A high-efficiency method for constructing functional bone marrow organoids by directional differentiation of human induced pluripotent stem cells

Abstract

The application discloses a kind of human induced pluripotent stem cell directional differentiation constructs the efficient method of functional bone marrow organoid, belongs to biomedical engineering and regenerative medicine field, to solve the problems such as low differentiation efficiency, imperfect hematopoietic function of existing method.The method uses human induced pluripotent stem cell as seed cell, induces formation cell aggregate, after mesoderm induction and stromal cell, hematopoietic cell differentiation, induces blood vessel sprouting and hematopoietic microenvironment formation with the culture medium containing multiple cytokines;Again separate single organoid and transfer into low adhesion 96 well plate culture, obtain functional bone marrow organoid.The application cell source is stable, can individualized preparation, and optimized culture scheme significantly improves the maturity of organoid;The obtained organoid contains complex stromal cell network, supports hematopoietic stem cell long-term maintenance and differentiation, step is clear, component is clear, is convenient for standardization production, can be used in blood disease model construction, drug screening, hematopoietic stem cell in vitro expansion and other fields.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering, regenerative medicine, and tissue engineering. Specifically, it relates to a method for efficiently and stably constructing functional human bone marrow-like organoids (BM-organoids) using human induced pluripotent stem cells (hiPSCs), and the applications of the resulting organoids in drug screening, disease modeling (such as hematological diseases, immune-related diseases, and bone marrow failure syndromes), in vitro expansion of hematopoietic stem cells (HSCs), toxicology studies, cell therapy, and personalized medicine. Background Technology

[0002] Bone marrow is the core hematopoietic organ in the human body, continuously producing various blood cells such as red blood cells, white blood cells, and platelets, as well as immune cells. Simultaneously, bone marrow possesses a unique hematopoietic microenvironment, providing crucial signal regulation and structural support for the self-renewal, directed differentiation, homing and colonization, and maintenance of stem cell function in hematopoietic stem cells (HSCs). Abnormal bone marrow function can induce various malignant and refractory hematological diseases, including leukemia, lymphoma, aplastic anemia, and myelodysplastic syndromes, seriously threatening human health.

[0003] Traditional two-dimensional (2D) cell culture systems struggle to replicate the complex three-dimensional spatial structure and dynamic intercellular interactions of bone marrow, making it impossible to maintain the biological function and stemness characteristics of HSCs long-term. Animal models, due to significant interspecies physiological and genetic differences, cannot accurately simulate the physiological and pathological state of human bone marrow, resulting in substantial biases in disease mechanism analysis, drug efficacy prediction, and toxicity assessment. Primary bone marrow cell or tissue in vitro culture methods suffer from problems such as scarcity of healthy donor samples, significant individual heterogeneity, and limited in vitro expansion capacity. Furthermore, precise genetic modification is difficult, leading to low model standardization and failing to meet the needs of large-scale research.

[0004] Organoids are three-dimensional structures formed in vitro through the self-assembly of pluripotent stem cells and adult stem cells. They can mimic the tissue structure and physiological function of corresponding organs to a certain extent, showing great application potential in areas such as in vitro disease modeling, high-throughput drug screening, and developmental mechanism research. HiPSCs possess both unlimited proliferative potential and multi-lineage differentiation capabilities, and can be obtained from individualized sources for patients, making them ideal seed cells for constructing human bone marrow organoids. However, at present, there are still many technical bottlenecks in using hiPSCs to construct efficient, stable, and functional bone marrow organoids with complete microenvironments and long-term hematopoietic function. These problems generally include low differentiation efficiency, lengthy induction cycles, easy disintegration of three-dimensional structures, and imperfect hematopoietic function, which greatly limits their application in basic hematological research and clinical translation.

[0005] Therefore, breaking through the technical barriers to constructing hiPSC-derived bone marrow organoids, establishing efficient, stable, and standardized induction differentiation protocols, and constructing human functional bone marrow organoids containing a complete hematopoietic support microenvironment and possessing long-term hematopoietic activity are of significant theoretical and clinical value for overcoming the inherent defects of traditional research models, elucidating the pathogenesis of human hematopoietic development and blood diseases, and realizing personalized drug screening and regenerative medicine research. Summary of the Invention

[0006] To address the problems of low efficiency, long cycle, and imperfect structure and function in the construction of functional human bone marrow organoids using hiPSC in existing technologies, this invention provides a highly efficient, stable, and reproducible method to improve the yield and quality of bone marrow stromal cells and hematopoietic progenitor cells, and ultimately form 3D human bone marrow organoids with a functional bone marrow microenvironment that can support hematopoiesis.

[0007] Technical solution:

[0008] A highly efficient method for constructing functional human bone marrow organoids using human induced pluripotent stem cells, characterized by the following steps:

[0009] 1. Inducing cell aggregates: Take hiPSCs with a confluence of 60% to 70%, digest them into single cells, resuspend them at a ratio of 1:3 in hiPSC complete medium supplemented with 50 μM Y27632, and seed them in 6-well plates with very low adhesion for culture.

[0010] 2. Induction of mesoderm differentiation: After 24 h of culture, the medium was replaced with STEMdiff APEL2 medium containing 3 μM CHIR99021, 50 ng / ml BMP4, 50 ng / ml VEGFA, and 50 ng / ml FGF2 for induction culture.

[0011] 3. Directed differentiation of stromal cells and hematopoietic cells: After 72 h of induction, the medium was replaced with STEMdiff APEL2 medium containing 50 ng / ml BMP4, 50 ng / ml VEGFA, 50 ng / ml FGF2, 25 ng / ml hSCF, and 25 ng / ml Flt3, and incubated for another 48 h.

[0012] 4. Hydrogel embedding and vascular budding induction: Cell aggregates were collected by gravity sedimentation, mixed with Matrigel-Collagen I solution, and then inoculated. After gelation, STEMdiff APEL2 medium containing 5% KO serum and 5 U / mL heparin was added, along with cytokines at final concentrations of 100 ng / mL VEGFA, 50 ng / mL VEGFC, FGF2, TPO, EPO, Flt3, hSCF, G-CSF, BMP4, and 20 ng / mL IL-3 and IL-6. After 48 h of culture, STEMdiff APEL2 medium with the same basic components and cytokine ratio was added. After 72 h of culture, half the medium was replaced with STEMdiff APEL2 medium containing 2% KO serum and 25 ng / mL VEGFC, FGF2, TPO, EPO, Flt3, and hSCF.

[0013] 5. Isolation and individual culture of bone marrow organoids: After 48 hours of culture, the entire gel was transferred to a culture dish, and individual bone marrow organoids were aseptically isolated under a dissecting microscope and seeded into 96-well round-bottom plates with very low adhesion. StemPro-34 medium containing 2% KO serum, CD lipids, 25 ng / ml VEGFA, VEGFC, FGF2, hSCF, Flt3, and 10 ng / ml TPO, EPO, IL-3, and IL-6 was added, and the plates were incubated at 37°C. The medium was changed every 3 days.

[0014] 6. Functional Validation and Characterization of Organoids: The obtained bone marrow organoids were identified from three aspects: ① Morphology: H&E staining and immunofluorescence detection of markers such as CD34, CD45, CD41, CD71, CD144, and CD140b were used; ② Molecular level: Mesodermal-related gene expression was detected by qPCR or RNASeq; ③ Hematopoietic function: Exogenous CD34⁺ hematopoietic stem cells or hiPSC-derived hematopoietic progenitor cells were seeded into organoids, and flow cytometry and colony formation assays were used to evaluate cell homing, survival, proliferation, and myeloid and lymphoid differentiation capabilities.

[0015] Beneficial effects:

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) High efficiency: By optimizing the differentiation schemes at each stage and the 3D assembly medium, the differentiation efficiency and organoid formation rate were significantly improved.

[0018] (2) High yield: It can produce a large number of bone marrow organoids with good structural uniformity.

[0019] (3) Strong functionality: The generated organoids contain a complex network of stromal cells, such as osteoblasts, adipocytes, endothelial cells, and mesenchymal stromal cells, which simulate the key components of the bone marrow microenvironment and have a strong ability to support hematopoiesis, especially to support the maintenance and differentiation of long-term hematopoietic stem cells (LT-HSCs), which is difficult to achieve in many existing models.

[0020] (4) Stable and personalized source: Based on hiPSC, an unlimited amount of starting materials can be provided, and disease models can be built directly using patient-derived hiPSC.

[0021] (5) Standardization potential: The methods and steps are clear and the components of the culture medium are well-defined, which is conducive to achieving standardized production and quality control.

[0022] (6) Wide range of applications: It provides a powerful new platform for research on blood diseases, drug development and toxicity testing, immunological research, HSC in vitro amplification, personalized medicine, etc. Attached Figure Description

[0023] Appendix Figure 1 Representative images of induced hematopoietic and stromal cells.

[0024] Appendix Figure 2 Validation of mesoderm-specific gene expression.

[0025] Appendix Figure 3 Representative images of induced hematopoietic cell, angiogenesis, and bone marrow organoid culture in isolation.

[0026] Appendix Figure 4 Immunofluorescence staining images of key cell types (CD45+ hematopoietic cells, CD144+ endothelial cells, CD71+ early / intermediate / late erythroblasts, CD41+ megakaryocytes, etc.) in bone marrow organoids.

[0027] Appendix Figure 5 Transmission electron microscopy results of bone marrow organoids. Appendix Figure 6 Bone marrow organoid culture flowchart Detailed Implementation

[0028] (a) Induced aggregates

[0029] 1. Digest hiPSCs with a confluence of 60%-70% into single cells.

[0030] 2. The digested single cells were resuspended at a ratio of 1:3 in hiPSC complete medium supplemented with 50 μM Y27632 and seeded in 6-well plates with very low adhesion.

[0031] (ii) Induction of mesodermal layer

[0032] 1. After incubation for 24 hours, replace the medium with STEMdiff APEL2 medium supplemented with 3 μM CHIR99021, 50 ng / ml BMP4, VEGFA, and FGF2, and continue induction for 72 hours.

[0033] (III) Differentiation of stromal cells and hematopoietic cells

[0034] 1. After 72 hours, the medium was replaced with STEMdiff APEL2 medium containing 50 ng / ml BMP4, VEGFA, FGF2, 25 ng / ml hSCF, and Flt3, and incubated for 48 hours.

[0035] (iv) Inducing vascular budding

[0036] 1. Cell aggregates were allowed to settle to the bottom of a 15ml centrifuge tube by gravity, mixed with Matrigel-Collagen I solution, and seeded into 12 wells. After the Matrigel-Collagen I solution solidified, STEMdiff APEL2 medium containing 5% KO serum and 5 U / mL heparin was added.

[0037] 2. Add cytokines at concentrations of 100 ng / ml for VEGFA, 50 ng / ml for VEGFC, FGF2, TPO, EPO, Flt3, hSCF, G-CSF, and BMP4, and 20 ng / ml for IL-3 and IL-6.

[0038] 3. After 48 hours, add STEMdiff APEL2 medium containing 5% KO serum, 5 U / mL heparin, 100 ng / mL VEGFA, 50 ng / mL VEGFC, FGF2, TPO, EPO, Flt3, hSCF, BMP4, G-CSF, and 20 ng / mL IL-3 and IL-6 cytokines. 4. After 72 hours, replace half of the medium with STEMdiff APEL2 medium containing 2% KO serum and 25 ng / mL VEGFC, FGF2, TPO, EPO, Flt3, and hSCF cytokines.

[0039] (v) Extraction and separate culture of bone marrow organoids

[0040] 1. After 48 hours, the entire gel was transferred to a 10cm culture dish using a sterile spoon. The bone marrow organoids were then isolated individually under a dissecting microscope using two 30-gauge sterile needles, which may reduce the amount of matrix gel around the vascular network.

[0041] 2. Individual organoids were seeded one by one into a 96-well plate with very low adhesion using a Parsley tube. Fresh StemPro-34 medium containing 2% KO serum, CD lipids, 25 ng / ml VEGFA, VEGFC, FGF2, hSCF, Flt3, and 10 ng / ml TPO, EPO, IL3, and IL6 was added and incubated at 37°C. The medium was changed every 3 days.

[0042] (vi) Functional verification

[0043] 1. Characterize the obtained bone marrow organoids, including: morphology: H&E staining, immunofluorescence (detection of CD34, CD45, CD41, CD71, CD144, CD140b, etc.).

[0044] 2. Molecular level: qPCR / RNASeq detection of mesoderm-related gene expression.

[0045] 3. Hematopoietic support capacity: Exogenous CD34+ HSCs or hiPSC-derived hematopoietic progenitor cells are seeded onto organoids, and their homing, survival, proliferation, and differentiation (myeloid and lymphoid) capabilities are evaluated (through flow cytometry, colony formation assays, CFU, etc.).

Claims

1. A highly efficient method for constructing functional bone marrow organoids through directed differentiation of human induced pluripotent stem cells, characterized in that, Includes the following steps: (1) Induced aggregates: hiPSCs with a fusion degree of 60%-70% were digested and resuspended in hiPSC complete medium with 50 μM Y27632 at a ratio of 1:3, and inoculated into 6-well plates with very low adhesion for culture. (2) Induction of mesoderm: After incubation for 24 hours, the medium was replaced with STEMdiff APEL2 medium supplemented with 3 μM CHIR99021, 50 ng / ml BMP4, VEGFA, and FGF2 for further culture. (3) Differentiation of stromal cells and hematopoietic cells: After culturing for 72 hours, the medium was replaced with STEMdiff APEL2 medium containing 50 ng / ml BMP4, VEGFA, FGF2, 25 ng / ml hSCF, and Flt3, and incubated for another 48 hours. (4) Encapsulation with hydrogel to induce vascular budding: The cell aggregates cultured in step (3) were settled by gravity to the bottom of a 15ml centrifuge tube, mixed with Matrigel-Collagen I mixture, and seeded into a 12-well plate. The mixture was incubated until it solidified. Then, STEMdiff APEL2 medium containing 5% KO serum, 5 U / mL heparin, and 100 ng / ml VEGFA, 50 ng / ml VEGFC, FGF2, TPO, EPO, Flt3, hSCF, G-CSF, BMP4, 20 ng / ml IL-3, and IL-6 cytokines was added. After 48 hours of culture, the medium was replaced with STEMdiff APEL2 medium containing 5% KO serum, 5 U / mL heparin, 100 ng / ml VEGFA, 50 ng / ml VEGFC, FGF2, TPO, EPO, Flt3, hSCF, BMP4, G-CSF, and 20 ng / ml heparin. STEMdiff APEL2 medium containing IL-3 and IL-6 cytokines; after 72 hours of culture, half of the medium was replaced with STEMdiff APEL2 medium containing 2% KO serum, 25 ng / ml VEGFC, FGF2, TPO, EPO, Flt3, and hSCF cytokines. (5) Extraction and separate culture of bone marrow organoids: After 48 hours of culture, the whole gel was transferred to a 10cm culture dish using a sterile spoon. The bone marrow organoids were separated individually under a dissecting microscope using two 30-gauge sterile needles, minimizing the amount of matrix gel around the vascular network. The individual organoids were seeded one by one into a 96-well plate with very low adhesion using a Pasteur tube. StemPro-34 medium containing 2% KO serum, CD lipids, 25ng / ml VEGFA, VEGFC, FGF2, hSCF, Flt3, 10ng / ml TPO, EPO, IL3, and IL6 was added and incubated at 37°C. The culture medium was changed every 3 days.

2. The method according to claim 1, characterized in that, In step (2), the culture was carried out using STEMdiff APEL2 medium containing 3 μM CHIR99021, 50 ng / ml BMP4, VEGFA, and FGF2.

3. The method according to claim 1, characterized in that, In step (3), differentiation culture was induced using STEMdiff APEL2 medium containing 50 ng / ml BMP4, VEGFA, FGF2, 25 ng / ml hSCF, and Flt3.

4. The method according to claim 1, characterized in that, In step (4), cell aggregates were embedded using Matrigel-Collagen I mixture, and cultured stepwise in STEMdiff APEL2 medium containing KO serum, heparin and various concentrations of cytokines, with half-volume medium replacement.

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