Human bone marrow stromal cell line and application thereof

Abstract

The invention provides a human bone marrow stroma cell line and application thereof. The invention successfully establishes the human bone marrow stromal cell line hFSC022 capable of efficiently promoting in-vitro hematopoietic generation and induced differentiation of hematopoietic cells for the first time. The human bone marrow stromal cell line established by the invention is derived from waste human bone marrow tissues, and compared with mouse trophoblast cells, the human bone marrow stromal cell line has no species crossing risk, has higher safety and is more suitable for clinical research and use; compared with mouse trophoblast cells or stromal cells derived from other bone marrow tissues, the stromal cells have higher hematopoietic generation promoting capacity; in-vitro culture is free of animal-derived components, cell therapy level requirements of clinical application are met, and the method is more suitable for clinical research and application. Meanwhile, the cell line established by the invention can be used as a trophoblast or a combined medicine, is used for supporting in-vitro maintenance or proliferation of human hematopoietic stem / progenitor cells and inducing differentiation of human pluripotent stem cells to hematopoietic cells, and has a wide application prospect in the fields of in-vitro hematopoietic generation and cell therapy.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cells, and specifically relates to a human bone marrow stromal cell line and application thereof. Background Art

[0002] More and more studies have shown that the in vitro proliferation and differentiation of human hematopoietic stem / progenitor cells and the process of inducing human pluripotent stem cells to differentiate into hematopoietic cells require trophoblasts to provide a microenvironment. At present, it has been reported that many mouse-derived trophoblasts can support in vitro hematopoiesis and hematopoietic cell induction differentiation, such as OP9 cells, AFT024 cells, MS5 cells, etc. However, these cell lines are all mouse-derived cells, and have very large limitations for the application needs of human hematopoiesis and differentiation, especially in clinical applications, mainly considering the safety of xenogeneic cells. Of course, there are also reports on the establishment of human bone marrow stromal cell lines to support hematopoiesis, such as HS-5 cells. The above-mentioned mouse-derived cell lines are all derived from cells at different embryonic development stages, while HS-5 cells are a cell line derived from adult bone marrow stroma. Adult bone marrow-derived stromal cells have not been shown to be able to efficiently promote in vitro hematopoiesis and hematopoietic cell induction differentiation.

[0003] According to the law of hematopoiesis during the development of organisms, there are fundamental differences in the cell composition and molecular interactions of the bone marrow in different growth periods or under different growth conditions, especially the bone marrow microenvironment in the early growth stage is very different from the bone marrow microenvironment in the late growth stage. In addition, recent studies have found that in the early stages of human growth and development, human hematopoietic stem cells (HSC) colonize in the bone marrow, and their microenvironment has been established before HSC colonizes the bone marrow. Therefore, it is urgent to develop a human trophoblast cell line that can efficiently promote in vitro hematopoiesis and induce differentiation of hematopoietic cells and bone marrow tissue to solve the above problems. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a human bone marrow stromal cell line and application thereof.

[0005] In the first aspect of the present invention, a human bone marrow stromal cell line hFSC022 is provided. The human bone marrow stromal cell line hFSC022 was deposited on November 28, 2024 at the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 46248.

[0006] Furthermore, the cell line can proliferate indefinitely when cultured in vitro and has bone marrow stromal cell-like morphological characteristics.

[0007] Furthermore, the cell line has a spindle-shaped or irregular triangle shape when attached to the wall, an oval nucleus, a large nuclear-cytoplasmic ratio, and protrusions of different lengths extending outward from the cytoplasm, growing like fibroblasts.

[0008] Furthermore, the cell doubling time of the cell line is 28 hours.

[0009] Furthermore, the cell line expresses CD29, CD44, CD105, CD271, CD73 molecules, but does not express CD45 molecules.

[0010] In the second aspect of the present invention, the use of the human bone marrow stromal cell line hFSC022 described in the first aspect of the present invention is provided. The cell line can be used as a trophoblast to support the maintenance and expansion of human CD34+ hematopoietic stem / progenitor cells in vitro.

[0011] Furthermore, the human CD34+ hematopoietic stem / progenitor cells are derived from umbilical cord blood tissue, peripheral blood mobilization, bone marrow, hPSC induced differentiation sources, etc.

[0012] Further preferably, the human CD34+ hematopoietic stem / progenitor cells are derived from umbilical cord blood tissue.

[0013] In the third aspect of the present invention, the use of the human bone marrow stromal cell line hFSC022 described in the first aspect of the present invention is provided. The cell line can be used as a trophoblast cell to induce hematopoietic differentiation of human pluripotent stem cells (hPSC) to obtain hematopoietic stem / progenitor cells (iHPC).

[0014] Furthermore, the obtained hematopoietic stem / progenitor cells have the ability to differentiate into multiple hematopoietic lineages.

[0015] Furthermore, the obtained hematopoietic stem / progenitor cells can differentiate into downstream lineage cells such as lymphoid progenitor cells, myeloid progenitor cells, natural killer cells (NK cells), T cells, B cells, macrophages, neutrophils, monocytes, and erythrocytes.

[0016] Further preferably, the obtained hematopoietic stem / progenitor cells can differentiate into natural killer cells (NK cells), T cells, and macrophages.

[0017] In the fourth aspect of the present invention, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the cell line described in the first aspect of the present invention.

[0018] Furthermore, the pharmaceutical composition may be in any form known in the medical field, preferably an injection (including injection solution and lyophilized powder).

[0019] Furthermore, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.

[0020] Further, the general principles of the preparation of the pharmaceutical composition can be referred to in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000.

[0021] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0022] Compared with the prior art, the present invention has the following advantages and progress: The present invention successfully established for the first time a human bone marrow stromal cell line hFSC022 that can effectively promote in vitro hematopoiesis and induce differentiation of hematopoietic cells. The human bone marrow stromal cell line established by the present invention is derived from discarded human bone marrow tissue. Compared with mouse trophoblasts, it has no species cross-risk, has higher safety, and is more suitable for clinical research use. The human bone marrow stromal cell line established by the present invention has a stronger ability to promote hematopoiesis compared with mouse trophoblasts or stromal cells derived from other human bone marrow tissues. The cell line established by the present invention has no animal-derived components in in vitro culture, meets the cell therapy level requirements for clinical applications, and is more suitable for clinical research applications. At the same time, the cell line established by the present invention can be used as a trophoblast cell or combination drug to support the maintenance and proliferation of human hematopoietic stem / progenitor cells in vitro, and to induce human pluripotent stem cells to differentiate into hematopoietic cells. In summary, the present invention separates stromal cells from discarded human bone marrow tissue, introduces and overexpresses immortalization genes, further establishes a line through single-cell cloning, and establishes a cell line through multiple rounds of functional screening, thereby obtaining a human bone marrow stromal cell line hFSC022 that can efficiently promote in vitro hematopoiesis and induce differentiation of hematopoietic cells, which has broad application prospects in the fields of hematopoiesis and cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 It is a schematic diagram of the functional evaluation of the human bone marrow stromal cell line hFSC022 and its application of the present invention.

[0025] Figure 2 This is a cell morphology diagram of the human bone marrow stromal cell line hFSC022 of the present invention.

[0026] Figure 3 It is a flow cytometric phenotype diagram of the molecular markers expressed by the human bone marrow stromal cell line hFSC022 of the present invention.

[0027] Figure 4 Schematic diagram of the co-culture experiment of hFSC022 cells and human umbilical cord blood CD34+ cells (UCB-CD34).

[0028] Figure 5 This is the flow cytometry phenotype of CD45+CD34+CD38- cells after hFSC022 cells were co-cultured with human umbilical cord blood CD34+ cells for 7 days.

[0029] Figure 6 This is a statistical chart of the absolute number of CD45+CD34+ cells after hFSC022 cells were co-cultured with UCB-CD34+ cells for 7 days.

[0030] Figure 7 This is a statistical chart of the absolute number of CD45+CD34+CD38- cells after hFSC022 cells were co-cultured with UCB-CD34+ cells for 7 days.

[0031] Figure 8 This is a representative CFU colony image of CD34+ cells formed after 7 days of co-culture of hFSC022 cells and UCB-CD34+ cells.

[0032] Fig. 9 This is a representative statistical graph of the absolute number of CFU colonies formed by CD34+ cells after 7 days of co-culture of hFSC022 cells and UCB-CD34+ cells.

[0033] Fig.10 This is a flow chart of using hFSC022 cells as trophoblasts to induce differentiation of human pluripotent stem cells to obtain hematopoietic stem / progenitor cells (iHPCs).

[0034] Fig.11 This is the flow cytometry phenotype of iHPC cells (CD45+CD34+CD43+) obtained by inducing hPSC differentiation from hFSC022 cells.

[0035] Fig.12 This is a representative CFU colony image of hFSC022-induced differentiation of hPSC-derived iHPC cells.

[0036] Fig.13 This is the flow cytometry phenotype of iNK cells obtained by further differentiation of iHPC cells derived from hPSC induced by hFSC022.

[0037] Fig.14 This is a schematic diagram and flow cytometry phenotype diagram of iT cells obtained by further differentiation of hPSC-derived iHPC cells induced by hFSC022.

[0038] Fig.15 This is the flow cytometry phenotype of macrophages obtained by further differentiation of iHPC cells derived from hPSC induced by hFSC022. DETAILED DESCRIPTION

[0039] The present invention provides a human bone marrow stromal cell line and its application. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and they are all carried out according to the general technical conditions described in the literature of the field or the relevant product instructions.

[0040] Human bone marrow stromal cell line hFSC022: The human bone marrow stromal cell line hFSC022 was deposited on November 28, 2024 at the General Microbiology Center of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 46248. The human bone marrow stromal primary cells of the cell line were isolated from discarded human bone marrow tissue. The human bone marrow stromal cell line hFSC022 has a spindle-shaped or irregular triangle shape when attached to the wall, an oval nucleus, a large nuclear-cytoplasmic ratio, and the cytoplasm extends outward with protrusions of different lengths, growing like fibroblasts ( Figure 2 ). The human bone marrow stromal cell line hFSC022 expresses CD29, CD44, CD105, CD271, and CD73 molecules; it does not express CD45 molecules ( Figure 3 ).

[0041] Bone marrow stromal cell culture medium: DMEM medium containing 20 ng / mL bFGF (Novoprotein, GMP-C046), 20 μg / mL ascorbic acid (Sigma-Aldrich, A-5960) and 5% EliteGro (EliteCell, EPAGMP-050).

[0042] NK cell differentiation medium: DMEM medium containing 5% serum replacement, 24 μM β-mercaptoethanol, 50 μM ethanolamine, 20 μg / mL ascorbic acid, 50 ng / mL sodium selenite, 10 ng / mL IL-15 (PeproTech), 20 ng / mL IL-7 (PeproTech), 20 ng / mL SCF (PeproTech), and 10 ng / mL Flt3L (PeproTech).

[0043] T cell differentiation medium: RPMI1640 medium containing 5% serum replacement, 24 μM β-mercaptoethanol, 30 μg / mL ascorbic acid, 20 ng / mL IL-7 (PeproTech), 20 ng / mL SCF (PeproTech) and 5 ng / mL Flt3L (PeproTech).

[0044] Macrophage differentiation medium: RPMI-1640 medium containing 5% EliteGro (EliteCell, EPAGMP-050), 2 µM L-Glutamax, 1% non-essential amino acids, 0.055 mM β-mercaptoethanol, 50 ng / mL Flt3L (PeproTech), 50 ng / mL M-CSF (PeproTech) and 25 ng / mL IL-3 (PeproTech).

[0045] hFSC022 cells were seeded into 24-well plates in advance (mouse AFT024 cells that can maintain CD34+ HSPCs in vitro were used as control cells), and irradiation pretreatment was performed when the cell density grew to 90%. Day -1, hFSC022 and AFT024 cells were irradiated at a dose of 20 Gy (2 Gy / min, 10 min). After being cultured in a carbon dioxide incubator for 1 day of recovery, they were used for co-culture. Day 0, enriched UCB-CD34+ cells were seeded into 24-well plates at a number of 50,000 cells per well, and co-cultured using SFEM stem cell expansion medium (StemSpan SFEM Medium) + 100 ng / mL SCF + 100 ng / mL TPO + 50 ng / mL Flt3L + UM171 (50nM) ( Figure 4 Half of the medium was changed every other day, and cells were collected on Day 7 to detect the phenotypes of CD34+ and CD34+CD38- cells ( Figure 5 ) and absolute number ( Figure 6 and Figure 7 ). Mouse AFT024 cells are commonly used in the field to maintain / expand CD34+ cells in vitro. The results show that hFSC022 can achieve a CD34+ cell expansion number comparable to that of AFT024, and can replace mouse-derived cells AFT024.

[0046] hFSC022 cells were used as trophoblasts to expand human umbilical cord blood CD34+ cells. The cell density was adjusted to 1×10^4 / ml with IMDM (containing 2% FBS), and then mixed according to the ratio of 100μl cell suspension + 1ml H4434 semi-solid medium. After sufficient oscillation and mixing, the mixture was left to stand at room temperature for 10 min, and then transferred to a 35 mm culture dish and placed in a carbon dioxide incubator for culture. After 14 days of culture, the culture dish was removed and representative colonies were photographed under a microscope according to their morphological characteristics ( Figure 8 ) and statistical counting of each colony ( Fig. 9 The colony forming assay (CFU) is often used to detect the multi-lineage differentiation function of hematopoietic stem / progenitor cells in vitro. The results showed that the cord blood CD34+ cells expanded by hFSC022 cells can successfully form BFU-E (burst-forming unit of erythrocytes), CFU-G (granulocyte colony-forming unit), CFU-M (macrophage colony-forming unit), CFU-GM (granulocyte / macrophage colony-forming unit) and CFU-Mix (mixed cell line colony-forming unit), indicating that the cord blood CD34+ cells expanded by hFSC022 cells still have the potential for multi-lineage differentiation.

[0047] Using in vitro organoid induction method ( Fig.10): First, the pluripotent stem cells (hESC) cultured in monolayer to 80% adhesion density were digested with Accutase (Sigma-Aldrich, A6964) for 1 min, resuspended into a single cell suspension with E8 medium (Gibco, A1517001), added with 2 µM ROCK inhibitor thiazovivin (Selleck, S1459), and then transferred to a cell culture dish covered with VTN (Animal-Free Human Vitronectin Matrix, PeproTech, AF-VMB-220), and cultured in an incubator for 24 h (this time was defined as Day 0 of the induction procedure). The E8 medium in the culture dish was aspirated, and the lateral plate mesoderm differentiation medium was added (differentiation culture conditions were: day 1 using Essential 6 medium containing 40 ng / mL BMP4, 30ng / mL ACTIVIN A, 20 ng / mL bFGF, 6 µM CHIR-99021 and 100 nM PIK-90; day 2 using Essential 6 medium containing 30 ng / mL BMP4, 1 µM A-83-01 and 1 µM C59), and induced differentiation culture was performed in the incubator to obtain lateral plate mesoderm cells. Then, the lateral plate mesoderm cells were digested to prepare a single cell suspension, and mixed with OP9 trophoblast cells or human bone marrow stromal cell line hFSC022, respectively, at a mixing ratio of 1:25, and the total number of cells was adjusted to 500,000 according to the pore size of the culture dish to prepare hematopoietic differentiation cell capsules. Under hematopoietic differentiation induction culture conditions (differentiation culture conditions: using Essential 6 medium containing 10 µM SB431542, 10 µM Hydrocortisone, 5 ng / mL Flt3L, 5 ng / mL TPO, 50 ng / mL SCF, 50 ng / mL EGF, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL IGF-1, 50 μg / mL ascorbic acid and 2% SUPERGROW cell culture supplement (Dakoway, 6122011)), continue induction for 14-16 days, changing the medium every 1-3 days, hematopoietic stem / progenitor cells (CD45+CD34+CD43+, such as Fig.11 ). The results showed that hFSC022 could successfully induce human pluripotent stem cells (hPSC) to differentiate into hematopoietic stem / progenitor cells (iHPC), and the proportion and number of iHPC were significantly higher than those in the OP9 control group, and it could replace mouse OP9 cells for the hematopoietic differentiation of hPSC.

[0048] hFSC022 cells were used to induce hPSC-derived iHPC cells. The cell density was adjusted to 2×10^4 / ml using IMDM (containing 2% FBS). Then, 100µl of cell suspension was mixed with 1ml of H4434 semi-solid medium. After sufficient oscillation, the mixture was allowed to stand at room temperature for 10 min, and then transferred to a 35 mm culture dish and placed in a carbon dioxide incubator for culture. After 14 days of culture, the culture dish was removed and representative colonies were photographed under a microscope according to their morphological characteristics ( Fig.12 The results showed that the iHPC cells derived from hPSCs induced by hFSC022 cells could successfully form BFU-E (burst-forming unit of erythrocytes), CFU-G (granulocyte colony-forming unit), CFU-M (macrophage colony-forming unit), CFU-GM (granulocyte / macrophage colony-forming unit) and CFU-Mix (mixed cell line colony-forming unit), suggesting that the iHPCs derived from hPSCs induced by hFSC022 cells showed hematopoietic multi-lineage differentiation ability.

[0049] hFSC022 cells were used to induce hPSC-derived iHPC cells and mixed with OP9 trophoblasts at a mixing ratio of 1:25. The total number of cells was adjusted to 500,000 according to the pore size of the culture dish to prepare hematopoietic differentiation cell capsules. NK cell induction differentiation medium was used to continue induction for 14 days, and the medium was changed every 1-3 days to obtain NK cells (CD45+CD3-CD56+CD16+ / -). The above cells were subjected to flow cytometry detection, and the phenotype was as follows Fig.13 The results showed that hFSC022 cells induced hPSC-derived iHPC cells to differentiate into iNK cells.

[0050] The hFSC022 cells were used to induce the iHPC cells derived from hPSCs and mixed with human thymus tissue blocks, and then placed in 3D microcarriers for induction differentiation. The total amount of iHPC cells can be adjusted to 500,000 (a 24-well plate was used in this example). T cell induction differentiation medium was used to continue induction for 28 days, and half of the medium was changed every 1-3 days to obtain T cells (CD45+CD3+CD4+ / CD8+). The above cells were subjected to flow cytometry detection, and the phenotype was as follows Fig.14 The results showed that hFSC022 cells induced hPSC-derived iHPC cells to differentiate into CD45+CD3+CD4+ iT cells and CD45+CD3+CD8+ iT cells.

[0051] Adjust the cell density of hFSC022 cells to induce hPSC-derived iHPC cells, adjust the seeding density to 500,000 according to the size of the culture dish (a 6-well plate is used in this example), and continue inducing with macrophage differentiation medium for 7-21 days, changing the medium every 1-3 days to obtain macrophages (CD45+CD14+CD11b+). The above cells were subjected to flow cytometry detection, and the phenotype was as follows Fig.15 The results showed that hFSC022 cells could efficiently induce hPSC-derived iHPC cells to differentiate into macrophages.

[0052] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A human bone marrow stromal cell line hFSC022, characterized in that: The human bone marrow stromal cell line hFSC022 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC NO. 46248.

2. The human bone marrow stromal cell line hFSC022 according to claim 1, characterized in that: The cell line has a spindle-shaped or irregular triangle shape when attached to the wall, an oval nucleus, a large nuclear-cytoplasmic ratio, and protrusions of different lengths extending outward from the cytoplasm, and grows like a fibroblast.

3. The human bone marrow stromal cell line hFSC022 according to claim 1, characterized in that: The cell line expresses CD29, CD44, CD105, CD271, and CD73 molecules; but does not express CD45 molecules.

4. The use of the human bone marrow stromal cell line hFSC022 according to any one of claims 1 to 3, characterized in that: The cell line is used as a trophoblast cell to support the maintenance and expansion of human CD34+ hematopoietic stem / progenitor cells in vitro.

5. The use according to claim 4, characterized in that: The human CD34+ hematopoietic stem / progenitor cells are derived from umbilical cord blood tissue, peripheral blood mobilization, bone marrow, and hPSC induced differentiation sources.

6. The use of the human bone marrow stromal cell line hFSC022 according to any one of claims 1 to 3, characterized in that: The cell line is used as a trophoblast cell to induce hematopoietic differentiation of human pluripotent stem cells (hPSC) to obtain hematopoietic stem / progenitor cells (iHPC).

7. The use according to claim 4, characterized in that: The obtained hematopoietic stem / progenitor cells have the ability to differentiate into multiple hematopoietic lineages.

8. The use according to claim 4, characterized in that: The obtained hematopoietic stem / progenitor cells can differentiate into lymphoid progenitor cells, myeloid progenitor cells, natural killer cells (NK cells), T cells, B cells, macrophages, neutrophils, monocytes, and erythrocytes.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the human bone marrow stromal cell line hFSC022 according to any one of claims 1 to 3.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension or emulsion.

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