A method for efficient expansion of cd34 + hematopoietic stem cells and inducing their differentiation into cdc1 and applications thereof

Abstract

The application belongs to the technical field of cell biology and immunology, and particularly relates to a method for efficiently amplifying CD34 + hematopoietic stem cells and inducing differentiation of the hematopoietic stem cells into type 1 classical dendritic cells (cDC1) and application thereof. The application provides a first medium composition for amplifying CD34 + hematopoietic stem cells, which comprises SCF, Flt3L, TPO, IL-3 and IL-6; and the application also provides a second medium composition for inducing differentiation of the amplified CD34+ cells into cDC1, which comprises SCF, Flt3L, GM-CSF, IL-4, IFN-gamma, IFN-beta and TNF-alpha. The first medium composition of the application can significantly improve the amplification multiple of CD34+ cells, and the amplified cells have stronger potential for differentiation into cDC1; the cDC1 after induction and differentiation has significantly enhanced T cell activation function. Moreover, the cDC1 prepared by the method of the application has sufficient quantity and good function, and can be used for preparing anti-tumor drugs or tumor vaccines after loading tumor antigens, and has broad clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and immunology, specifically relating to a method for efficiently amplifying CD34 through optimized cytokine combinations. + This invention relates to a method for cultivating hematopoietic stem cells, and a method for efficiently inducing the expanded cells to differentiate into type 1 classical dendritic cells (cDC1). The invention also relates to the application of the above method in the preparation of cell therapy products, tumor vaccines, and drug screening. Background Technology

[0002] Dendritic cells (DCs) are the most potent professional antigen-presenting cells in the body, playing a central role in initiating and regulating the immune response. Among them, type 1 classical dendritic cells (cDC1) possess a unique cross-presentation capability, enabling them to efficiently present exogenous antigens to CD8 cells. + T cells, which induce specific anti-tumor immune responses, have become a research hotspot in the field of tumor immunotherapy.

[0003] However, cDC1 is scarce in vivo, and there is a lack of efficient and stable in vitro amplification methods, which severely restricts its clinical application. Currently, the in vitro preparation of cDC1 mainly relies on isolating CD34 from umbilical cord blood or peripheral blood. + Hematopoietic stem cells are then induced to differentiate through a combination of cytokines. Existing techniques have reported the use of cytokines such as SCF, Flt3L, and TPO to expand CD34+ cells and the use of factors such as GM-CSF, IL-4, and IFN-γ to induce their differentiation into cDC1. However, the expansion and differentiation efficiency still need to be improved, making it difficult to meet the needs of large-scale clinical applications.

[0004] Therefore, it is necessary to develop a method that can efficiently amplify CD34. + The method of stimulating hematopoietic stem cells and significantly improving their differentiation efficiency into cDC1 has important scientific value and clinical application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient method for amplifying CD34. + Culture medium composition for inducing hematopoietic stem cells to differentiate into cDC1 and its application.

[0006] In a first aspect, the present invention provides a first culture medium composition for efficient amplification of CD34. + Hematopoietic stem cells, wherein the first culture medium composition comprises a basal culture medium and a cytokine combination, the cytokine combination comprising: (a) Stem cell factor (SCF) 20-200 ng / mL; (b) Fms-like tyrosine kinase 3 ligand (Flt3L) 20-200 ng / mL; (c) Thrombopoietin (TPO) 5-150 ng / mL; (d) Interleukin-3 (IL-3) 5-50 ng / mL; (e) Interleukin-6 (IL-6) 5-100 ng / mL; The basal culture medium is StemSpan. TM SFEM or SFEM II medium.

[0007] Preferably, the concentration of SCF is 40-120 ng / mL, the concentration of Flt3L is 40-120 ng / mL, the concentration of TPO is 10-100 ng / mL, the concentration of IL-3 is 10-40 ng / mL, and the concentration of IL-6 is 10-80 ng / mL.

[0008] Preferably, the concentration of SCF is 50-100 ng / mL, the concentration of Flt3L is 50-100 ng / mL, the concentration of TPO is 20-50 ng / mL, the concentration of IL-3 is 15-30 ng / mL, and the concentration of IL-6 is 15-60 ng / mL.

[0009] Preferably, the concentration of SCF is 100 ng / mL, the concentration of Flt3L is 100 ng / mL, the concentration of TPO is 25 ng / mL, the concentration of IL-3 is 20 ng / mL, and the concentration of IL-6 is 20 ng / mL.

[0010] Preferably, the concentration of SCF is 50 ng / mL, the concentration of Flt3L is 50 ng / mL, the concentration of TPO is 25 ng / mL, the concentration of IL-3 is 20 ng / mL, and the concentration of IL-6 is 20 ng / mL.

[0011] Secondly, the present invention provides a second culture medium composition for incorporating CD34 + Hematopoietic stem cells are induced to differentiate into cDC1. The second culture medium composition comprises a basal culture medium and a cytokine combination, wherein the cytokine combination includes: (a) Stem cell factor (SCF) 20-200 ng / mL; (b) Fms-like tyrosine kinase 3 ligand (Flt3L) 20-200 ng / mL; (c) Granulocyte-macrophage colony-stimulating factor (GM-CSF) 1-20 ng / mL; (d) Interleukin-4 (IL-4) 1-20 ng / mL; (e) Interferon-γ (IFN-γ) 2-50 ng / mL; (f) Interferon-β (IFN-β) 2-50 ng / mL; (g) Tumor necrosis factor-α (TNF-α) 2-50 ng / mL; The preferred basal culture medium is StemSpan. TM SFEM or SFEM II medium.

[0012] Preferably, the concentration of SCF is 40-120 ng / mL, the concentration of Flt3L is 40-120 ng / mL, the concentration of GM-CSF is 2-10 ng / mL, the concentration of IL-4 is 2-10 ng / mL, the concentration of IFN-γ is 3-20 ng / mL, the concentration of IFN-β is 3-20 ng / mL, and the concentration of TNF-α is 3-20 ng / mL.

[0013] Preferably, the concentration of SCF is 50-100 ng / mL, the concentration of Flt3L is 50-100 ng / mL, the concentration of GM-CSF is 2.5-5 ng / mL, the concentration of IL-4 is 2.5-5 ng / mL, the concentration of IFN-γ is 5-10 ng / mL, the concentration of IFN-β is 5-10 ng / mL, and the concentration of TNF-α is 5-10 ng / mL.

[0014] Preferably, the concentration of SCF is 100 ng / mL, the concentration of Flt3L is 100 ng / mL, the concentration of GM-CSF is 2.5 ng / mL, the concentration of IL-4 is 2.5 ng / mL, the concentration of IFN-γ is 5 ng / mL, the concentration of IFN-β is 5 ng / mL, and the concentration of TNF-α is 5 ng / mL.

[0015] Preferably, the first culture medium composition and the second culture medium composition further contain antibiotics.

[0016] Preferably, the antibiotics are penicillin and streptomycin.

[0017] Thirdly, this invention provides a method for efficiently expanding CD34+ hematopoietic stem cells, comprising the following steps: Separate CD34 +Hematopoietic stem cells were seeded into a culture vessel containing the first culture medium composition and cultured under suitable conditions to obtain CD34. + Hematopoietic stem cells.

[0018] Preferably, the culture conditions are 5-15 days in an incubator at 37°C and 5% CO2, more preferably 8-12 days, and even more preferably 9-10 days.

[0019] Preferably, the CD34 + The seeding density of hematopoietic stem cells is 1×10⁻⁶. 5 per mL.

[0020] Preferably, the culture container is a 24-well plate, a 6-well plate, or a culture flask.

[0021] Preferably, during the culture process, half or all of the medium is replaced every 2-4 days.

[0022] In one specific embodiment, after culturing with the first culture medium composition of the present invention (G4 group: SCF 50 ng / mL + Flt3L 50 ng / mL + TPO 25 ng / mL + IL-3 20 ng / mL + IL-6 20 ng / mL) for 4 days, the CD34+ cell expansion reached more than 2.34 times, which was significantly higher than the control group without IL-3 and IL-6.

[0023] Fourthly, the present invention provides a method for amplifying CD34 + A method for inducing hematopoietic stem cells to differentiate into cDC1 includes the following steps: CD34 amplified by the method described in the third aspect + Hematopoietic stem cells were seeded in a culture vessel containing a second culture medium composition and cultured under suitable conditions to obtain cDC1.

[0024] Preferably, the culture conditions are 7-14 days in an incubator at 37°C and 5% CO2, more preferably 10-12 days.

[0025] Preferably, the CD34 + The seeding density of hematopoietic stem cells is 5×10⁻⁶. 5 per mL.

[0026] Preferably, during the culture process, half or all of the medium is replaced every 3-5 days.

[0027] In one specific embodiment, after culturing for 10 days using the second culture medium composition of the present invention, cDC1 (LIVEHLA-DR) + Lin - XCR1 +CD141 + The proportion of induced blasts was more than 1.53 times that of the conventional induction group, which was significantly higher than that of the control group induced by conventional methods.

[0028] Fifthly, the present invention provides cDC1 cells prepared by the above method.

[0029] In a sixth aspect, the present invention provides the application of the above-mentioned cDC1 cells in activating T cells.

[0030] In a seventh aspect, the present invention provides the application of the above-mentioned cDC1 cells in the preparation of antitumor drugs or tumor vaccines.

[0031] Preferably, the cDC1 cells are loaded with tumor antigens.

[0032] Preferably, the tumor antigen is selected from tumor cell lysates, inactivated whole-cell tumor antigens, tumor-specific peptides, or mRNA encoding tumor antigens.

[0033] The first culture medium composition of the present invention can significantly increase the expansion fold of CD34+ cells, and the expanded cells have a stronger potential to differentiate into cDC1. Moreover, the cDC1 cells prepared by the method of the present invention are sufficient in quantity and have good function, and can be loaded with tumor antigens for the preparation of anti-tumor drugs or tumor vaccines, which has broad clinical application prospects.

[0034] The following will further explain the concept, specific structure, and technical effects of the present invention in order to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0035] Figure 1 Different combinations of cytokines affect CD34 + The effect of hematopoietic stem cell expansion fold.

[0036] Figure 2 This relates to the effect of different amplification conditions on the subsequent cDC1 differentiation rate.

[0037] Figure 3 This is a functional evaluation of cDC1 activation of T cells after induced differentiation. Detailed Implementation

[0038] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0039] Example 1: Isolation and Expansion of CD34+ Hematopoietic Stem Cells 1.1 Isolation of CD34+ hematopoietic stem cells Freshly collected umbilical cord blood was diluted 1:1 with PBS. Lymphoprep (stemcell, 18061) was used as the separation medium, and the diluted blood sample was slowly added to the top layer of the separation medium. The mixture was centrifuged at 1200g for 20 minutes. The white membrane layer (monuclear cell layer) was collected and washed twice with PBS. CD34 positive sorting kit (EasySep Hu CB CD34 PosSel Kit II, stemcell, 17896) was used according to the manufacturer's instructions to obtain CD34. + Hematopoietic stem cells. Flow cytometry analysis showed CD34 levels. + Cell purity >90%.

[0040] 1.2 CD34 + Expansion of hematopoietic stem cells The sorted CD34 + Cells were resuspended in StemSpan™ SFEM II medium (containing 1% penicillin / streptomycin) and the cell density was adjusted to 1×10⁶ cells / year. 5 1 mL / well was inoculated into a 24-well plate.

[0041] Set up the following four different combinations of cytokines: Group G1: SCF 100 ng / mL + Flt3L 100 ng / mL + TPO 100 ng / mL Group G2: SCF 50 ng / mL + Flt3L 50 ng / mL + TPO 25 ng / mL Group G3: SCF 100 ng / mL + Flt3L 100 ng / mL + TPO 100 ng / mL + IL-3 20 ng / mL + IL-6 20 ng / mL Group G4: SCF 50 ng / mL + Flt3L 50 ng / mL + TPO 25 ng / mL + IL-3 20 ng / mL + IL-6 20 ng / mL Cells were incubated at 37℃ in a 5% CO2 incubator for 4 days. After 4 days, cells from each well were collected and viable cell counts were performed using trypan blue staining. The relative fold increase of each group was calculated using the fold increase of group G1 as a baseline (set as 1). The results are shown in Table 1 and... Figure 1 As shown.

[0042] Table 1. Effects of different cytokine combinations on CD34 + Effect of cell expansion fold

[0043] The above results indicate that the amplification folds of groups G3 and G4 were significantly higher than those of groups G1 and G2, suggesting that the addition of IL-3 and IL-6 synergistically promotes the expansion of CD34+ cells. Furthermore, even with reduced SCF, Flt3L, and TPO concentrations (G4 group), the amplification effect was superior to the high-concentration group (G3 group). This demonstrates that the addition of IL-3 and IL-6 can compensate for the decreased amplification efficiency caused by reducing SCF, Flt3L, and TPO, and may even produce a synergistic effect. The addition of IL-3 and IL-6 can significantly promote the expansion of CD34+ cells. + Cell expansion.

[0044] Example 2: Amplified CD34 + Induction of cell differentiation into cDC1 CD34 samples were collected from each group 4 days after amplification in Example 1. + Cells were washed once with PBS. The cells were then resuspended in StemSpan. TM In SFEM II medium (containing 1% penicillin / streptomycin), adjust the cell density to 5 × 10⁶ cells / year. 5 1 mL / well was inoculated into a 24-well plate.

[0045] Add the following cytokine combination to each well: SCF 100 ng / mL + Flt3L 100 ng / mL + GM-CSF 2.5 ng / mL + IL-4 2.5 ng / mL + IFN-γ 5 ng / mL + IFN-β 5 ng / mL + TNF-α 5 ng / mL. (This combination is the second culture medium composition described in this invention.) Incubate at 37℃ in a 5% CO2 incubator for 10 days, changing half the medium every 4 days.

[0046] Cells were collected after 10 days for flow cytometry analysis. The following antibody combination was used: LIVE / DEAD live / dead dye, anti-HLA-DR, Lineage antibody mixture (CD3 / CD19 / CD20 / CD56), anti-XCR1, and anti-CD141. cDC1 was defined as LIVE HLA-DR. + Lin - XCR1 + CD141 + .

[0047] Using the proportion of cDC1 cells induced from differentiation in group G1 as a baseline (set as 1), the relative proportion of cDC1 cells in each group was calculated. The results are shown in Table 2 and... Figure 2 As shown.

[0048] Table 2. Effects of different amplification conditions on the subsequent cDC1 differentiation rate

[0049] The above results show that regardless of the amplification conditions (G1-G4 group) from which CD34+ cells originate, the second culture medium composition of the present invention can successfully induce them to differentiate into cDC1, proving that the composition has good differentiation support ability and universality.

[0050] In addition, CD34 amplified with added IL-3 and IL-6 was used + Cells (G3 and G4 groups) showed significantly higher efficiency in differentiating into cDC1 than cells expanded without IL-3 / IL-6 (G1 and G2 groups). In particular, the G4 group (expanded with low concentrations of SCF / Flt3L / TPO + IL-3 / IL-6) exhibited an approximately 53% higher cDC1 differentiation rate compared to the G1 group. This demonstrates that the expansion method of this invention not only increases the number of CD34+ cells but also enhances their potential to differentiate into cDC1, and the second culture medium composition efficiently converts this potential into functional cDC1 cells.

[0051] Example 3: Functional evaluation of cDC1 in vitro activation of T cells Natural cDC1 (n-cDC1) and cDC1 induced from the G4 group in Example 2 were isolated from peripheral blood of healthy individuals. After collection, the cDC1 samples were washed twice with PBS and resuspended in RPMI 1640 medium containing 10% FBS. CD3 groups were isolated from peripheral blood of healthy individuals using a magnetic bead sorting kit. + T cells were then sorted and incubated with 5 μM CFSE at 37°C in the dark for 10 minutes. A stop label was added with 5 volumes of pre-cooled complete culture medium, and the cells were washed twice and resuspended for later use. Co-culture systems were set up as follows: PBS group (T cells cultured alone with an equal volume of PBS), n-cDC1 group (T cells: n-cDC1 = 10:1), and i-cDC1 group (T cells: i-cDC1 = 10:1). Each group was seeded into 96-well U-shaped plates and co-cultured for 5 days at 37°C in a 5% CO2 incubator. After co-culture, cells from each group were collected, and surface staining was performed using flow cytometry. CFSE dilution (FITC channel) was detected to assess T cell proliferation.

[0052] The results show that ( Figure 3 (a and b) Compared with the PBS group, both the n-cDC1 group and the i-cDC1 group were able to induce CD4+. + and CD8 +T cell proliferation was observed, with the i-cDC1 group showing significantly higher proliferation efficiency than the n-cDC1 group. To further evaluate T cell function, brefidobacterium A (BFA, final concentration 10 μg / mL) was added during the last 6 hours of co-culture to block cytokine secretion. After cell collection, surface staining (anti-CD8-BV711) was performed, followed by fixation, membrane rupture, and intracellular staining (anti-IFN-γ-BV605 and anti-Ki67-FITC). Flow cytometry results showed that ( Figure 3 cd), i-cDC1 group CD8⁺ T cells IFN-γ + Ratio and Ki67 + The proportions were significantly higher than those in the n-cDC1 group and the PBS group. These results indicate that the cDC1 prepared in this invention has a stronger stimulatory effect on T cell proliferation and induces CD8+. + The ability of T cells to produce IFN-γ and express Ki67 suggests that they have superior immune activation function in anti-tumor immune responses.

[0053] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A first culture medium composition for efficient amplification of CD34 + Hematopoietic stem cells, wherein the first culture medium composition comprises a basal culture medium and a cytokine combination, the cytokine combination comprising: (a) Stem cell factor (SCF) 20-200 ng / mL; (b) Fms-like tyrosine kinase 3 ligand (Flt3L) 20-200 ng / mL; (c) Thrombopoietin (TPO) 5-150 ng / mL; (d) Interleukin-3 (IL-3) 5-50 ng / mL; (e) Interleukin-6 (IL-6) 5-100 ng / mL; The basal culture medium is StemSpan. TM SFEM or SFEM II medium.

2. The first culture medium composition according to claim 1, characterized in that, The concentration of SCF is 40-120 ng / mL, the concentration of Flt3L is 40-120 ng / mL, the concentration of TPO is 10-100 ng / mL, the concentration of IL-3 is 10-40 ng / mL, and the concentration of IL-6 is 10-80 ng / mL.

3. A second culture medium composition for incorporating CD34 + Hematopoietic stem cells are induced to differentiate into cDC1. The second culture medium composition comprises a basal culture medium and a cytokine combination, wherein the cytokine combination includes: (a) Stem cell factor (SCF) 20-200 ng / mL; (b) Fms-like tyrosine kinase 3 ligand (Flt3L) 20-200 ng / mL; (c) Granulocyte-macrophage colony-stimulating factor (GM-CSF) 1-20 ng / mL; (d) Interleukin-4 (IL-4) 1-20 ng / mL; (e) Interferon-γ (IFN-γ) 2-50 ng / mL; (f) Interferon-β (IFN-β) 2-50 ng / mL; (g) Tumor necrosis factor-α (TNF-α) 2-50 ng / mL; The preferred basal culture medium is StemSpan. TM SFEM or SFEM II medium.

4. The second culture medium composition according to claim 3, characterized in that, The concentrations of the SCF, Flt3L, GM-CSF, IL-4, IFN-γ, IFN-β, and TNF-α are 40-120 ng / mL, 40-120 ng / mL, 2-10 ng / mL, 2-10 ng / mL, 3-20 ng / mL, 3-20 ng / mL, and 3-20 ng / mL, respectively.

5. The first culture medium composition according to claim 1 or the second culture medium composition according to claim 3, characterized in that, The first and second culture medium compositions further contain antibiotics; preferably, the antibiotics are penicillin and streptomycin.

6. A highly efficient method for amplifying CD34 + The hematopoietic stem cell method includes the following steps: Separate CD34 + Hematopoietic stem cells were seeded into a culture vessel containing the first culture medium composition and cultured under suitable conditions to obtain CD34. + Hematopoietic stem cells.

7. A method for amplifying CD34 + A method for inducing hematopoietic stem cells to differentiate into cDC1 includes the following steps: CD34 amplified by the method described in the third aspect + Hematopoietic stem cells were seeded in a culture vessel containing a second culture medium composition and cultured under suitable conditions to obtain cDC1.

8. cDC1 cells prepared by the method of claim 7.

9. The use of the cDC1 cells according to claim 8 in the preparation of antitumor drugs or tumor vaccines. The use of the cDC1 cells prepared by the method according to any one of claims 1-8 in the preparation of T cell activators or immune enhancers.

10. The application according to claim 9, characterized in that, The cDC1 cells are loaded with tumor antigens; the tumor antigens are selected from tumor cell lysates, inactivated whole-cell tumor antigens, tumor-specific peptides, or mRNA encoding tumor antigens.

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