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

Abstract

The present invention relates to a method for preparing a mammalian vascular network or mature mammalian bone marrow organoid. Also provided is the use of the prepared vascular network or mammalian bone marrow organoid in the treatment of bone marrow-related diseases, in vitro generation of BMO or mammalian blood cells, as a model system for the pathogenesis of bone marrow-related diseases, and as a system for identifying and / or testing pharmaceutically effective compounds for the treatment or prevention of bone marrow-related diseases.

Description

Technical Field

[0001] The present invention relates to a method for preparing a mammalian vascular network or a mature mammalian bone marrow organoid. In addition, the present invention provides the use of the prepared vascular network or mammalian bone marrow organoid in treating bone marrow-related diseases, preparing BMO or mammalian blood cells in vitro, as a model system for the pathogenesis of bone marrow-related diseases, and as a system for identifying and / or testing pharmaceutically effective compounds for treating or preventing bone marrow-related diseases. Background Art

[0002] Hematopoiesis occurs in the bone marrow after birth and involves a tightly regulated process in which hematopoietic stem cells (HSCs) continuously differentiate into mature blood cells and maintain the HSC pool through self-renewal. The surrounding microenvironment is called the bone marrow microenvironment, which is composed of a heterogeneous population of cells including mesenchymal cells (e.g., pericytes, adipocytes) and endothelial cells. The bone marrow microenvironment plays an important role in regulating and maintaining hematopoiesis throughout the life cycle (1).

[0003] The dense vascular network within the bone marrow is critical because it provides nutrients, growth factors, and critical cell-cell interactions to other microenvironmental cells. In addition, endothelial cells surrounded by perivascular PDGFRβ+ mesenchymal cells (pericytes) directly contribute to hematopoietic homeostasis by secreting a variety of factors; thus, HSCs are often located in close proximity to the perivascular area (2).

[0004] Impaired hematopoiesis can result from specific genetic mutations and manifest itself in the form of congenital bone marrow failure (IBMFS) such as severe congenital neutropenia in the case of germline mutations, or in the form of diseases such as myelodysplasia and leukemias due to somatic mutations. Interestingly, disruption of the bone marrow microenvironment has been shown to trigger myelodysplasia (3).

[0005] In recent years, human organoid models have been established as a model system for studying the occurrence of diseases in various tissues. Organoids are self-organizing 3D structures that simulate the most important functions and structural structures of organs and can be differentiated from induced pluripotent stem cells (iPSCs). Organoids are superior to traditional 2D cultures to a certain extent because they simulate the natural environment of certain cells through cell-cell interactions and cell-matrix interactions (7, Hofer, M., Lutolf, MP Engineering organoids. Nat Rev Mater 6, 402–420 (2021). https: / / doi.org / 10.1038 / s41578-021-00279-y).

[0006] Single cells or small clusters of cells expand and reorganize before organoids form and mature. There are two main categories of organoids based on the choice of stem cell. The first is derived from PSCs, including embryonic stem cells (ESCs) and iPSCs, and the second is derived from organ-specific adult stem cells (ASCs). Multiple workflows have been developed to generate organoids; however, specific organoid types require unique culture methods, and not all general workflows are suitable. The choice of cell culture conditions and 3D matrix is ​​critical for complex tissues.

[0007] WO2019122388A1 discloses co-cultures of organoids and immune cells, and methods of using these to identify drugs for treating diseases.

[0008] Breunig et al. (Differentiation of human pluripotent stem cells into pancreatic duct-like organoids. STAR Protoc. 2021 Dec 8; 2(4): 100913. doi: 10.1016 / j.xpro.2021.100913. PMID: 34917972; PMCID: PMC8669107) documented a scalable in vitro differentiation protocol to gradually guide human pluripotent stem cells into pancreatic duct-like organoids. This protocol simulates pancreatic duct development and has been successfully used to model the occurrence and development of pancreatic duct cancer; this method is suitable for a variety of downstream applications. However, this protocol is time-consuming and laborious.

[0009] Vallmajo-Martin, Q. et al. (in PEG / HA Hybrid Hydrogels for Biologically and Mechanically Tailorable Bone Marrow Organoids. Adv. Funct. Mater. 2020, 30, 1910282. https: / / doi.org / 10.1002 / adfm.201910282) disclose that bone marrow (BM) organoids provide a powerful tool for studying the important interactions between BM microenvironment and resident cells. A transglutaminase (TG) cross-linking system that tightly binds polyethylene glycol (PEG) and hyaluronic acid (HA) into hybrid hydrogels to form BM analogs is presented. The application of TG-PEG / HA hybrid hydrogels in maintaining, expanding or differentiating human bone marrow-derived stromal cells and human hematopoietic stem / progenitor cells in vitro is shown. And it is believed that TG-PEG / HA hybrid hydrogels are superior to the natural biomaterials currently used when forming humanized BM organoids in xenotransplantation models. The engineered humanized BM organoids presented can be an effective tool to study this complex organ.

[0010] Isern J et al. (Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion. Cell Rep. 2013 May 30; 3(5): 1714-24. doi: 10.1016 / j.celrep.2013.03.041. Epub 2013 Apr 25. PMID: 23623496) discussed strategies for expanding hematopoietic stem cells (HSCs), including co-culture with cells that recreate their natural microenvironment, such as bone marrow stromal stem / progenitor cells (BMSCs). BMSCs attached to plastic may not be sufficient to maintain primitive HSCs. They describe a method for isolating and culturing human BMSCs as non-adherent mesenchymal spheres. Human mesenchymal spheres were obtained from CD45 - CD31 - CD71 - CD146 + CD105 + nestin + Human mesenchymal spheres can also be simply grown from fetal and adult BM CD45-enriched cells. Human mesenchymal spheres stably differentiate into the mesenchymal lineage. In culture conditions where they exhibit a relatively undifferentiated phenotype, reduced plastic attachment, and increased self-renewal, they promote the proliferation of cord blood CD34 + Enhanced cell expansion. Expanded HSCs can be serially transplanted into immunodeficient mice and significantly increase long-term human hematopoietic engraftment. They discuss culture techniques that maintain the self-renewal capacity of human BMSCs and their ability to support functional HSCs.

[0011] Sun et al. (Generation of vascularized brain organoids to studyneurovascular interactions. Elife. 2022 May 4; 11: e76707. doi: 10.7554 / eLife.76707. PMID: 35506651; PMCID: PMC9246368) induced vascular and brain organoids separately, and then fused the two types of organoids to obtain vascularized brain organoids.

[0012] Janagama and Hui (in 3-D Cell Culture Systems in Bone Marrow Tissue and Organoid Engineering, and BM Phantoms as In Vitro Models of Hematological Cancer Therapeutics-A Review. Materials (Basel). 2020; 13(24): 5609. Published 2020Dec 9. doi: 10.3390 / ma13245609), in view of the recent interest in the bone marrow environment and blood cancer pathology, reviewed the current state of the art in bone marrow tissue engineering (BMTE) and blood cancer tissue engineering (HCTE). They focused on engineered BM tissues and organoids as in vitro models for blood cancer therapy, as well as the identification of BM components and their integrity as synthetic engineered BM mimetic scaffolds. In addition, the review specifically describes the dynamics of the interactions between multiple BM and blood cancer (HC) cell types in functionalized BM tissue / mimic co-culture systems, as well as their relationship with drug tolerance and cytotoxicity. The interaction between blood cancer cells and their microenvironment and the differences from the healthy microenvironment are described. The future prospects 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 explained. They conclude by outlining the clinical applications of biomaterials in BM and HC pathology and their challenges and opportunities.

[0013] Bessy T (Bioengineering the Bone Marrow Vascular Niche. Front Cell Dev Biol. 2021; 9:645496. Published 2021 Apr 28. doi:10.3389 / fcell.2021.645496) provided another comprehensive review focusing on the engineering of vascularized BM microenvironment models and summarizing current approaches, including bioengineered microfluidic chips.

[0014] Cornelia Lee-Thedieck et al. (in The extracellular matrix of hematopoieticstem cell niches, Advanced Drug Delivery Reviews, Volume 181, 2022, 114069, https: / / doi.org / 10.1016 / j.addr.2021.114069) reviewed that hematopoietic stem cells (HSCs) are the lifelong source of all types of blood cells. Their functions are controlled by their immediate microenvironment, the HSC microenvironment in the bone marrow. Although the importance of the extracellular matrix (ECM) in the microenvironment to coordinate microenvironment architecture and cell function is well known, it is still under exploration. In this review, they provide a comprehensive review of the ECM in the HSC microenvironment. For this purpose, they briefly outlined the biology of the HSC microenvironment, and then reviewed the roles of different types of ECM molecules in the microenvironment and how they are sensed by cells. Matrix remodeling and the increasing importance of biophysics in the function of the HSC microenvironment were discussed. Finally, we review the application of the current knowledge of the ECM in the microenvironment in the form of artificial HSC microenvironments for HSC expansion or targeted differentiation, as well as drug testing.

[0015] Due to differences in hematopoiesis between mice and humans, mouse models often fail to fully recapitulate human phenotypes, and alternative approaches are needed to study the genetic causes and mechanisms of bone marrow disease. Previous studies on modeling the in vitro microenvironment of human bone marrow have relied on the use of primary endothelial and mesenchymal cells, but these do not reflect the multicellular complexity of the natural microenvironment system, and their wider application is limited by the supply and limited lifespan of these cells (4-6).

[0016] As can be seen from the above, the demand for in vitro methods that can replicate hematopoiesis in a complex bone marrow-like microenvironment system to study hematopoietic diseases and develop new therapies has not been met. Therefore, the purpose of the present invention is to provide various approaches and methods for establishing a suitable system to study hematopoietic diseases and develop new therapies. After studying the current specification of the present invention, other objects and advantages will become clear. Summary of the invention

[0017] In its first aspect, the present invention solves the above-mentioned problems by providing a method for preparing mature mammalian bone marrow organoids, the method comprising the following steps, a) generating embryoid bodies from substantially single induced pluripotent stem cells (iPSCs) obtained from at least one mammal, comprising culturing the single iPSC in a polymerizing medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day, and thereafter culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells in the absence of the ROCK inhibitor for about 48 hours; b) inducing mesoderm in the embryoid bodies formed in step a), comprising culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells for about 48 hours, and resuspending the mesoderm-induced embryoid bodies every about 24 hours, wherein the culture 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 culture medium in b) with a medium supplemented with 80 ng / ml VEGF, 25ng / ml fibroblast growth factor (FGF)-2, 50ng / ml stem cell factor (SCF), and 2μM SB431542 in Essential6 medium for about 48 hours, resuspending the embryoid bodies and gently shaking them about every 24 hours; d) embedding the mesoderm-induced embryoid bodies in step c) into appropriate polymerized 3D collagen I / The medium was then supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and 2 μM SB431542. The medium is covered with the matrix for about 48 hours, and then the cytokines are replaced with 50ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours, and the cytokines are boosted to 25ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours; e) extracting the individual vascular networks generated in step d) and growing them in medium supplemented with 25ng / ml VEGF, 50ng / ml SCF, 50ng / ml IL-3, 50ng / ml Flt-3L and 5ng / ml TPO. The cells are cultured in a culture medium, and the culture medium is refreshed every 3-4 days for about 7-11 days, thereby generating mature mammalian bone marrow organoids, wherein preferably germline-directed cytokines such as EPO, IL-6 and / or G-CSF are not used in the method. Preferably, the mammalian bone marrow organoids are mature organoids, that is, they display the main in vivo physiological characteristics of the respective tissue.

[0018] In its second aspect, the present invention solves the above problems by providing a vascular network or mammalian bone marrow organoid, preferably a mature organoid, prepared 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. Also provided is the use of the vascular network or mammalian bone marrow organoid, preferably a mature organoid, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid of the present invention in the treatment of diseases.

[0019] In its third aspect, the present invention solves the above-mentioned problem by providing the use of the vascular network or mammalian bone marrow organoid or pharmaceutical composition of the present invention as a model system for the pathogenesis of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

[0020] In its fourth aspect, the present invention solves the above problems by providing the use of the vascular network or mammalian bone marrow organoid or pharmaceutical composition of the present invention as a model system for identifying and / or testing pharmaceutically effective compounds for treating or preventing bone marrow-related diseases, wherein bone marrow-related diseases are such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, diseases associated with HIV, sickle cell disease, and chemotherapy or infusion complications. Another aspect of this embodiment is a method for screening pharmaceutically effective compounds for treating or preventing bone marrow-related diseases, comprising using the vascular network or mammalian bone marrow organoid or pharmaceutical composition of the present invention as a model system, wherein bone marrow-related diseases are such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, diseases associated with HIV, sickle cell disease, and chemotherapy or infusion complications.

[0021] In its fifth aspect, the present invention solves the above-mentioned problem by providing an assembly comprising the vascular network or mature mammalian bone marrow organoid of the present invention, and at least one other organoid derived from iPSCs.

[0022] In its sixth aspect, the present invention solves the above problems by providing the use of the vascular network or mammalian bone marrow organoid or pharmaceutical composition of the present invention for preparing BMO or mammalian blood cells, especially autologous BMO or mammalian blood cells in vitro for transplantation purposes.

[0023] In its seventh aspect, the present invention solves the above problems by providing a pharmaceutically effective amount of the vascular network or mature mammalian bone marrow organoid or assembly or pharmaceutical composition of the present invention for use in the treatment of bone marrow-related diseases, preferably in transplantation, wherein the bone marrow-related diseases are such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, diseases associated with HIV, sickle cell disease, and chemotherapy or transfusion complications. Another aspect of the current embodiment is a method for treating bone marrow-related diseases, comprising administering a pharmaceutically effective amount of the vascular network or mammalian bone marrow organoid or pharmaceutical composition of the present invention to a subject in need thereof, wherein the bone marrow-related diseases are such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, diseases associated with HIV, sickle cell disease, and chemotherapy or transfusion complications. Preferably, the vascular network or mammalian bone marrow organoid or assembly or pharmaceutical composition of the present invention is administered as a transplant, such as an autologous transplant. Another aspect of the current embodiments is a method for transplanting a vascular network or mammalian bone marrow organoid or pharmaceutical composition of the present invention into a mammalian subject in need thereof, preferably to treat bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immune deficiencies, HIV-related diseases, sickle cell disease, and / or chemotherapy or infusion complications.

[0024] The present invention presents a new approach for preparing bone marrow organoids (BMO) (the term will include prepared bone marrow organoids and mature bone marrow organoids when it appears), which simulates the important structures and cell features of the human bone marrow microenvironment. These BMOs are generated only by human induced pluripotent stem cells (hiPSC), with hematopoiesis, matrix (mesenchyma) and vascular cavity. The inventors show that the system induces myeloid blood cells such as neutrophils, eosinophils and basophils and monocytes, as well as megakaryocyte lineages, mast cells, dendritic cells, lymphoid progenitors and early erythroid progenitors. In addition, the inventors can show that the matrix compartment is composed of mesenchymal stem cells and progenitor cells, such as reticular (CAR) cells and perivascular cells rich in CXCL12, and the vascular compartment is composed of self-assembled, connected and lumen-forming endothelial cells.

[0025] Khan et al. (Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discov. 2023Feb6; 13(2): 364-385. doi: 10.1158 / 2159-8290. CD-22-0199. PMID: 36351055; PMCID: PMC9900323) describe a step-by-step directed differentiation protocol in which organoids are generated from iPSCs differentiated into mesenchymal, endothelial, and hematopoietic lineages. These 3D structures reportedly capture the main features of human bone marrow stroma, lumen-forming sinusoidal vessels, and bone marrow cells, including platelet-forming megakaryocytes. These organoids reportedly support the implantation and survival of cells derived from patients with hematologic malignancies, including cancer types that are notoriously difficult to maintain in vitro. Fibrosis of organoids occurred after TGFβ stimulation and implantation of cells derived from myelofibrosis rather than healthy donors. Khan et al. did not mention the exact concentration used in their protocol.

[0026] Importantly, no Wnt-activators (e.g., CHIR99021) or Nodal inhibitors (e.g., SB431542) were used, which have been shown to be important for mesodermal patterning leading to the induction of definitive hematopoiesis. + ) depends on stage-specific activin-nodal signaling and inhibition of the Wnt-β-catenin pathway, which determines the progenitor cells (KDR+CD235a - ) requires Wnt-β-catenin signaling in the 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 plu-ripotent stem cells. Nat. Biotechnol. 32, 554–561 (2014)).

[0027] At the same time, the use of lineage-directed cytokines such as EPO, IL-6, and G-CSF in Khan et al. hinders the possibility of studying intrinsic cytokine signaling within bone marrow organoids and may pose an obstacle for modeling inherited bone marrow failure syndromes due to some possible rescue effects of these cytokines.

[0028] In the present invention, the maturation of neutrophils within BMOs was performed without the addition of recombinant cytokines, reminiscent of the in vivo situation (see below).

[0029] Therefore, structurally, the present invention is different from Khan et al. in that, for example, blood vessels are composed of arterial endothelial cells rather than sinusoids. This is crucial because long-term HSC has been shown to be produced by arterial hemogenic endothelial cells (Calvanese et al., Nature 2022). For the mesenchymal cell compartment, the desired heterogeneous mesenchymal cell type composition was found, including mesenchymal stem / progenitor cells that produce osteoblasts, cartilage and fat precursor cells. For hematopoietic cells, scRNA sequencing was found to be possible to differentiate into all hematopoietic lineages, rather than Khan et al.'s clear evidence of only differentiating into red bone marrow cells. Finally, Khan et al. did not transplant, and relatively, the present application organoids can be transplanted into NOD-SCID mice and show human CD45+ implantation in mouse bone marrow, indicating the HSC characteristics of BMO-derived cells.

[0030] The organoids according to the present invention exhibit a consistent spherical morphology during differentiation (see Figure 1 ). This could also be reproduced using a different iPS cell line derived from kidney epithelial cells.

[0031] Therefore, the inventors developed an improved protocol that allows for the reliable preparation of mammalian bone marrow organoids, in particular mature mammalian bone marrow organoids derived solely from iPSCs and composed of blood cells and various microenvironmental cells (e.g., endothelial cells and mesenchymal cells). The method according to the present invention comprises the following steps: a) generating embryoid bodies from substantially single induced pluripotent stem cells (iPSCs) obtained from at least one mammal, comprising culturing the single iPSC in a polymerizing medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day, and then culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells for about 48 hours in the absence of the ROCK inhibitor; b) inducing mesoderm in the embryoid bodies formed in step a), comprising culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells for about 48 hours, resuspending the mesoderm-induced embryoid bodies every about 24 hours with gentle shaking, wherein the culture 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 culture medium in b) with a medium supplemented with 80 ng / ml VEGF, 25ng / ml fibroblast growth factor (FGF)-2, 50ng / ml stem cell factor (SCF), and 2μM SB431542 in Essential 6 medium for about 48 hours, resuspending the embryoid bodies about every 24 hours; d) embedding the mesoderm-induced embryoid bodies in step c) into a suitable polymerized 3D collagen I / The cells were then treated with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and 2 μM SB431542. The medium is covered with the matrix for about 48 hours, followed by replacement of cytokines with 50ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours, and cytokine boost to 25ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours; e) extracting the individual vascular networks generated in step d) and growing them in medium supplemented with 25ng / ml VEGF, 50ng / ml SCF, 50ng / ml IL-3, 50ng / ml Flt-3L, and 5ng / ml TPO; The mammalian bone marrow organoids are cultured in a medium, and the medium is refreshed every 3-4 days for about 7-11 days, thereby generating mammalian bone marrow organoids, preferably without the use of germline-directed cytokines such as EPO, IL-6 and / or G-CSF. Preferred is a method for preparing a mammalian bone marrow organoid of the present invention, further comprising the step of culturing the mature organoids for up to about 47 days, preferably about 60 days.

[0032] The present invention provides a vascularized BMO preparation or system derived only from iPSC (particularly hiPSC), which simulates hematopoiesis in a multicellular environment containing blood cells and various bone marrow "microenvironment cells" (e.g., endothelial cells and mesenchymal cells) of natural bone marrow. The system makes it possible to model bone marrow-related diseases, i.e., to study the possibility of interaction between microenvironment cells and hematopoietic cells in the pathogenesis of blood diseases. This can also be achieved in the context of drug testing, i.e., to study the effects of drugs on the system, which makes it possible to test new therapies in a complex mammalian (e.g., human) in vitro model system of the present invention. It can also be performed in a high-throughput format, such as in a 96-well plate format. In addition, the prepared "product" can be used for therapeutic applications, i.e., bone marrow-related diseases, such as blood diseases. The method provides the possibility of transplanting the generated human BMO or improving the preparation of human blood cells (e.g., autologous patient-specific preparation) in vitro by iPSC and subsequent infusion or transplantation.

[0033] Ex vivo expansion of HSCs for stem cell transplantation has not been feasible so far. Advantageously, the vascular networks or mature mammalian bone marrow organoids according to the present invention retain the stemness of hematopoietic stem cells (HSCs), allowing the HSCs to be maintained in vitro for further quality control studies and to be sorted for therapeutic applications for only those cells that exhibit the desired molecular changes without any adverse side effects.

[0034] Preferred is a method for preparing mammalian bone marrow organoids according to the invention, further comprising the steps of fixing the vascular network in step e) and performing immunofluorescence analysis, and / or separating the vascular network into single cells and performing flow cytometry analysis and / or live cell imaging. This can be performed to characterize the composition of the developed network, and / or to study any effects of drugs to be tested (see also below).

[0035] Preferred is a method for preparing mammalian bone marrow organoids according to the invention, further comprising the steps of fixing the generated (mature) organoids and performing immunofluorescence analysis, and / or separating the mature organoids into single cells and performing flow cytometry analysis and / or live cell imaging. This can be performed to characterize the composition of the developed organoids (e.g. mature), and / or to study any effects of drugs to be tested (see also below).

[0036] In the first step of the method of the present invention, embryoid bodies are generated from essentially single / individual induced pluripotent stem cells (iPSCs) obtained from at least one mammal. Individual cells can be obtained in some way, such as by mechanical dissociation (e.g., using a pipette, etc.). Nevertheless, it is preferred that the method for preparing mammalian bone marrow organoids according to the present invention, wherein the single / individual iPSCs are obtained by using Digestion provides for the dissociation of iPSC cells into single cells, which is a gentler process. Gentle on cells, auto-inhibits at 37°C, and does not require neutralizing agents such as trypsin. Commercially available from Sigma, it is suitable for all mammalian iPSCs. The method further comprises culturing the individual / single iPSC in the polymerizing medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day.

[0037] Preferred is the method for preparing mammalian bone marrow organoids of the present invention, wherein the polymerization 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.

[0038] In the method for preparing mammalian bone marrow organoids according to the present invention, in the first step, cells are also cultured in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor. ROCK inhibition enables the maintenance of the stem cell phenotype; its effect on metabolism is not yet clear.

[0039] Preferred is a method for preparing mammalian bone marrow organoids according to the present invention, wherein the ROCK inhibitor is selected from Y-27632 ((1R, 4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide), fasudil and TS-f ROCK inhibitor disclosed by Shen et al. (Shen, M., Tian, ​​S., Pan, P. et al. Discovery of Novel ROCK1 Inhibitors via Integrated Virtual Screening Strategy and Bioassays. Sci Rep 5, 16749 (2015). https: / / doi.org / 10.1038 / srep16749, 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 corresponding literature.

[0040] Typically and preferably, step a) is performed for 60 to 84 hours, preferably about 72 hours, ie about 3 days, as described above, and after about 24 hours the culture medium is replaced with a suitable culture medium without Rock inhibitor (eg TeSRplus).

[0041] In the context of the present invention, the term "about" means a deviation of + / - 10% from a given value, unless otherwise stated.

[0042] In the context of the present invention, cultivation is generally performed at about 37°C, unless otherwise stated.

[0043] In the second step of the method of the present invention, mesoderm is induced in the embryoid bodies formed in step a). This step includes culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells and human iPSCs.

[0044] Preferably, the serum-free stable cell culture medium suitable for maintaining and expanding human embryonic stem cells without a feeder layer is Plus medium (STEMCELL Technologies, Fisher Scientific). The medium was 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).

[0045] For example, GSK3 inhibition by CHIR99021 promoted mESC self-renewal by stabilizing cytoplasmic β-catenin, an essential component of the canonical Wnt signaling pathway, and abolished TCF3-mediated transcriptional repression of pluripotency-associated genes, including Oct4, Nanog, Tfcp2l1, and Esrrb. Therefore, preferred is the method for preparing mammalian bone marrow organoids of the present invention, wherein the glycogen synthase kinase (GSK) 3 inhibitor is CHIR99021 or NPD13432, an auron derivative (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 used in combination with MEK inhibitors (such as PD0325901, referred to as "2i") that support long-term self-renewal of mouse embryonic stem cells (mESCs), and blockade of the MEK / ERK pathway by PD0325901 increases the expression of Nanog, Tfcp2l1, and Klf4 in mESCs, thereby promoting self-renewal.

[0046] Typically and preferably, step b) is performed for 36 to 60 hours, preferably about 48 hours, ie about 2 days, wherein the mesoderm-induced embryoid bodies are gently mixed or resuspended about every 24 hours.

[0047] In the third step of the method of the present invention, the medium in 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 TGF-β / activin / NODAL pathway inhibitor, preferably 2 μM SB431542 (STEMCELL Technologies). SB431542 is a selective and potent inhibitor of the TGF-β / activin / NODAL pathway, which inhibits ALK5 (IC) by competing for ATP binding sites. 50 =94nM), ALK4 (IC 50=140 nM) and ALK7. It does not inhibit the BMP type I receptors ALK2, ALK3, and ALK6. Essential 6 Medium (Thermo Fisher) is a feeder-free, xeno-free medium that supports the reprogramming of somatic cells and the spontaneous or directed differentiation of human pluripotent stem cells (PSCs).

[0048] Typically and preferably, step c) is performed for 36 to 60 hours, preferably about 48 hours, ie about 2 days, wherein the embryoid bodies are gently mixed or resuspended about every 24 hours and placed on a rocking shaker.

[0049] The fourth step of the method of the present invention includes embedding the mesoderm-induced embryoid bodies generated in step c). Embedding the embryoid bodies in a suitable polymeric 3D matrix can prevent the embryoid bodies from sinking to the bottom of the culture dish or well, which will hinder or affect their further development into mature embryoid bodies and vascular structures. The extracellular matrix (ECM) also provides biochemical cues and structural support, such as porosity and hardness, which mediate signal transduction for cell migration, cell behavior, and polarization of organoid structures. In general, any suitable polymeric 3D matrix can be used. The present invention preferably uses collagen I / in a culture container (e.g., a vial, a culture dish, or a well). For example, first prepare 500 μl / well of collagen The mixture was polymerized at 37°C for about 1 hour as the bottom layer. The embryoids were then resuspended in 500 μl / well of the same collagen I-Matrigel mixture to prepare the second layer, which was also polymerized at 37°C for about 1 hour. The two layers were then covered with an appropriate culture medium, such as Culture medium as described above supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and an appropriate TGF-β / activin / NODAL pathway inhibitor, preferably 2 μM SB431542 (STEMCELL Technologies). SFM (e.g. from Thermo Fisher) is a serum-free medium specifically formulated to support the development of human hematopoietic cells in culture (Burridge PW, et al. A universal system for highly efficient cardiac differentiation of human induced pluripo-tent stem cells that eliminates interline variability. PLoS One. 2011 Apr 8; 6(4): e18293. doi: 10.1371 / journal.pone.0018293. PMID: 21494607; PMCID: PMC3072973).

[0050] Typically and preferably, the first part of step d) is carried out for 36 to 60 hours, preferably about 48 hours, i.e. about 2 days. Then, cytokine replacement is carried out with the same culture medium containing 50ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L and 5ng / mL TPO. Typically and preferably, the second part of step d) is carried out for 36 to 60 hours, preferably about 48 hours, i.e. about 2 days. Finally, in the same culture medium, cytokines are strengthened to 25ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L and 5ng / mL TPO. Typically and preferably, the third part of step d) is carried out for 36 to 60 hours, preferably about 48 hours, i.e. about 2 days. At the end of the fourth step of the method of the present invention, a vascular network is generated. These also already contain hematopoietic cells and are therefore referred to as "germinating embryoid bodies".

[0051] In the fifth step of the method of the present invention, the individual vascular networks generated in step d) are extracted / isolated, for example, by transferring them to one well of a low-attachment 96-well plate and then incubating in a flask 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 vascular network is cultured in the culture medium for about 6 to 8 days, preferably about 7 days. The culture medium is renewed every about 3 to 4 days.

[0052] Then, mature mammalian bone marrow organoids according to the present invention are generated. The present method advantageously uses Wnt activators (e.g., CHIR99021) and Nodal inhibitors (e.g., SB431542), which are important for causing the mesoderm patterning that determines hematopoietic induction, and avoids lineage-directed cytokines, such as EPO, IL-6, and G-CSF. However, EPO can be used in the later stage to generate more mature erythroid cells.

[0053] Preferably, the method for preparing mammalian bone marrow organoids of the present invention, wherein the matrix used contains 3D collagen I and The ratio of is about 1:1 to 4:1, preferably about 3:1 or 1.2:1. Derived from the mouse Engelbreth-Holm-Swarm sarcoma cell line, which currently hinders cGMP-compliant BMO manufacturing. Xeno-free synthetic scaffold materials 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), which can be advantageously included in the methods of the present invention.

[0054] Also preferred is the method for preparing mammalian bone marrow organoids according to the present invention, wherein the resuspension of the mesoderm-induced embryoid bodies comprises the use of a pipette.

[0055] Preferred is a method for preparing mammalian bone marrow organoids according to the invention, wherein the dissociation and / or extraction and separation of individual vascular networks is mechanical and preferably comprises the use of sterile dissection tools.

[0056] In the method for preparing mammalian bone marrow organoids of the present invention, the mammal is preferably selected from humans, mice, monkeys, rats, pigs, dogs, cats, rabbits, sheep, cattle, horses, and goats. Most preferably, it is humans, thereby providing hiPSCs.

[0057] The methods of the invention may also be performed in a high-throughput format, for example in a 96-well plate format.

[0058] The methods of the present invention may also include the step of testing and / or analyzing the generated BMO for its suitability as a pharmaceutical product for transplantation and other therapeutic purposes.

[0059] The method according to the invention may also include the step of isolating cells and cell types from the generated BMO. In this case, the BMO can be used as a source of functional endothelial cells, pericytes, hematopoietic stem cells and mesenchymal stem cells. Preferably, the method according to the invention further includes isolating mesenchymal stem cells (CD45 - CD31 - CD34 - CD90 + CD105 + CD271 + CD73 + ) or HSC. This can preferably be done by cell sorting, such as FACS. In the context of the present invention, FACS sorted MSCs were found to have the ability to differentiate into osteoblasts, chondrocytes and adipocytes in a tri-lineage differentiation assay, which was visualized by Alizarin Red-S, Alcian Blue and Oil Red-O staining, respectively. Sorted BMO-derived MSCs expanded in culture and showed continuous re-cultivation ability.

[0060] Another aspect of the invention relates to preparations of generated HSCs or MSCs.

[0061] Another aspect of the present invention relates to a vascular network or mature mammalian bone marrow organoid generated according to the method of the present invention, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid.

[0062] Another aspect of the present invention relates to an assembly comprising a vascular network or a mature mammalian bone marrow organoid according to the present invention, and at least one additional iPSC-derived organoid. Preferred is an assembly according to the present invention comprising immune cells, blood vessels and pericytes.

[0063] Since the lack of immune cells and blood vessels is a common limitation of organoid models, the present invention includes combining current BMOs with other iPSC-derived organoids to form so-called assemblies that integrate immune cells, blood vessels, and pericytes (see, e.g., Sharma, A., Sances, S., Workman, M. J. & Svendsen, CN Multi-lineage Human iPSC-Derived Platforms for Disease Modeling and Drug Discovery. Cell Stem Cell 26, 309–329 (2020)., Kanton, S. & SPHuman assembloids. Development 149, dev201120 (2022)). Although previous methods require mixing these cell types (for example, see Wang, L. et al. A human three-dimen-sional 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 more reasonably models developmental and functional interactions in a spatial context. In addition, the combination of bone marrow organoids with other organoids has the advantage of containing not only vascular cells but also hematopoietic cells.

[0064] Also provided is the use of a vascular network or mammalian bone marrow organoid, preferably a mature organoid, generated according to the method of the invention, an assembly according to the invention, or a pharmaceutical composition of the invention comprising a vascular network and / or mature mammalian bone marrow organoid and / or assembly, in the treatment of a disease.

[0065] The pharmaceutical composition used may optionally contain a pharmaceutically acceptable carrier. Those skilled in the art are aware of suitable formulations for cells and cell products, and can easily select suitable pharmaceutically acceptable carriers or excipients, for example, according to the formulation and route of administration of the pharmaceutical composition.

[0066] Pharmaceutically acceptable carriers or excipients include diluents (fillers, expanders, such as lactose, microcrystalline cellulose), disintegrants (such as sodium starch glycolate, crosslinked sodium carboxymethyl cellulose), binders (such as PVP, HPMC), lubricants (such as magnesium stearate), glidants (such as colloidal SiO2), solvents / cosolvents (such as aqueous carriers, propylene glycol, glycerol), buffers (such as citrate, gluconate, lactate), preservatives (such as sodium benzoate, parabens (Me, Pr and Bu), BKC), antioxidants (such as BHT, BHA, ascorbic acid), wetting agents (such as polysorbates, sorbitan esters), thickeners (such as methylcellulose, or hydroxyethylcellulose), sweeteners (such as sorbitol, saccharin, aspartame, acesulfame), flavoring agents (such as mint, lemon oil, butterscotch, etc.), humectants (such as propylene glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable excipients are described in particular in Remington's Pharmaceutical Sciences, 15 thEd., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5 th Ed., Govi-Verlag Frankfurt (1997).

[0067] The pharmaceutical composition 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 infusion, for example in the form of injection or drip.

[0068] In addition to the above-mentioned products of the present invention, the pharmaceutical composition may also contain other conventional, usually inert carrier materials or excipients. Therefore, the pharmaceutical preparation may also contain additives, such as fillers, extenders, disintegrants, binders, glidants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatics, buffer substances, and solvents or solubilizers or agents for achieving storage effects, and salts, coating agents or antioxidants for changing osmotic pressure. They may also contain other therapeutically active substances as described herein.

[0069] The present invention provides a vascularized BMO preparation or system (e.g., an assembly according to the present invention) derived solely from iPSCs (particularly hiPSCs) that mimics hematopoiesis in the multicellular environment of natural bone marrow comprising blood cells and a variety of bone marrow "microenvironmental cells" (e.g., endothelial cells and mesenchymal cells).

[0070] The prepared products can be used in two strategies; as a research tool, such as studying the interaction between microenvironment cells and hematopoietic cells in the pathogenesis of blood diseases. 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 be further used to test cell therapies, such as CAR-T cells or antibodies or combinations of T cells and antibodies (BiTe) for, for example, leukemia stem cells (LSC), or its effects on HSC. In addition, the system can be used as a tool for screening and identifying new drugs for blood diseases and other diseases disclosed herein. In the second strategy, the generated "products" and compositions themselves can be used as therapeutic agents, i.e., for preventing and / or treating bone marrow-related diseases, such as blood diseases and other diseases disclosed herein. This strategy involves the transplantation of generated human BMOs, or the improved generation of human blood cells (e.g., autologous patient-specific production) by iPSC in vitro and the possibility of subsequent infusion or transplantation. The present invention has the advantage of maintaining the stemness of hematopoietic stem cells, which are usually lost when cultured in vitro with cytokines. When stem cell properties are retained, single cell studies can be performed and stem cells with desired genetic modifications can be selected.

[0071] Preferred are vascular networks or mature mammalian bone marrow organoids or assemblies prepared according to the present invention, which are models for bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immune deficiencies, diseases associated with HIV, sickle cell disease, and chemotherapy or infusion complications. In these cases, the model is generated on iPSCs that are mutated or modified to reflect the origin of the bone marrow-related disease, and then these cells are used to generate organoids. However, the organoid or assembly or network or certain cellular components thereof can also be modified to reflect the origin of the bone marrow-related disease to be analyzed.

[0072] Therefore, another aspect of the present invention relates to a method for identifying a pharmaceutically active compound for a bone marrow-related disease, comprising the steps of: a) providing a vascular network or mature mammalian bone marrow organoid or assembly prepared according to the present invention, which is a model for at least one bone marrow-related disease, b) contacting the vascular network or mature mammalian bone marrow organoid or assembly of step a) with at least one potential pharmaceutically active compound, and c) identifying a physiological effect that reflects or indicates treatment or improvement of the bone marrow-related disease in the presence of the at least one potential pharmaceutically active compound when compared to a control in the absence of the at least one potential pharmaceutically active compound, wherein the effect identifies a pharmaceutically active compound for the bone marrow-related disease. The bone marrow-related disease can be, for example, a blood disease, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital hematopoietic and immune deficiencies, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

[0073] Preferred are methods or uses according to the invention, wherein the contacting is in vivo or in vitro, in solution or comprises the candidate compound bound or conjugated to a solid support. Various formats are also described in the art and are known to those skilled in the art.

[0074] In the context of the present invention, anti-aging candidate compounds can be selected from any suitable appropriate molecules, such as chemical organic molecules, molecules selected from organic small molecule libraries (molecular weight less than 500 Da), molecules selected from combinatorial libraries, cell extracts, especially plant cell extracts, small molecule drugs, proteins, protein fragments, molecules selected from peptide libraries, antibodies or fragments thereof.

[0075] These candidate molecules can also be used as the basis for screening for improved compounds (see below).

[0076] In the context of the present invention, any method suitable for detecting the effect of a compound may be used. Corresponding methods are known to those skilled in the art and are disclosed in the art. The components of the assays disclosed herein may be labeled, for example using radioactive or fluorescent labels or with antigenic labels.

[0077] The present invention also relates to identifying the improved compounds identified in the first round of screening / identification. After providing the identified compound, the compound can be modified. Generally speaking, many methods of how to modify the compounds of the present invention are known to those skilled in the art and are recorded in the literature. The modification of the compound is generally divided into several categories, such as a) chemical modification, such as 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, joints or carriers such as chelating agents). All of these modifications are presented as a new strategy, any of which or a combination thereof ultimately leads to the "rational design" of the improved compounds used in the context of the present invention. The present invention also includes further strategies for improving compounds that are only partially subjected to "directed evolution" or "directed mutagenesis", i.e., the compound can undergo multiple continuous rounds of the above-mentioned method.

[0078] In the next step, the modified compound is tested for a change in the physiological effect reflecting or indicating the treatment or improvement of the bone marrow-related disease in the presence of the at least one potential pharmaceutically active compound compared to the absence of the at least one potential pharmaceutically active compound, compared to the unmodified pharmaceutically active compound or a control.

[0079] Therefore, another aspect of the present invention relates to a pharmaceutically active compound for bone marrow-related diseases identified according to the present invention, or a pharmaceutical composition comprising the pharmaceutically active compound. This aspect also includes a method for preparing a pharmaceutical composition containing a pharmaceutically active compound for bone marrow-related diseases identified according to the present invention, comprising formulating the compound with a suitable diluent and / or carrier. Generally speaking, the same conditions are applied to the pharmaceutical composition.

[0080] Another aspect of the invention relates to the use of the vascular network or mature mammalian bone marrow organoid or assembly or pharmaceutical composition of the invention (comprising the identified pharmaceutically active compound) as a model system for identifying and / or testing pharmaceutically effective compounds for treating or preventing bone marrow related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immune deficiencies, diseases associated with HIV, sickle cell disease, and chemotherapy or infusion complications. Identification and testing are generally as described above.

[0081] Another aspect of the invention relates to the use of the vascular network or mature mammalian bone marrow organoid or assembly or pharmaceutical composition of the invention (comprising the identified pharmaceutically active compound) as a model system for the pathogenesis of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications. As described above, in these cases, the model used can be generated on iPSCs that are mutated or modified to reflect the source of the bone marrow-related disease, and then these cells are used to generate organoids. However, the organoid or assembly or network or certain cellular components thereof can also be modified to reflect the source of the bone marrow-related disease to be analyzed. The model system can also be used to test the suitability of BMOs as pharmaceutical preparations for transplantation and other therapeutic purposes.

[0082] Another aspect of the present invention relates to the use of the vascular network or mature mammalian bone marrow organoid or assembly or pharmaceutical composition of the present invention for the in vitro preparation of BMO or mammalian blood cells, especially autologous BMO or mammalian blood cells, especially for transplantation.

[0083] Another aspect of the present invention relates to the use of the vascular network of the present invention or mature mammalian bone marrow organoids or assemblies in maintaining the stemness of hematopoietic stem cells (HSC). It is advantageous to keep HSC in vitro for further quality control studies and to sort out only cells that show the desired molecular changes without any adverse side effects (such as off-target editing or oncogene activation, etc.) for therapeutic applications. The advantage of the present invention is to maintain the stemness of hematopoietic stem cells, which are usually lost when cultured in vitro with cytokines. When stemness is retained, single cell studies can be performed and stem cells with desired genetic modifications can be selected.

[0084] Another aspect of the present invention relates to the use of a pharmaceutically effective amount of the vascular network or mature mammalian bone marrow organoid or assembly or pharmaceutical composition of the present invention (including the identified pharmaceutically active compound) in the treatment or prevention of bone marrow-related diseases, preferably in transplantation, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

[0085] Another aspect of the present invention relates to a method for preventing or treating a bone marrow-related disease in a subject, comprising administering to the subject an effective amount of the vascular network or mature mammalian bone marrow organoid or assembly of the present invention or a pharmaceutical composition (comprising the identified pharmaceutically active compound), wherein the bone marrow-related disease is, for example, a blood disease, severe congenital neutropenia, myelofibrosis, blood cell cancer, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications. Preferably, the method comprises transplanting an effective amount of the vascular network or mature mammalian bone marrow organoid or assembly of the present invention.

[0086] It should be understood that the product, compound and / or pharmaceutical composition can be used for administration to human patients. The term "administering" refers to the administration of a single therapeutic agent or a combination with another therapeutic agent. Therefore, it is envisioned that the pharmaceutical composition of the present invention can be used in a combined treatment method, i.e., co-administered with other drugs or medicaments and / or any other therapeutic agents that may be beneficial in the context of the present method. However, if desired, other drugs or medicaments and / or any other therapeutic agents can be administered separately from the compound of use, as long as they act in conjunction with the compound of the current use (i.e., directly and / or indirectly, preferably synergistically).

[0087] The compounds of the invention can therefore be used alone or in combination with other active compounds - for example with known drugs and therapies for the treatment of the above-mentioned diseases, in which case advantageous additive, potentiating or preferably synergistic effects are observed.

[0088] As described herein, the product or compound is administered to the subject in an effective dose. The dose can vary over a wide range and is suitable for the individual condition of each case. For the above-mentioned purposes, the appropriate dose will vary according to the mode of administration, the specific condition to be treated, and the desired effect. However, in general, satisfactory results are achieved at the dose rate as described above, such as about 1 to 100 mg / kg animal body weight, particularly 1 to 50 mg / kg. For larger mammals, such as humans, the appropriate dose rate is conveniently administered once or in divided doses, such as 2 to 4 times a day, or in a sustained release form, at the order of about 10 mg to 3 g / day. In general, in the case of oral administration, each human individual daily dose of about 10 mg to 100 mg, particularly 10 to 50 mg is suitable. The effective concentration to be achieved at the cellular level can be set between 50 and 200 μM, preferably about 100 μM.

[0089] Another aspect of the present invention relates to a kit, such as a diagnostic kit, comprising materials for implementing the method of the present invention. The materials are a set of culture media, such as the polymerized culture medium containing a Rho-associated protein kinase (ROCK) inhibitor in step a) herein, the serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells supplemented in step b) herein, the Essential 6 culture medium supplemented in step c) herein, and the polymerized 3D collagen I / I supplemented in step d) herein. The material of the substrate and Culture medium.

[0090] The kit may also include relevant antibodies, dyes and other labels that bind to cell markers of BMO, as well as buffers and matrices for performing the above methods.

[0091] The kit can be used in the methods of the present invention, i.e. for identifying and / or testing pharmaceutically effective compounds for treating or preventing bone marrow-related diseases, as a model system for the pathogenesis of bone marrow-related diseases, and / or for preparing BMO or mammalian blood cells in vitro, in particular autologous BMO or mammalian blood cells, in particular for transplantation.

[0092] In summary, the present invention provides a vascularized BMO system derived exclusively from iPSCs and mimics hematopoiesis in the multicellular environment of natural bone marrow.

[0093] A method for generating a complex self-organizing bone marrow organoid containing a de novo synthesized vascular network, hematopoietic cells, and stromal microenvironment cells by co-differentiation of human iPSC is also provided. BMO presents the key cellular, structural, and molecular characteristics of the natural human bone marrow microenvironment. They contain not only multipotent hematopoietic stem / progenitor cells (HSPC), but also mesenchymal stem / progenitor cells (MSPC), and model the development of fetal bone marrow. This novel organoid system can serve the study of hematopoietic development and disease evolution. As described above, a method for generating a complex assembly according to the present invention is also provided, such as (further) comprising iPSC-derived immune cells, blood vessels, and pericytes.

[0094] The present invention relates to the following items.

[0095] Item 1. A method for preparing mammalian bone marrow organoids, comprising a) generating embryoid bodies from essentially single induced pluripotent stem cells (iPSCs) obtained from at least one mammal, comprising culturing the single iPSC in a polymerizing medium in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor for about 1 day, and then culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells in the absence of the ROCK inhibitor for about 48 hours; b) inducing mesoderm in the embryoid bodies formed in step a), comprising culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells for about 48 hours, and resuspending the mesoderm-induced embryoid bodies approximately every 24 hours, wherein the culture 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 culture medium in b) with a medium supplemented with 80 ng / ml VEGF, 25ng / ml fibroblast growth factor (FGF)-2, 50ng / ml stem cell factor (SCF), and 2μM SB431542 in Essential 6 medium for about 48 hours, resuspending the embryoid bodies about every 24 hours; d) embedding the mesoderm-induced embryoid bodies in step c) into a suitable polymerized 3D collagen I / The cells were then supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and 2 μM SB431542. The medium is covered on the matrix for about 48 hours, followed by replacement of cytokines with 50ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours, and cytokine boost to 25ng / mL VEGF, 50ng / mL SCF, 50ng / mL IL-3, 50ng / mL Flt-3L, and 5ng / mL TPO for about 48 hours; e) extracting the individual vascular networks generated in step d) and growing them in medium supplemented with 25ng / ml VEGF, 50ng / ml SCF, 50ng / ml IL-3, 50ng / ml Flt-3L, and 5ng / ml TPO. The cells are cultured in culture medium, with the culture medium refreshed every 3-4 days for about 7-11 days, thereby generating mammalian bone marrow organoids, preferably without the use of germline-directed cytokines such as EPO, IL-6 and / or G-CSF.

[0096] Item 2. The method for preparing 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 single cells and performing flow cytometric analysis and / or live cell imaging.

[0097] Item 3. The method for preparing mammalian bone marrow organoids according to Item 1 or 2, further comprising the steps of fixing the generated organoids and performing immunofluorescence analysis, and / or dissociating the organoids into single cells and performing flow cytometric analysis and / or live cell imaging.

[0098] Item 4. The method for preparing mammalian bone marrow organoids according to any one of Items 1 to 3, further comprising a step of culturing the organoids for up to about 47 days, preferably about 60 days.

[0099] Item 5. A method for preparing a mammalian bone marrow organoid according to any one of Items 1 to 4, wherein the single iPSC is obtained by using Digestion is used to dissociate iPSC cells into single cells.

[0100] Item 6. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 5, wherein the ROCK inhibitor is selected from Y-27632 ((1R, 4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide) and fasudil.

[0101] Item 7. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 6, wherein the polymerization 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.

[0102] Item 8. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 7, wherein the serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells is Plus medium.

[0103] Item 9. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 8, wherein the glycogen synthase kinase (GSK) 3 inhibitor is CHIR99021.

[0104] Item 10. A method for preparing a mammalian bone marrow organoid according to any one of Items 1 to 9, wherein the 3D collagen I / The ratio of the matrix is ​​about 1:1 to 4:1, preferably about 3:1 or 1.2:1.

[0105] Item 11. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 10, wherein the resuspension of embryoid bodies / mesoderm-induced embryoid bodies comprises using a pipette.

[0106] Item 12. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 11, wherein the dissociation or extraction of individual vascular networks comprises extraction using a sterile dissection tool.

[0107] Item 13. A method for preparing mammalian bone marrow organoids according to any one of Items 1 to 12, wherein the mammal is selected from humans, mice, monkeys, rats, pigs, dogs, cats, rabbits, sheep, cows, horses and goats.

[0108] Item 14. A method for preparing mesenchymal stem cells (MSC) and / or hematopoietic stem cells (HSC), comprising performing the method of any one of Items 1 to 13 and appropriately isolating MSC or HSC from a vascular network or a mammalian bone marrow organoid.

[0109] Item 15. A vascular network or mature mammalian bone marrow organoid prepared by the method of any one of Items 1 to 13, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid.

[0110] Item 16. An assembly comprising the vascular network or mature mammalian bone marrow organoid of Item 15, and at least one other iPSC-derived organoid.

[0111] Item 17. The assembly according to 16, which comprises immune cells, blood vessels and pericytes.

[0112] Item 18. Use of the vascular network or mature mammalian bone marrow organoid of Item 15, or the assembly of Item 16 or 17, as a model system for bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and complications of chemotherapy or infusion.

[0113] Item 19. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of Item 15, or the assembly of Item 16 or 17, as a model system in the pathogenesis of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

[0114] Item 20. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of Item 15, or the assembly of Item 16 or 17, as a model system for identifying and / or testing pharmaceutically effective compounds for treating or preventing bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

[0115] Item 21. The vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of Item 15, for use in the in vitro preparation of BMO or mammalian blood cells, especially autologous BMO or mammalian blood cells, especially for use in transplantation.

[0116] Item 22. Use of a pharmaceutically effective amount of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of Item 15, or the assembly of Item 16 or 17, in the treatment of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications, preferably for use in transplantation.

[0117] Item 23. An MSC or HSC prepared by Item 14, or a pharmaceutical composition comprising the MSC or HSC.

[0118] Item 24. Use of a pharmaceutically effective amount of MSCs or HSCs or pharmaceutical compositions of Item 23 in the treatment of bone marrow-related diseases, spinal cord-related diseases such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications, preferably for use in transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] The present invention will be further described in the following examples with reference to the accompanying drawings and sequence listing, but the present invention is not limited thereto. For the purpose of the present invention, all references cited herein are incorporated by reference in their entirety.

[0120] Figure 1Figure 2 shows the generation of human iPSC-derived bone marrow organoids and analysis of cellular composition via flow cytometry. a) Schematic diagram of the process of BMO generation. b) Representative bright field 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 cytometric data of 105,000 cells stained by protein expression from three independent differentiations. d) Overlay of manually gated populations on tSNE projections. Subsets defined as CD45 - CD31 + Endothelial cells, CD45 + Hematopoietic cells, CD45 + CD11b + Myeloid cells and CD45 - CD31 - CD271 + Mesenchymal cells. CD45 + CD11b - CD34 + For HSPC, CD45 - CD31 - CD271 + CD90 + CD105 + CD73 + e) Frequency of manually gated cell types, n = 5 independent differentiations. f) Sorted and May-Gruenwald-Giemsa stained HSPC and CD45 + CD11b + Bright field microscopy of myeloid cells. High nuclear to cytoplasmic ratio typical of HSPCs. Morphology of phase contrast sorted MSPCs in culture. Representative images of n=6 experiments using two iPSC lines. iPSC, induced pluripotent stem cell; EB, embryoid body. Scale bars in b) 500 μm, day 6 inset 100 μm, f) 20 μm.

[0121] Figure 2 Figure 3 shows that the spatial architecture of BMOs recapitulates key features of the human bone marrow microenvironment. a) Confocal imaging of whole organoids reveals a densely interconnected network of CD31-expressing blood vessels and CD271-expressing stromal cells, which contain CD45-labeled hematopoietic cells. b) CD31 is shown in a) + and CD271 + CD45 in microenvironmental cells + c) Surface rendering of a 3D z-reconstruction of an organoid stained for endothelial cells (CD31) and pericytes (PDGFRβ) imaged by confocal microscopy to visualize the vascular network and PDGFRβ.+ d) Pericyte-endothelial cell association at day 10 and day 21 of differentiation. Top: PDGFRβ at day 10 of differentiation + Finger-like extensions of pericytes. Bottom: PDGFRβ at day 21 of differentiation + Pericytes are tightly associated with endothelial cells. e) The microenvironmental marker CXCL12 is expressed in specific cell subsets throughout the organoids as visualized by confocal microscopy. f) Pericytes expressing CXCL12 are tightly associated with endothelial cells. g) Nestin + Cells arrayed on CD31 + Blood vessels and CD45 + In hematopoietic cells, CD45 高 The expressing cells have ribbon-shaped nuclei. h) Two-photon microscopy shows the spatial structure of the entire organoid. The expression of the key microenvironment marker Nestin is related to CD31 + Blood vessels and CD45 + Spatial correlation of 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.

[0122] Figure 3 The vascular structure showing the main characteristics of blood vessels and surrounding hematopoietic cells. a) Endothelial cells are covered by collagen IV + (ColIV) Basement membrane coverage. b) TEM image of pericytes and endothelial cells bound by tight junctions (TJs). c) Figure 2 d) Orthogonal 2Dz projection of the vascular structure in the xz and yz directions of the bottom image. Note the lumen formation indicated by the arrow. d) TEM image of an organoid section showing capillary-like structures of endothelial cells (E) forming the lumen (L) and surrounded by pericytes (P). Asterisks mark rod-shaped (Weibel-Palade) bodies. e) Lined with Nestin-expressing cells and surrounded by CD45 + CD31 on hematopoietic cells + Confocal imaging of blood vessels. f) Surface rendering of the fluorescent image in e) to show the vascular structure. Transparent surfaces of CD31 and Nestin show CD45 in the blood vessels. + Cells. g) Tissue sections stained with hematoxylin-eosin neutrophil precursors (HE) show morphological hematopoietic cells within the lumen of vascular-like structures. h) TEM image of organoid sections showing endothelial cells (E) surrounding round cells resembling myeloid cells (M). Scale bars are, a) 10 μm, b) 1 μm, c) 20 μm, d) 2 μm, e) f) 10 μm, g) 20 μm, h) 2 μm.

[0123] Figure 4 Showing that bone marrow organoids are capable of granulopoiesis. a) Expression of S100A8 / A9 and b) myeloperoxidase (MPO) in cells with bands / segmented nuclei. Magnified images of the rectangular areas are depicted in the inset. c) MPO expression confirmed by immunohistochemistry of organoid sections. Magnified images of the rectangular areas are depicted in the inset. d) Representative TEM images of typical morphology of neutrophil-like cells within organoids. e) Gating scheme for analysis of neutrophil differentiation in dissociated BMOs at day 21 of differentiation by flow cytometry. f) Histogram of surface marker expression of different neutrophil precursor cell stages before BMO-derived neutrophils reach a mature neutrophil-like state. g) CD45 + Frequency of neutrophil precursor cell subsets in the population, n = 6 independent experiments. h) Magerge staining of sorted BMO-derived neutrophil precursor cells shows that they are highly similar in morphology to human neutrophil precursor cells in vivo. Scale bars a), b) 10 μm, c) 50 μm, inset 10 μm, d) 2 μm, h) 20 μm.

[0124] Figure 5 Single cell transcriptome analysis of BMOs identifies distinct cell populations. a) Coarse-grained clustering of scRNA-seq data reveals three major populations, including endothelial, hematopoietic, and mesenchymal cells. b) Expression of signature markers indicative of the three major populations. c) UMAP projection of a total of 31,040 cells colored according to detailed cell type annotations. d) Expression of marker genes used to annotate the hematopoietic lineage. e) Expression of marker genes for endothelial cell subtypes. f) UMAP projection of arterial endothelial cells and pre-HE cells co-expressing the displayed marker genes. g) Expression of marker genes for mesenchymal cell clusters. h) UMAP projection of different mesenchymal subpopulations co-expressing marker genes. DC, dendritic cell; ELP, early lymphoid progenitor; eo / baso, eosinophil / basophil; GMP, granulocyte / monocyte progenitor; HE, hematopoietic endothelial cell; HSC / MPP, hematopoietic stem cell / multipotent progenitor; LMPP, lymphoid primed multipotent progenitor; MEP, megakaryocyte / erythroid progenitor; MK, megakaryocyte; mono.mac., monocytic-like macrophage; PS, primitive streak.

[0125] Figure 6In vitro and in vivo functional properties of BMOs and modeling of monogenic bone marrow failure syndromes are shown. 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 Alizarin Red S, Oil Red O, or Alcian Blue staining, respectively; n=4 independent experiments using two iPS cell lines. b) FACS-sorted BMO-derived CD45 + CD11b - CD34 + Colony forming unit assay of HSPCs. CFU-GM, granulocyte-macrophage colony forming unit, BFU-E, burst erythroid forming unit, CFU-GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte colony forming unit; representative phase contrast microscopy colony morphology (left) and Magerge staining of selected colonies (right). c) Frequency of BMO HPSC-derived colonies, n=4 independent experiments using two iPS cell lines, showing the number of FiPSC-derived colonies. d) BMO xenografts performed under the kidney capsule of NSG mice. e) Macroscopic morphology showing further growth in vivo after 3.5 months. f) Hematoxylin-eosin stained section of paraffin-embedded organoids 3.5 months after transplantation, showing blood vessels filled with erythrocytes within the BMO. g) Representative images of flow cytometric analysis showing human CD45 in mouse bone marrow in transplanted mice but not in control mice. + , n=2 mice. h) Modeling of VPS45 deficiency in bone marrow organoids. Schematic diagram of gene editing and experimental setup for creating VPS45 mutant iPS cell lines with a syngeneic background. i) Histological comparison of control and VPS45 mutant BMOs by HE and Gomori staining reveals reticular fibrosis in VPS45 mutant BMOs. Two batches, n=8 organoids per condition. j) Alpha smooth muscle actin (SMA) expression in control and VPS45 mutant BMOs by immunofluorescence analysis. k) Mean fluorescence intensity (MFI) quantification of SMA expression, two batches, n=5 organoids per condition, four different areas. l) Flow cytometry shows increased expression of Annexin V on mature neutrophils (Neu) in VPS45 mutant BMOs, n=3. m) MFI quantification of Annexin on mature Neu, n=3, *p<0.05, ****p<0.0001, unpaired two-tailed t-test. Scale bars are a) 100 μm, b) 200 μm (left) and 50 μm (right), f) 20 μm, i) and j) 50 μm. DETAILED DESCRIPTION

[0126] Example

[0127] background

[0128] In 2011, a 2D system for hematopoietic differentiation was disclosed in which blood progenitor cells were generated from human induced pluripotent stem cells (hiPSCs) by continuous cytokine induction (8). At that time, a single-layer culture without blood was established that could track the in vivo hematopoietic pathway from ES / iPS cells via mesodermal progenitors to functionally committed blood cells, and exogenous cytokine mixtures were gradually adjusted to induce hematopoietic mesodermal progenitors via primitive streak cells. These progenitor cells then differentiated into various cell lineages depending on the hematopoietic cytokines present.

[0129] The protocol of (8) was modified considerably to differentiate hematopoietic progenitor cells into functional neutrophils and macrophages. To generate vascularized bone marrow organoids, the inventors implemented portions of a recently published protocol by Wimmer et al. to generate vascularized organoids (9). These hiPSC-derived vascularized organoids consisted not only of functional endothelial cells and pericytes but also contained minimal amounts of hematopoietic cells and mesenchymal stromal cells (10).

[0130] The inventors concluded that the combination of the above-described hematopoietic differentiation protocol with a three-dimensional vascularized culture system would promote the formation of complex cellular interactions leading to the formation of an architectural tissue microenvironment similar to the native bone marrow microenvironment, which surprisingly proved to be true.

[0131] Summary of the Preferred Methods of the Invention

[0132] Bone marrow organoids were generated from iPSCs by mesoderm induction and subsequent hematopoiesis induction in a 3D stromal cytokine induction system. Human iPSC embryoid bodies were cultured in low-adhesion plates with Y-27632 and grown for 2-3 days until aggregates reached a size of 0.5 μm ( Figure 1 A). 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-βRI kinase inhibitor VISB431521 (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 / (Sigma) solution to induce vascular sprouting and promote 3-dimensional cell organization. Thereafter, hematopoietic differentiation was induced with VEGF, SCF, Fms-related receptor tyrosine kinase 3 ligand (Flt-3L), interleukin 3 (IL-3) and thrombopoietin (TPO). On day 10, the vascular network was isolated / extracted and the individual networks were transferred to 96-well low-attachment plates. From day 10 to day 17, vascular networks formed into spherical BMOs and 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 ).

[0133] Detailed optimization method for preparing bone marrow organoids

[0134] Bone marrow organoids (BMOs) were generated in a 3D culture system by sequential addition of growth factors as described below.

[0135] like Figure 1 As an example, on day -3 of the method, using (Sigma) at 37°C for 5 minutes to dissociate iPSCs into single cells and generate embryoid bodies. Use a P1000 pipette to mechanically disrupt cell aggregates. The dissociation reaction was stopped with ELISA Plus (Fisher Scientific), and the cells were then collected at 300 g for 3 min at room temperature (RT).

[0136] Resuspend the cells 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 count the cells using a hemocytometer. 6 Cells were resuspended in aggregation medium supplemented with 50 μM Y-27632 and plated in low-adhesion dishes.

[0137] On day 0, cells were supplemented with 80 ng / ml BMP4, 4 μM CHIR99021, and 80 ng / ml VEGF. Plus induction of mesoderm. To replace the medium, the embryoid bodies were collected by gravity (15-30 minutes) in a 15 ml standard tube. To avoid over-fusion, the embryoid bodies were resuspended once a day and placed on a shaker. On day 2, the medium was replaced with Essential 6 medium supplemented with 80 ng / mL VEGF, 25 ng / mL FGF-2, 50 ng / mL SCF and 2 μM SB431542.

[0138] On day 4, 30-60 embryoid bodies were embedded in 1 ml / well collagen I / Collagen I solution (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 type I collagen) was prepared according to the manufacturer's protocol, and 1N NaOH was added dropwise to adjust the solution pH to 7.4.

[0139] For embedding, first prepare 500 μl / well of collagen The mixture was layered and polymerized at 37°C for 1 hour to prevent the embryoid bodies from sinking to the bottom of the culture dish. The embryoid bodies were then resuspended in 500 μl / well of collagen I-Matrigel mixture to prepare the second layer, which was polymerized at 37°C for 1 hour. Finally, the cells were covered with prewarmed 5% paraformaldehyde supplemented with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF, and 2 μM SB431542. The culture medium covers the embedded embryoid bodies.

[0140] On day 6, the 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, the 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.

[0141] On day 10, vascular networks generated by individual embryoid bodies were extracted using sterile dissection tools under laminar flow at the lowest magnification of an inverted light microscope. Individual vascular networks were then transferred and further cultured in 96-well low attachment plates. Thereafter, organoids were cultured in 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. Medium (Fisher Scientific) was added, with half of the medium replaced every 3 to 4 days. On day 10, vascular networks and on day ≥17, mature organoids were fixed for immunofluorescence analysis or dissociated into single cells for flow cytometry analysis. In this case, organoids were cultured until day 45.

[0142] Heterogeneous cell type composition

[0143] To investigate the cell type composition in detail, we analyzed the dissociated BMOs at day 17 of differentiation by flow cytometry. Figure 1 c). To visualize subset heterogeneity, we performed dimensionality reduction on flow cytometry data using t-distributed stochastic neighbor embedding (t-SNE) ( Figure 1 a). The inventors integrated the data from three independent differentiations into a joint t-SNE visualization to create a representative map across different experiments ( Figure 1 a). Overlay of manually gated populations showing three major cell clusters ( Figure 1 e). Based on CD45 as a common leukocyte antigen marker, CD31 (PECAM1) as a vascular endothelial cell (EC) marker, and CD271 (NGFR) as a mesenchymal cell marker, these clusters were assigned as endothelial cells (CD45 - CD31 + ), hematopoietic cells (CD45 + ) and mesenchymal stromal cell clusters (CD45 - CD31 - CD271 + ).

[0144] Hematopoietic cells are further defined as HSPC (CD45 + CD11b - CD34 + ) and myeloid cells (CD45 + CD11b + ). According to the minimum standard of multipotent MSPC, CD45 - CD31 - CD271 + CD90 + CD105 + CD73 +The cells are characterized as MSPC. Quantitative analysis of the cell composition on the 17th day of culture showed that each BMO (n=5) contained an average of 39.3% hematopoietic cells, 41.3% mesenchymal cells, 6% EC, 1.42% HSPC and 0.96% MSPC. This relative cell type composition remained unchanged at different time points of differentiation. To determine the reproducibility of the protocol, the inventors also used an iPS cell line derived from renal epithelial cells shed in urine (the so-called urine iPSC (UiPSC)) to generate BMO. UiPSC-derived BMO exhibited a morphology highly similar to that of fibroblast-derived iPSC (FiPSC). The main composition of hematopoietic cells, mesenchymal cells and endothelial cells was consistent, but compared with FiPSC-derived BMO, UiPSC-derived BMO showed higher endothelial cells (average content of 12.47%) and mesenchymal cells (49.15%), as well as lower hematopoietic cell (23.42%) percentages. Next, the inventors used fluorescence activated cell sorting (FACS) to sort defined cell subsets and analyzed their morphology by microscopy. + CD11b - CD34 + and CD45 + CD11b + Magerge staining of cell smears from the populations showed typical morphology of myeloid progenitor cells and monocyte-like cells, respectively. - CD31 - CD90 + CD105 + CD271 + CD73 + MSPCs adhered to plastic and had a typical spindle-like morphology, which is characteristic of MSPCs. Furthermore, these cells expanded in culture with serial reculture capacity up to 15 passages. Thus, this suggests that BMOs are composed of endothelial, hematopoietic, and mesenchymal cells, contain phenotypically defined HSPCs and MSPCs, and can be generated from both FiPSCs and UiPSCs.

[0145] Space Architecture

[0146] The inventors then analyzed the spatial distribution of different cell subsets through a series of microscopic studies. Confocal imaging showed that spherical CD45 + Hematopoietic cells embed into CD31 + Vascular structure and CD271 + To increase the imaging depth, the inventors used two-photon microscopy, which enabled the inventors to capture fluorescence 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 essential for supporting the hematopoietic microenvironment by providing nutrients and growth factors to HSCs and other microenvironmental cells in the BM. The BM microenvironment consists of different types of vessels and, therefore, different perivascular areas. Platelet-derived growth factor β (PDGFRβ) surrounding the vessels + Pericytes cover ECs, thereby supporting and promoting angiogenesis. These PDGFRβ + Pericytes are spatially associated with HSCs and osteoprogenitors and surround arteries and H-type vessels in the metaphyseal and endosteal regions of mice. Confocal imaging and 3D surface rendering of BMOs revealed that CD31 + ECs form a network mediated by PDGFRβ + A vascular network covered by pericytes ( Figure 3 ). PDGFR-β + The formation of pericytes changes over time. On day 10 of differentiation, PDGFR-β + The finger-like extensions of pericytes were only adjacent to ECs, whereas PDGFR-β was found at day 21 when organoids were fully mature. + Pericytes are wrapped with CD31 + This spatial association is therefore similar to that of endosteal arteries and type H vessels.

[0147] The chemokine CXCL12, which signals through the CXCR4 receptor, is a major retention and maintenance factor for HSCs and hematopoietic progenitors in the BM microenvironment. 32, 33 CXCL12-enriched reticular (CAR) cells are characterized by high expression of CXCL12 and their close association with ECs. In addition, CXCL12 is expressed in ECs themselves. Notably, BMOs contain perivascular CXCL12-expressing protrusions extending toward endothelial cells. + - A mesh network of cells ( Figure 2 e, f), reminiscent of CAR cells ( Figure 2 f). Nestin-expressing perivascular mesenchymal stem cells known to support and regulate hematopoiesis are located in the endosteal region, spatially associated with HSCs, and have multi-lineage and self-renewal capabilities in mouse and human fetal BM. Notably, the inventors identified CD31 in their BMOs using confocal and two-photon microscopy. + Blood vessels and CD45 + Hematopoietic cells have spatial relationships with Nestin + Confocal z-stack imaging reveals Nestin in perivascular mural cells that line the vascular structure. + Changes, like those seen in the bone marrow in the body.

[0148] When the inventors explored the vascular architecture in more detail, their findings indicated that the endothelial and mesenchymal BMO compartments recapitulated key architectural features and cellular composition of the human BM microenvironment.

[0149] Characteristics and local distribution of microenvironmental cells and hematopoietic cells in the generated BMOs

[0150] The current analysis shows that BMOs are composed of hematopoietic, vascular, and stromal (mesenchymal) compartments. Confocal microscopy revealed a self-assembled, interconnected, and partially luminal vascular network composed of a layer of Col IV + CD31 on basement membrane + Endothelial cells and PDGFR-β + In addition, flow cytometric analysis showed that the relative composition of endothelial cells and mesenchymal stem cells was comparable to that in native bone marrow (11). Interestingly, the inventors found that CXCL12 + The cells were located in a network-like structure that was tightly connected to the endothelial network, reminiscent of the native bone marrow architecture. This strongly suggests that bone marrow-specific CAR cells were formed in the current differentiation system. This is particularly noteworthy because CAR cells are major producers of CXCL12 and SCF, signaling molecules that are essential for HSC maintenance and homing and for the proliferation of lymphoid and erythroid progenitor cells (12). The inventors found that CD45 + Hematopoietic cells were clustered and evenly distributed throughout the organoids. This partial clustering could be explained by increased localization of certain microenvironmental cells and their regulatory cytokines to these areas. It is known that hematopoietic stem and progenitor cells are located near blood vessels, as growth factors and products produced by microenvironmental cells are important regulators of HSC maintenance (11,13).

[0151] Differentiation of myeloid cells in BMO

[0152] Furthermore, the inventors could show that the current differentiation protocol promotes spontaneous differentiation of hematopoietic progenitor cells into myeloid cells within the BMO structure, indicating a facilitatory interaction between hematopoietic progenitor cells and microenvironmental 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 and segmented nuclei, indicating that monocytes and granulocytes formed at different stages of maturation. FACS sorted myeloid populations (CD45 + CD34 - CD11b +) also showed the presence of macrophages, monocytes, and granulocytes. These results are particularly interesting because HSC differentiation into granulocytes and macrophages is dependent on GCSF and GM-CSF signaling, but these cytokines are not included in the cytokine cocktail of the current differentiation system. Therefore, these results suggest that the microenvironmental cells within the BMO system intrinsically produce sufficient amounts of these cytokines. Although the inventors have not been able to demonstrate the presence of other lineages such as lymphocytes, erythroid cells, or platelets, FACS-sorted hematopoietic progenitor cells (CD45 + CD34 + CD11b - ) showed the potential of these cells to differentiate into erythrocytes and megakaryocytes. Since the cytokines used in the inventors' system induce a shift in hematopoiesis toward differentiation of myeloid cell lineages, future adjustments to the cytokine composition may make differentiation of erythroid or even lymphoid cells possible.

[0153] Neutrophils and their precursors constitute the largest fraction of nucleated BM cells. The inventors investigated whether the BMO microenvironment would promote maturation of blood progenitor cells into myeloid mature cells without the addition of lineage-directing cytokines such as G-CSF or GM-SCF.

[0154] Organoids were stained for key myeloid markers S100A8 / A937 and myeloperoxidase (MPO). Cells expressing S100A8 / A9 and MPO were found in BMOs, some of which exhibited banded or segmented nuclei, indicating myeloid maturation. Immunohistochemical studies confirmed MPO expression in myeloid cells ( Figure 4 c), TEM showed cells with electron-dense cytoplasmic granules, segmented nuclei and heterochromatin formation at the nuclear margins, which are characteristic of neutrophils. Importantly, the inventors documented the orderly maturation of neutrophils by flow cytometry: based on the expression of cell surface markers, neutrophil precursor cell stages (ProNeu1, ProNeu2, PreNeu) as well as immature Neu and mature Neu were identified in both FiPSC- and UiPSC-derived BMOs. Optical microscopy analysis of flow-sorted and Magerge-stained cells showed that specific stages of BMO-derived neutrophils were morphologically similar to corresponding cells in human BM. Therefore, neutrophil maturation in BMO (which can be performed without the addition of recombinant cytokines) is reminiscent of the in vivo situation.

[0155] Mesenchymal stem cells obtained from BMO meet the criteria for mesenchymal stem cells

[0156] Mesenchymal stem cells are one of the major cellular components of the bone marrow microenvironment. MSCs maintain bone marrow tissue homeostasis by differentiating into adipocytes, osteocytes, and chondrocytes, while primitive mesenchymal cells and their progeny directly regulate hematopoiesis by secreting cytokines (14). In other in vitro bone marrow microenvironment studies, donor primary mesenchymal cells are used to generate the stromal (mesenchymal) compartment of the bone marrow microenvironment, which are then co-cultured with donor hematopoietic cells to mimic the bone marrow hematopoietic compartment. In this system, in contrast, iPSCs simultaneously differentiate into hematopoietic cells, endothelial cells, and, most notably, into mesenchymal progenitor cells such as PDGFRβ. + Vascular pericytes and CXCL12 + CAR cells. In addition, it was shown that this system induces the formation of truly functional mesenchymal stem cells, because BMO-derived MSCs meet the minimum criteria for mesenchymal stem cells proposed by ISCT: MSCs are negative for the hematopoietic and endothelial surface molecules CD45, CD34, and CD31, but positive for CD90, CD105, CD271, and CD73. MSCs adhere to plastic and can differentiate into osteoblasts, adipocytes, and chondrocytes in vitro (15, 16).

[0157] Modeling Development Process

[0158] Having identified pre-arterial HE-like cells by scRNA-seq, the inventors next set out to investigate the 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 cells expressing RUNX1 were located in distinct clusters within the organoids. Some of these cells co-expressed CD31 and remained attached to the endothelial wall. Immunofluorescence of immature organoids (day 10) showed that CD31 + Cells aggregated from the vascular lining, which was also seen in CD34 and H / E stained BMO histology sections, mirroring the in vivo findings of EHT. Consistent with these protein expression data, we found PECAM1 co-expressing RUNX1 in endothelial clusters in our scRNA-seq dataset. + cell.

[0159] To capture the intermediate states of cell differentiation in our flow cytometry data, we analyzed protein expression by unsupervised clustering using the FlowSOM algorithm. We found that the previously assigned endothelial cell compartment was heterogeneous and contained additional subpopulations. The surface marker expression profile of cells in cluster 3 was related to human HE as well as HSC, as they were CD45 negative and co-expressed CD31, CD34, CD90 (Thy1), and CD105 (endoglin). The adjacent cluster 4 consisted of CD31 低 CD34 + and CD45+ Cell definition, constituting manually gated HSPC clusters. As the expression of CD34 and CD45 increases, CD31 expression continues to decrease, reflecting the immunophenotypic order of cells undergoing EHT. It is noteworthy that in the inventors' scRNA-seq dataset, the inventors also identified rare cell populations expressing RUNX1, MECOM, HLF, SPINK2, and MLLT3 (characteristic genes of human fetal HSC / MPP). These data indicate that BMOs produce hemogenic endothelium, which is transformed into human HSC / MPP-like cells through EHT. Interestingly, unsupervised clustering also revealed cell clusters co-expressing CD271, CD31, and CD105. This marker spectrum overlaps with endothelial-derived BM stromal cells (eBMSCs) that have been identified in human fetuses and regenerated BM. EBMSCs undergo endothelial-mesenchymal transition (EndoMT) and show the ability to reconstruct the entire hematopoietic microenvironment after transplantation, including osteoprogenitor cells, thereby exhibiting MSC characteristics. Confocal imaging confirmed the co-expression of CD271, CD31, and CD105 in the EC subset, indicating that the cells were undergoing EndoMT.

[0160] Features

[0161] The phenotypic identification of HSPC and MSPC prompted the inventors to study their functional properties. To evaluate the pluripotency of BMO-derived MSPC, the inventors performed a three-line differentiation assay. It is noteworthy that when placed in each conditioned medium, the separation MSPC from FiPSC and UiPSC-derived BMO has the ability to differentiate into osteoblasts, adipocytes and chondrocytes, as visualized by the Alcian blue staining of the Alizarin red staining of calcium deposits, the Oil Red O staining of lipid vacuoles and glycosaminoglycans, respectively. To check the multi-line differentiation ability of BMO-derived HSPC, the inventors performed a colony forming unit (CFU) test. FACS-sorted HSPC, mainly produces granulocyte-macrophage progenitor cells (CFU-GM), but also produces multipotential granulocytes, erythrocytes, macrophages and megakaryocyte progenitor cells (CFU-GEMM) and erythrocyte progenitor cells (BFU-E). The separation HSPC from UiPSC-derived BMO also produces CFU-GEMM and CFU-GM, but does not produce typical BFU-E colonies. Colony characteristics were verified by Meggi staining of selected colonies. These findings indicate that BMOs contain multipotent MSPCs and HSPCs. To determine the functional properties of BMOs in vivo, the inventors transplanted BMOs at day 21 of differentiation under the renal capsule of immunodeficient NOD / SCID / IL2Rγ-deficient (NSG) mice. Analysis 3.5 months after transplantation showed further growth of organoids. Histological analysis of organoid sections after transplantation showed blood vessels filled with red blood cells. Surprisingly, the inventors found human CD45 in the BM of transplanted mice. + These data demonstrate that organoids can enter the mouse vasculature and that BMO-derived human CD45 + Cells can home to mouse BM.

[0162] Disease Modeling

[0163] To test the suitability of the generated BMOs for modeling bone marrow-related diseases, the inventors prepared VPS45-deficient BMOs in a proof-of-concept manner. Inherited VPS45 deficiency in patients results in a disease associated with severe congenital neutropenia and myelofibrosis (17). Although there were no significant differences in the relative composition of hematopoietic and microenvironmental cells between WT and VPS45 BMOs, preliminary histological results of VPS45 BMOs showed increased reticular fiber formation in VPS45 mutant BMOs, a sign of myelofibrosis.

[0164] Finally, the inventors tested whether our new BMOs can indeed be used as a model system to recapitulate the phenotypes of monogenic BM diseases. Children with vacuolar protein sorting 45 homolog (VPS45) deficiency develop neutropenia and myelofibrosis in the first year of life and develop BM failure. Their BM cells are excessive and show myeloproliferation associated with increased neutrophil apoptosis and functional defects. The inventors generated BMOs using a gene-edited iPSC-derived line homozygous for the Thr224Asn mutation of VPS4514 and compared them with isogenic control BMOs (wild type, WT). No major differences in the composition of hematopoietic cells and microenvironment cells were found. However, VPS45-deficient BMOs showed increased deposition of reticular fibers, reminiscent of myelofibrosis in BM biopsies of VPS45-deficient patients. This was accompanied by the expansion of myofibroblast-like stromal cells expressing alpha-smooth muscle actin (SMA), which has previously been described as a key driver of myelofibrosis.

[0165] Flow cytometric analysis of VPS45-deficient BMOs revealed a higher number of mature Neu compared with myeloproliferative controls, and significantly increased Annexin V expression on VPS45 mutant mature Neu, indicating increased apoptosis in this subpopulation, a feature also found in patients. Thus, BMOs may provide a new model system for dissecting genes and pathways in the pathomechanisms of BM failure diseases that were previously difficult to resolve.

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Claims

1. A method for preparing mammalian bone marrow organoids, comprising: a) generating embryoid bodies from substantially single induced pluripotent stem cells (iPSCs) obtained from at least one mammal, comprising culturing the single iPSC in a polymerizing medium for about 1 day in the presence of at least one Rho-associated protein kinase (ROCK) inhibitor, followed by culturing the embryoid bodies in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells in the absence of the ROCK inhibitor for about 48 hours; b) inducing mesoderm in the embryoid bodies formed in step a), comprising culturing the embryoid bodies for about 48 hours in a suitable serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells, and resuspending the mesoderm-induced embryoid bodies about every 24 hours, wherein the culture 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 in 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, and resuspending the embryoid bodies about every 24 hours; d) embedding the mesoderm-induced embryoid bodies in step c) into a suitable polymerized 3D collagen The cells were then treated with 80 ng / ml VEGF, 25 ng / ml FGF-2, 50 ng / ml SCF and 2 μM SB431542. -34 medium overlays the matrix for approximately 48 hours, followed by 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 approximately 48 hours, and 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 approximately 48 hours; e) extracting the individual vascular networks generated in step d) and lysing them in a culture 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. -34 medium, with the medium refreshed every 3-4 days for about 7-11 days, thereby generating mature mammalian bone marrow organoids, preferably without the use of germline-directed cytokines such as EPO, IL-6 and / or G-CSF.

2. The method for preparing mammalian bone marrow organoids according to claim 1, further comprising fixing the vascular network in step e) and performing immunofluorescence analysis, and / or dissociating the vascular network into single cells and performing flow cytometry analysis and / or live cell imaging.

3. The method for preparing mammalian bone marrow organoids according to claim 1 or 2, further comprising the steps of fixing the generated organoids and performing immunofluorescence analysis, and / or dissociating the organoids into single cells and performing flow cytometry analysis and / or live cell imaging.

4. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 3, further comprising the step of culturing the organoids for up to about 60 days.

5. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 4, wherein the single iPSC is Digestion is provided to dissociate iPSC cells into single cells.

6. A method for preparing mammalian bone marrow organoids according to any one of claims 1 to 5, wherein the ROCK inhibitor is selected from Y-27632 ((1R, 4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide) and fasudil.

7. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 6, wherein the polymerization 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.

8. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 7, wherein the serum-free stable cell culture medium suitable for feeder-free maintenance and expansion of human embryonic stem cells is Plus medium.

9. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 8, wherein the glycogen synthase kinase (GSK) 3 inhibitor is CHIR99021.

10. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 9, wherein the 3D collagen The ratio of substrate is about 1:1 to 4:1, preferably about 3:1 or 1.2:

1.

11. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 10, wherein the resuspending of the embryoid bodies / mesoderm-induced embryoid bodies comprises using a pipette.

12. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 11, wherein the dissociation or extraction of the single vascular network comprises extraction using a sterile dissection tool.

13. The method for preparing mammalian bone marrow organoids according to any one of claims 1 to 12, wherein the mammal is selected from humans, mice, monkeys, rats, pigs, dogs, cats, rabbits, sheep, cows, horses and goats.

14. A vascular network or mature mammalian bone marrow organoid prepared by the method of any one of claims 1 to 13, or a pharmaceutical composition comprising the vascular network and / or mature mammalian bone marrow organoid. 15 . An assembly comprising the vascular network or mature mammalian bone marrow organoid according to claim 14 , and at least one other iPSC-derived organoid. The assembly according to claim 15 , comprising immune cells, blood vessels and pericytes.

17. Use of the vascular network or mature mammalian bone marrow organoid of claim 14, or the assembly of claim 15 or 16, as a model system for bone marrow related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immune deficiencies, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

18. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of claim 14, or the assembly of claim 15 or 16 as a model system for the pathogenesis of bone marrow related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

19. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of claim 14, or the assembly of claim 15 or 16 as a model system for identifying and / or testing pharmaceutically effective compounds for treating or preventing bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications.

20. Use of the vascular network or mature mammalian bone marrow organoid or pharmaceutical composition of claim 14, or the assembly of claim 15 or 16, in the in vitro generation of BMO or mammalian blood cells, especially autologous BMO or mammalian blood cells, in particular for transplantation.

21. Use of a pharmaceutically effective amount of the vascular network or mature mammalian bone marrow organoid of claim 14, or the assembly of claim 15 or 16, or a pharmaceutical composition comprising at least one of them, in the treatment of bone marrow-related diseases, such as blood diseases, severe congenital neutropenia, myelofibrosis, blood cell cancers, anemia, thrombocytopenia, congenital hematopoietic and immunodeficiency, HIV-related diseases, sickle cell disease, and chemotherapy or infusion complications, preferably in transplantation.

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