system. Human iPSC embryoid bodies were cultured in low-attachment plates containing Y-27632 and grown for 2-3 days until the aggregates reached a size of 0.5 μm (Figure 1A). Mesoderm was induced with bone morphogenetic protein 4 (BMP4), GSK-3 inhibitor CHIR99021 (Sigma), and vascular endothelial growth factor (VEGF) on day 0 and patterned with VEGF, TGF-β RI kinase inhibitor VI SB431521 (Sigma), fibroblast growth factor 2 (FGF2), and stem cell factor (SCF) on days 2 and 4. On day 4, embryoid bodies were embedded in collagen I / Matrigel™ (Sigma) solution to induce vascular sprouting and promote cell organization in a three-dimensional form. Hematopoietic differentiation was then induced with VEGF, SCF, Fms-related receptor tyrosine kinase 3 ligand (Flt-3L), interleukin 3 (IL-3) and thrombopoietin (TPO). On day 10, vascular networks were isolated / excised and individual vascular networks were transferred to 96-well low-attachment plates. From days 10 to 17, vascular networks formed into spherical BMOs, which were collected on days 17, 21 and 45 for further analysis. The resulting BMOs were characterized by histological methods, confocal microscopy, flow cytometry and differentiation assays (Figure 1).

[0127] Detailed Preferred Method for Producing Bone Marrow Organoids Bone marrow organoids (BMOs) were generated in a 3D culture system using sequential addition of growth factors as follows:

[0128] As exemplarily shown in Figure 1, on day -3 of the method, embryoid bodies were generated by dissociating iPSCs into single cells with Accutase™ (Sigma) for 5 min at 37°C. Cell aggregates were mechanically disrupted using a P1000 pipette. The dissociation reaction was stopped with mTeSR™ Plus (Fisher Scientific), and then cells were harvested at 300 g for 3 min at room temperature (RT).

[0129] Cells were resuspended in aggregation medium (KnockOut DMEM / F12 containing 20% ​​serum replacement, 1% L-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin and 100 μM β-mercaptoethanol) and cells were counted and processed using a hemocytometer. 6 pieces~3×10 6 Cells were resuspended in aggregation medium supplemented with 50 μM Y-27632 and plated onto low-adherence Petri dishes.

[0130] Mesoderm was induced on day 0 with mTeSR™ Plus supplemented with 80 ng / ml BMP4, 4 μM CHIR99021, and 80 ng / ml VEGF. Embryoid bodies were collected by gravity (15-30 min) into 15 ml canonical tubes for medium changes. To avoid excessive fusion, embryoid bodies were resuspended once a day and placed on a rocking shaker. On day 2, medium was changed to Essential 6 medium supplemented with 80 ng / mL VEGF, 25 ng / mL FGF-2, 50 ng / mL SCF, and 2 μM SB431542.

[0131] On day 4, 30-60 embryoid bodies were embedded in 12-well plates containing 1 ml / well of collagen I / Matrigel™ mixture. Collagen I solution was prepared according to the manufacturer's protocol (50 μl 10×DMEM, 137.5 μl ddH2O, 12.5 μl 7.5% sodium bicarbonate, 235.9 μl Hams-F12, 9.45 μl HEPES, 4.6 μl Glutamax, 300 μl 5 mg / ml collagen type I) and the solution was brought to pH 7.4 by dropwise addition of 1N NaOH.

[0132] For embedding, a layer of 500 μl / well of collagen I-Matrigel™ mixture was first prepared and polymerized for 1 h at 37° C. to prevent the embryoid bodies from sinking to the bottom of the dish. Then, the embryoid bodies were resuspended in 500 μl / well of collagen-I-Matrigel mixture to prepare a second layer, which was polymerized for 1 h at 37° C. Finally, the embedded embryoid bodies were covered with pre-warmed StemPro™-34 medium supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and 2 μM SB431542.

[0133] On day 6, cytokines were replaced with 50 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO. On day 8, cytokines were changed to 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO.

[0134] On day 10, the vascular networks arising from the individual embryoid bodies were excised using a sterile dissection tool under the laminar flow unit at the lowest magnification of an inverted light microscope. The individual vascular networks were then transferred to 96-well low-attachment plates and further cultured. The organoids were then cultured in StemPro™-34 medium (Fisher Scientific) supplemented with 25 ng / ml VEGF, 50 ng / ml SCF, 50 ng / ml IL-3, 50 ng / ml Flt-3L and 5 ng / ml TPO, refreshing half the medium every 3-4 days. The vascular networks on day 10 and the mature organoids from day 17 onwards were either fixed for immunofluorescence analysis or dissociated into individual cells for flow cytometry analysis. In this case, the organoids were cultured until day 45.

[0135] Heterogeneous cell type composition To investigate the cell type composition, we analyzed dissociated BMOs at day 17 of differentiation by flow cytometry (Fig. 1c). To visualize subset heterogeneity, we performed dimensionality reduction of the flow cytometry data by t-SNE (Fig. 1a). We integrated data from three independent differentiations into a joint t-SNE visualization to generate a representative map across different experiments (Fig. 1a). Overlaying the manually gated populations revealed three major cell clusters (Fig. 1e). Based on CD45 as a pan-leukocyte marker, CD31 (PECAM1) as a marker for vascular endothelial cells (ECs), and CD271 (NGFR) as a marker for mesenchymal cells, we assigned these clusters as endothelial cell cluster (CD45-CD31+), hematopoietic cell cluster (CD45+), and mesenchymal stromal cell cluster (CD45-CD31-CD271+), respectively.

[0136] Hematopoietic cells were further defined as HSPCs (CD45+CD11b-CD34+) and myeloid cells (CD45+CD11b+). CD45-CD31-CD271+CD90+CD105+CD73+ cells were characterized as MSPCs according to the minimal criteria for multipotent MSPCs. Quantitative analysis of the cell composition on day 17 of culture yielded an average content of 39.3% hematopoietic cells, 41.3% mesenchymal cells, 6% ECs, 1.42% HSPCs and 0.96% MSPCs per BMO (n=5). This relative cell type composition was constant at different time points of differentiation. To determine the reproducibility of the protocol, we also generated BMOs by using an iPS cell line derived from exfoliated renal epithelial cells present in urine, the so-called urinary iPSCs (UiPSCs). UiPSC-derived BMOs showed a morphology very similar to that of fibroblast-derived iPSCs (FiPSCs). The main composition of hematopoietic, mesenchymal and endothelial cells was consistent, but UiPSC-derived BMOs showed a higher percentage of endothelial cells (average content 12.47%) and mesenchymal cells (49.15%) and fewer hematopoietic cells (23.42%) compared to FiPSC-derived BMOs. We then used fluorescence-activated cell sorting (FACS) to sort defined cell subsets and analyzed their morphology by microscopy. May-Grünwald-Giemsa staining of cytospins of isolated CD45+CD11b-CD34+ and CD45+CD11b+ and populations showed typical morphology of myeloid progenitor and monocyte-like cells, respectively. Sorted BMO-derived CD45-CD31-CD90+CD105+CD271+CD73+MSPCs adhered to plastic and had the typical spindle-shaped morphology characteristic of MSPCs. Moreover, these cells expanded in culture and had the capacity for continuous reseeding for up to 15 passages. Thus, this demonstrates that BMOs consist of endothelial, hematopoietic and mesenchymal cells, contain phenotypically defined HSPCs and MSPCs, and can be generated from FiPSCs and UiPSCs.

[0137] spatial structure Next, we analyzed the spatial distribution of the different cell subsets by a series of microscopic studies. Confocal imaging revealed spherical CD45+ hematopoietic cells embedded in a meshwork of CD31+ vascular structures and CD271+ stromal cells. To increase the imaging depth, we used two-photon microscopy, allowing us to capture fluorescent signals up to 845 μm deep (Figure 2h). These studies confirmed the presence of vascular, hematopoietic and mesenchymal structures throughout the organoids. The dense vascular network is crucial for supporting the hematopoietic niche by providing nutrients and growth factors to HSCs and other niche cells in the BM. The BM niche is composed of various types of blood vessels and is therefore a distinct perivascular region. Perivascular platelet-derived growth factor beta (PDGFRβ)+ mural cells cover ECs, thereby supporting and promoting vasculogenesis. These PDGFRβ+ pericytes are spatially associated with HSCs and osteoprogenitor cells and surround arterioles and H-type vessels in the metaphyseal and endosteal regions of mice. Confocal imaging of BMOs followed by 3D surface rendering showed that CD31+ ECs form vessel-like meshworks covered by PDGFRβ+ pericytes (Figure 3). The formation of PDGFR-β+ pericytes changed over time. At day 10 of differentiation, finger-like extensions of PDGFR-β+ pericytes were only adjacent to ECs, whereas at day 21 of complete organoid maturation, PDGFR-β+ pericytes were found to envelop CD31+ cells. This spatial association is thus similar to endosteal arterioles and H-type vessels.

[0138] The chemokine CXCL12 signals through the CXCR4 receptor and is a major retention and maintenance factor for HSCs and hematopoietic progenitors in the BM niche. CXCL12-rich reticular (CAR) cells are characterized by high expression of CXCL12 and close association with ECs. Moreover, CXCL12 is expressed on ECs themselves. Notably, BMOs contain reticular structures of perivascular CXCL12+ cells (Fig. 2e, f), which extend processes toward the endothelium reminiscent of CAR cells (Fig. 2f). Nestin-expressing perivascular mesenchymal stem cells, known to support and regulate hematopoiesis, are located in the endosteal region in spatial association with HSCs and have multilineage and self-renewal potential in mouse and human fetal BM. Notably, we identified nestin+ stromal cells in spatial relationship with CD31+ vascular and CD45+ hematopoietic cells in our BMOs by confocal and two-photon microscopy. Confocal z-stack imaging revealed nestin+ processes in perivascular mural cells lining vascular structures similar to those seen in bone marrow in vivo.

[0139] When the inventors investigated the vascular constructs in more detail, findings showed that the endothelial and mesenchymal BMO compartments recapitulated key structural features and cellular composition of the human BM niche.

[0140] Characterization and local distribution of niche and hematopoietic cells within manufactured BMOs The present analysis shows that BMOs consist of hematopoietic, vascular, and stromal (mesenchymal) compartments. Confocal microscopy revealed self-organized, interconnected, and partially tube-forming vascular-like meshworks composed of CD31+ endothelial cells and PDGFR-β+ perivascular mesenchymal cells covered by Col IV+ basement membrane. Furthermore, flow cytometry analysis showed that the relative composition of endothelial cells and mesenchymal stem cells was comparable to that of native bone marrow (11). Interestingly, we found CXCL12+ cells in a meshwork-like structure closely connected to the endothelial meshwork, reminiscent of native bone marrow structure. This strongly suggests the formation of bone marrow-specific CAR cells in the present differentiation system. This is particularly noteworthy because CAR cells are the major producers of CXCL12 and SCF, signaling molecules essential for the maintenance and homing of HSCs and the proliferation of lymphoid and erythroid progenitors (12). We found that CD45+ hematopoietic cells were distributed uniformly in clusters throughout the organoids. This local clustering can be explained by the increased localization of certain niche cells and their regulatory cytokines in these regions. Hematopoietic stem and progenitor cells are known to localize near blood vessels, because growth factors and products produced by niche cells are important regulators for the maintenance of HSCs (11, 13).

[0141] Differentiation of bone marrow cells within the manufactured BMO Furthermore, we could show that the present differentiation protocol promotes the autologous differentiation of hematopoietic progenitor cells into myeloid cells within the BMO structure, indicating a promoting interaction between hematopoietic progenitor cells and niche cells. Immunofluorescence showed a population of cells positive for monocyte and neutrophil markers (S100A8 / A9, S100A8 and MPO). Interestingly, these cells also had bean-shaped, segmented nuclei, indicating the formation of monocytes and granulocytes at various stages of maturation. Wright-Giemsa staining of FACS-sorted myeloid populations (CD45+CD34-CD11b+) also revealed the appearance of macrophages, monocytes and granulocytes. These results are particularly interesting because the differentiation of HSCs into granulocytes and macrophages depends on GCSF and GM-CSF signaling, but these cytokines are not included in the cytokine cocktail in the present differentiation system. Thus, these results indicate sufficient endogenous production of these cytokines by niche cells within the BMO system. Although we have not yet been able to demonstrate the emergence of other blood lineages such as lymphocytes, erythrocytes or platelets, CFU assays of FACS-sorted hematopoietic progenitor cells (CD45+CD34+CD11b-) showed the potential of these cells to differentiate into erythrocytes and megakaryocytes. Because the cytokines used in our system induce a shift in hematopoietic differentiation towards the myeloid lineage, future adjustments of the cytokine composition will enable the differentiation of erythrocytes or even lymphoid cells.

[0142] Neutrophilic granulocytes and their precursors represent the majority of nucleated BM cells. We investigated whether the BMO niche promotes maturation of blood progenitor cells into mature cells of the myeloid lineage without the addition of lineage-instructing cytokines such as G-CSF or GM-SCF.

[0143] Organoids were stained for key myeloid markers S100A8 / A937 and myeloperoxidase (MPO). Cells expressing both S100A8 / A9 and MPO were found in BMO, some of which showed banded or lobulated nuclei indicative of myeloid maturation. Immunohistochemical studies confirmed the expression of MPO by myeloid cells (Fig. 4c), and TEM revealed cells with electron-dense cytoplasmic granules, lobulated nuclei, and heterochromatin formation at the nuclear periphery, characteristic of neutrophil granulocytes. Importantly, we documented the orderly maturation of neutrophil granulocytes by flow cytometry. Based on the expression of cell surface markers, neutrophil progenitor stages (ProNeu1, ProNeu2, PreNeu), as well as immature Neu and mature Neu, were identified in both FiPSC- and UiPSC-derived BMO. Flow sorting and light microscopy analysis of May-Grünwald-Giemsa stained cells showed that the defined stages of BMO-derived neutrophil granulocytes resembled the morphology of their counterparts in human BM. Thus, maturation of neutrophil granulocytes in BMOs proceeding without the addition of recombinant cytokines is reminiscent of the in vivo situation.

[0144] Mesenchymal stem cells derived from BMO meet the criteria for mesenchymal stem cells Mesenchymal stem cells are one of the major cellular components of the bone marrow niche. MSCs maintain bone marrow tissue homeostasis by differentiating into adipocytes, osteocytes, and chondrocytes, whereas primitive mesenchymal cells and their progeny directly regulate hematopoiesis by secreting cytokines (14). In other in vitro bone marrow niche studies, donor primary mesenchymal cells were used to generate the stromal (mesenchymal) compartment of the bone marrow niche, which were then co-cultured with donor hematopoietic cells to mimic the bone marrow hematopoietic compartment. In this system, iPSCs are rather differentiated simultaneously into hematopoietic cells, endothelial cells, and most notably, mesenchymal progenitor cells such as PDGFRβ+ perivascular cells and CXCL12+ CAR cells. Furthermore, it was demonstrated that this system induces the formation of functional bona fide mesenchymal stem cells, as BMO-derived MSCs meet the minimum criteria for mesenchymal stem cells proposed by ISCT. MSCs were negative for the hematopoietic and endothelial surface molecules CD45, CD34, and CD31, but positive for CD90, CD105, CD271, and CD73. MSCs adhered to plastic and could differentiate in vitro into osteoblasts, adipocytes, and chondroblasts (15, 16).

[0145] Modeling the developmental process Having identified arterial pre-HE-like cells by scRNA sequencing, we next set out to study endothelial hematopoietic transition (EHT), which is characterized by morphological changes in arterial ECs and expression of the transcription factor RUNX1. Confocal imaging revealed that round RUNX1-expressing cells were localized in distinct clusters within the organoids. Some of these cells co-expressed CD31 and were still attached to the endothelial wall. Immunofluorescence of immature organoids (day 10) revealed that CD31+ cells were clustered from the vascular lining, which was also seen in tissue sections of BMO stained for CD34 and H / E, thus mirroring the findings of EHT in vivo. Consistent with these protein expression data, we found PECAM1+ cells that co-expressed RUNX1 in the endothelial clusters of our scRNA sequencing dataset.

[0146] To capture intermediate states of cell differentiation in our flow cytometry data, we analyzed protein expression by unsupervised clustering using the FlowSOM algorithm. The previously assigned endothelial compartment was found to be heterogeneous and to contain additional subpopulations. The surface marker expression profile of cells in cluster 3 was related to human HE and HSC, they were negative for CD45 and co-expressed CD31, CD34, CD90 (Thy 1) and CD105 (endoglin). The adjacent cluster 4 was defined by CD31 low CD34+ and CD45+ cells and constituted the manually gated HSPC cluster. The decrease in CD31 expression accompanied by increased expression of CD34 and CD45 reflects the immunophenotypic sequence of cells undergoing EHT. Of note, in our scRNA sequencing dataset, we also identified rare populations of cells expressing RUNX1, MECOM, HLF, SPINK2 and MLLT3, which are hallmark genes of human fetal HSC / MPP. These data suggest that BMO give rise to hemogenic endothelium and transition to human HSC / MPP-like cells upon EHT. Interestingly, unsupervised clustering also revealed a cluster of cells co-expressing CD271, CD31 and CD105. This marker profile overlaps with endothelium-derived BM stromal cells (eBMSCs) that have been identified in human fetal and regenerating BM. EBMSCs demonstrate the ability to undergo endothelial-mesenchymal transition (EndoMT) and reconstitute the entire hematopoietic niche after transplantation, including osteoprogenitor cells, thereby displaying MSC characteristics. Confocal imaging confirmed the co-expression of CD271, CD31 and CD105 in a subset of ECs, indicative of cells undergoing EndoMT.

[0147] Functional properties The phenotypic identification of HSPCs and MSPCs prompted us to investigate their functional properties. To assess the multipotency of BMO-derived MSPCs, we performed a triple lineage differentiation assay. Notably, when placed in the respective conditioned media, MSPCs isolated from both FiPSC-derived BMOs and UiPSC-derived BMOs had the capacity to differentiate into osteogenic, adipogenic, and chondrogenic cells, as visualized by Alizarin Red staining for calcium deposits, Oil Red O staining for lipid vesicles, and Alcian Blue staining for glycosaminoglycans, respectively. To investigate the multilineage differentiation potential of BMO-derived HSPCs, we performed a colony-forming unit (CFU) assay. FACS-sorted HSPCs gave rise mainly to granulocyte-macrophage progenitors (CFU-GM), but also to multipotential granulocyte, erythroid, macrophage, and megakaryocytic progenitors (CFU-GEMM) and erythroid progenitors (BFU-E). HSPCs isolated from UiPSC-derived BMOs also gave rise to CFU-GEMM and CFU-GM, but not to typical BFU-E colonies. Colony identity was confirmed by May-Grünwald-Giemsa staining of harvested colonies. These findings demonstrate that BMOs contain multipotent MSPCs and HSPCs. To determine the functional properties of BMOs in vivo, we transplanted them under the kidney capsule of immunodeficient NOD / SCID / IL2Rγnull (NSG) mice on day 21 of differentiation. Analysis 3.5 months after transplantation revealed further growth of the organoids. Histological analysis of organoid sections after transplantation showed blood vessels filled with red blood cells.Strikingly, we found human CD45+ cells in the BM of transplanted mice (mic).These data indicate that organoids gain access to mouse vasculature and induce BMO-derived human CD45+ cells into mouse BM.

[0148] Disease modeling To test the suitability of the manufactured BMOs to model bone marrow-related diseases, we generated VPS45-deficient BMOs in a proof-of-concept approach. Genetic VPS45 deficiency in patients leads to disease associated with severe congenital neutropenia and myelofibrosis (17). Although there was no significant difference in the relative composition of hematopoietic and niche cells between WT and VPS45 BMOs, preliminary histological results of VPS45 BMOs showed increased formation of reticulin fibers as a sign of myelofibrosis in VPS45 mutant BMOs.

[0149] Finally, we tested whether our novel BMOs could actually be used as a model system to recapitulate the phenotype of monogenic BM diseases. Children with Vacuolar Protein Sorting 45 Homolog (VPS45) deficiency present with neutropenia and myelofibrosis in the first year of life, progressing to BM failure. Their BM is hyperplastic, showing myeloid hyperplasia with increased apoptosis and functional defects of neutrophils. We generated BMOs from a gene-edited iPSC derivative line carrying a homozygous Thr224Asn mutation in VPS4514 and compared them with isogenic control BMOs (wild type, WT). No major differences were found in the composition of hematopoietic and niche cells. However, VPS45-deficient BMOs showed increased deposition of reticulin fibers, reminiscent of myelofibrosis in BM biopsies from VPS45-deficient patients. This was accompanied by the proliferation of α-smooth muscle actin (SMA)-expressing myofibroblast-like stromal cells, previously described as a key promoter of myelofibrosis.

[0150] Flow cytometric analysis of VPS45-deficient BMOs showed higher numbers of mature Neu compared to controls resembling myeloid hyperplasia, and a significant increase in Annexin V expression in VPS45 mutant mature Neu, indicating enhanced apoptosis of this subpopulation, a feature also seen in patients. Thus, BMOs may provide a novel model system to dissect genes and pathways in the pathomechanisms of previously refractory BM failure diseases.

[0151] References 1. Pinho S, Frenette PS. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol. 2019 May l;20(5):303-20. 2. Asada N, Takeishi S, Frenette PS. Complexity of bone marrow hematopoietic stem cell niche. Int J Hematol. 2017 Jul 1; 106(l):45-54. 3. Raaijmakers MHGP, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA, et al. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature. 2010 Apr; 464(7290):852-7. 4. Giger S, Hofer M, Miljkovic-Licina M, Hoehnel S, Brandenberg N, Guiet R, et al. Microarrayed human bone marrow organoids for modeling blood stem cell dynamics [Internet], Bioengineering; 2021 May [cited 2021 Aug 28], Available from: httpV / biorxiv.org / lookup / doi / 10.1101 / 2021.05.26.445803 5. Chou DB, Frismantas V, Milton Y, David R, Pop-Damkov P, Ferguson D, et al. On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology. Nat Biomed Eng. 2020 Apr; 4(4):394-406. 6. Isern J, Martin-Antonio B, Ghazanfari R, Martin AM, Lopez JA, del Toro R, et al. Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion. Cell Rep. 2013 May 30; 3(5): 1714-24. 7. Li M, Izpisua Belmonte JC. Organoids - Preclinical Models of Human Disease. N Engl J Med. 2019 Feb 7; 380(6):569-79. 8. Niwa A, Heike T, Umeda K, Oshima K, Kato I, Sakai H, et al. A Novel Serum-Free Monolayer Culture for Orderly Hematopoietic Differentiation of Human Pluripotent Cells via Mesodermal Progenitors. PLOS ONE. 2011 Jul 27; 6(7):e22261. 9. Wimmer RA, Leopold! A, Aichinger M, Kerjaschki D, Penninger JM. Generation of blood vessel organoids from human pluripotent stem cells. Nat Protoc. 2019 Nov 1; 14(11):3082-100. 10. Wimmer RA, Leopold! A, Aichinger M, Wick N, Hantusch B, Novatchkova M, et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature. 2019 Jan l; 565(7740):505-10. 11. Crane GM, Jeffery E, Morrison SJ. Adult haematopoietic stem cell niches. Nat Rev Immunol. 2017 Sep; 17(9):573-90. 12. Nagasawa T, Omatsu Y, Sugiyama T. Control of hematopoietic stem cells by the bone marrow stromal niche: the role of reticular cells. Trends Immunol. 2011 Jul; 32(7):315-20. 13. Kokkaliaris KD, Kunz L, Cabezas-Wallscheid N, Christodoulou C, Renders S, Camargo F, et al. Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations. Blood. 2020 Nov 12; 136(20):2296-307. 14. Kfoury Y, Scadden DT. Mesenchymal cell contributions to the stem cell niche. Cell Stem Cell. 2015 Mar 5; 16(3):239-53. 15. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy. 2005; 7(5):393-5. 16. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8(4):315-7. 17. Vilboux T, Lev A, Malicdan MC, Simon AJ, Jarvinen P, Racek T, et al. A congenital neutrophil defect syndrome associated with mutations in VPS45. N Engl J Med. 2013 Jul 4; 369(l):54-65.

Explanation of symbols

[0152] Drawing translation Figure 1a EB formation EB formation Mesoderm induction hemogenic endothelium induction embedding hematopoietic induction hematopoietic induction sprouting separation separation maturity flow cytometry microscopy scRNA-seq scRNA sequencing Aggregation medium day-3 - Day 3 VEGF low SCF VEGF low SCF Figure 1b day 0 day 4 Day 6 Day 17 Day 17 Figure 1c Day 17 Day 17 flow cytometry Joint tSNE of n=3 experiments low High Figure 1d Overlay of gated populations ungated Ungated Figure 1e Frequency of live in % Figure 2d Organoid day 21 Organoid day 21 Sprouting EB day 10 Sprouting EB day 10 Figure 2g CD31 CD45 Nestin CD31 CD45 Nestin Figure 2h Two-photon microscopy CD31 CD45 Nestin DAPI CD31 CD45 Nestin DAPI Figure 3a basement membrane Figure 3b tight junctions Figure 3c lumen formation lumen formation Figure 3d lumen formation lumen formation Figure 3e CD31 CD45 Nestin DAPI CD31 CD45 Nestin DAPI Nestin Figure 3f 3D surface rendering CD31 CD45 Nestin CD31 CD45 Nestin Figure 3g hematopoietic cells inside vessel lumen Hematoxylin-Eosin Figure 4e flow cytometry Gated on single / live / CD14- / CD45+ / Siglec8- Gated on single / live / CD14- / CD45+ / Siglec8- ImmatureNeu ImmatureNeu matureNeu matureNeu Figure 4f normalized to mode immatureNeu immatureNeu matureNeu matureNeu Figure 4g Frequency of CD45+ in % immatureNeu immatureNeu matureNeu matureNeu Figure 4h FACS sort FACS sorting Cytospin Giemsa stain immatureNeu immatureNeu matureNeu matureNeu Figure 5a scRNA-seq scRNA sequencing hematopoietic endothelial endothelium Mesenchymal Figure 5b per cent of cells gene expression Figure 5c hematopoietic endothelial endothelium mono.mac. Monocyte-like macrophage monocyte Monocyte progenitor eo / baso Eosinophils / Basophils neutrophils mast mast cell neutrophil progenitor neutrophil progenitor cell megakaryocyte megakaryocyte Mesenchymal Figure 5d Neutr. prog. Neutrophil progenitor cells neutrophil mono. prog. Monocyte precursor cells monocyte mon. mac. monocyte-like macrophage eo / baso Eosinophils / Basophils mast mast cell endothelial endothelium Mesenchymal Figure 5e endothelial endothelium non-endothelial arterial endothelial Arterial pre HE per cent of cells gene expression Figure 5f endothelial endothelium Figure 5g Mesenchymal non-mesenchymal chondrogenic precursor chondrogenic precursor cell Osteogenic precursor vascular smooth muscle cells vascular smooth muscle cells pericytes per cent of cells gene expression Figure 5h Mesenchymal Figure 6a FACS-sorted MSPCs Trilineage differentiation assay osteogenic bone formation Adipogenic Lipid production chondrogenic cartilage formation Alizarin-Red stain Oil-red-O stain Alcian blue stain Figure 6b FACS-sorted HSPCs FACS-sorted HSPCs CFU-assay Brightfield Giemsa stain Figure 6c number of colonies / 1x10 3 Number of HSPCs colonies / 1×10 3 HSPC Figure 6d Xenotransplantation 3.5 months histology flow cytometry Figure 6e Control kidney kidney with transplant Figure 6f HE stain of BMO after transplantation vessel blood vessel erythrocytes Figure 6g Analysis of mouse bone marrow Control transplanted Figure 6h modeling VPS45 deficiency Control microscopy flow cytometry Figure 6i Control Gomori Figure 6j Control Figure 6k Control Figure 6l matureNeu matureNeu normalized to mode Control Figure 6m matureNeu matureNeu Fold change Annexin V MFI Fold change Annexin V MFI Control

Claims

1. A method for producing a mammalian bone marrow organoid, comprising: a) generating embryoid bodies from substantially single induced pluripotent stem cells (iPSCs) obtained from at least one mammalian species, culturing the single iPSCs in an aggregation medium for about 1 day in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor, and subsequently culturing the embryoid bodies for about 48 hours in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells without a ROCK inhibitor; b) inducing mesoderm in the embryoid bodies formed in step a), culturing the embryoid bodies for about 48 hours in a suitable serum-free stabilized cell culture medium suitable for feeder-free maintenance and proliferation of human embryonic stem cells, while resuspending the mesoderm-induced embryoid bodies every about 24 hours, wherein the medium is supplemented with 80 ng / ml bone morphogenetic protein 4 (BMP4), 4 μM glycogen synthase kinase (GSK) 3 inhibitor, and 80 ng / ml vascular endothelial growth factor (VEGF); c) replacing the medium of the culture of b) with Essential 6 medium supplemented with 80 ng / ml VEGF, 25 ng / ml fibroblast growth factor (FGF)-2, 50 ng / ml stem cell factor (SCF), and 2 μM SB431542, and culturing for about 48 hours while resuspending the embryoid bodies every about 24 hours; d) embedding the mesoderm-induced embryoid bodies of step c) in a suitable polymerized 3D collagen I / Matrigel (trademark) matrix, subsequently covering the matrix with StemPro (trademark)-34 medium supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and 2 μM SB431542 for about 48 hours, and then performing cytokine replacement with 50 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours, and cytokine boosting to 25 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL-3, 50 ng / mL Flt-3L, and 5 ng / mL TPO for about 48 hours; e) Excising the individual vascular networks generated in step d), and subsequently culturing for about 7 to 11 days while refreshing the medium every 3 to 4 days in StemPro™-34 medium supplemented with 25 ng / ml VEGF, 50 ng / ml SCF, 50 ng / ml IL-3, 50 ng / ml Flt-3L, and 5 ng / ml TPO, whereby the mammalian bone marrow organoids are produced, or lineage-directed cytokines such as EPO, IL-6, and / or G-CSF are not used; A method comprising the above steps. **Claim 2** The method for producing mammalian bone marrow organoids according to claim 1, further comprising the steps of fixing the vascular network in step e), performing immunofluorescence analysis, and / or dissociating the vascular network into individual cells and performing flow cytometry analysis and / or live cell imaging. **Claim 3** The method for producing mammalian bone marrow organoids according to claim 1 or 2, further comprising the steps of fixing the produced organoids, performing immunofluorescence analysis, and / or dissociating the organoids into individual cells and performing flow cytometry analysis and / or live cell imaging. **Claim 4** The method for producing mammalian bone marrow organoids according to claim 1, further comprising the step of culturing the organoids for up to about 60 days. **Claim 5** The method for producing mammalian bone marrow organoids according to claim 1, wherein the single iPSC is provided by dissociating iPSC cells into single cells by Accutase™ digestion. **Claim 6** The method for producing mammalian bone marrow organoids according to claim 1, wherein the ROCK inhibitor is selected from the group consisting of Y-27632 ((1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide) and Fasudil. **Claim 7** The method for producing mammalian bone marrow organoids according to claim 1, wherein the aggregation medium is KnockOut DMEM / F12 containing 20% serum replacement, 1% L-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin, and 100 μM β-mercaptoethanol. **Claim 8** The method for producing mammalian bone marrow organoids according to claim 1, wherein the serum-free stabilized cell culture medium suitable for the feeder-free maintenance and proliferation of human embryonic stem cells is mTeSR™ Plus medium. **Claim 9** The method for producing a mammalian bone marrow organoid according to claim 1, wherein the glycogen synthase kinase (GSK) 3 inhibitor is CHIR99021.

10. The method for producing a mammalian bone marrow organoid according to claim 1, wherein the ratio of the 3D collagen I / Matrigel (trademark) matrix is about 1:1 to 4:1, or about 3:1 or 1.2:

1.

11. The method for producing a mammalian bone marrow organoid according to claim 1, wherein the resuspension of the embryoid body / mesoderm-induced embryoid body includes the use of a pipette.

12. The method for producing a mammalian bone marrow organoid according to claim 1, wherein the dissociation or excision of individual vascular networks includes excision by using a sterile dissection tool.

13. The method for producing a mammalian bone marrow organoid according to claim 1, wherein the mammal is selected from the group consisting of human, mouse, monkey, rat, pig, dog, cat, rabbit, sheep, cow, horse, and goat.

14. A vascular network or a mature mammalian bone marrow organoid produced according to the method of claim 1.

15. A pharmaceutical composition comprising the vascular network and / or the mature mammalian bone marrow organoid according to claim 14.

16. An assembroid comprising the vascular network or the mature mammalian bone marrow organoid according to claim 14 and having at least one additional iPSC-derived organoid.

17. The assembroid according to claim 16, comprising immune cells, blood vessels, and pericytes.

18. Use of the vascular network or the mature mammalian bone marrow organoid according to claim 14 as a model system for bone marrow-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

19. Use of the assembroid according to claim 16 or 17 as a model system for bone marrow-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

20. Use of the vascular network or mature mammalian bone marrow organoid according to claim 14 or the pharmaceutical composition according to claim 15 as a model system in the development of myeloid-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

21. Use of the assembloid according to claim 16 or 17 as a model system in the development of myeloid-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

22. Use of the vascular network or mature mammalian bone marrow organoid according to claim 14 or the pharmaceutical composition according to claim 15 as a model system for identifying and / or testing pharmaceutically active compounds for the treatment or prevention of myeloid-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

23. Use of the assembloid according to claim 16 or 17 as a model system for identifying and / or testing pharmaceutically active compounds for the treatment or prevention of myeloid-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or blood transfusion.

24. Use of the vascular network or mature mammalian bone marrow organoid according to claim 14 or the pharmaceutical composition according to claim 15 for the in vitro production of BMO or mammalian blood cells or autologous BMO or autologous mammalian blood cells, or Use of the vascular network or mature mammalian bone marrow organoid according to claim 14 or the pharmaceutical composition according to claim 15 for the in vitro production of BMO or mammalian blood cells or autologous BMO or autologous mammalian blood cells for transplantation.

25. Use of the assembloid according to claim 16 or 17 for the in vitro production of BMO or mammalian blood cells or autologous BMO or autologous mammalian blood cells or Use of the assemblyoid according to claim 16 or 17 for the in-vitro production of BMOs or mammalian blood cells for transplantation or autologous BMOs or autologous mammalian blood cells. [

26. ] For use in the treatment or transplantation of myeloid-related diseases including blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital abnormalities of hematopoiesis and immunity, HIV-related conditions, sickle cell disease, and complications due to chemotherapy or transfusion A pharmaceutical composition comprising a pharmaceutically effective amount of the vascular network according to claim 14 or a mature mammalian bone marrow organoid or the assemblyoid according to claim 16 or 17, or at least one of them.

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