Culture medium for amplifying hematopoietic stem cells and use thereof

Abstract

The application belongs to the field of biological medicine, and relates to a culture medium for expanding hematopoietic stem cells and application thereof. The application provides a composition comprising DJ001, UM171, SRT3025, ALA and Trolox. The culture medium containing the composition can be used for expanding hematopoietic stem cells, and is especially suitable for expansion culture of LT-HSCs. Increasing the content of LT-HSC cells is the key to improving the long-term implantation success rate of hematopoietic stem cells. The application of the present application has invented a culture medium for specifically expanding LT-HSC cells, which can expand the LT-HSC cells by more than 500 times in 14 days, and has great clinical treatment value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a culture medium for expanding hematopoietic stem cells and an application thereof. Background Art

[0002] Hematopoietic stem cells (HSCs) are adult stem cells with the capacity for self-renewal and multipotential differentiation. They are primarily found in the bone marrow, umbilical cord blood, and mobilized peripheral blood. Research has shown that HSCs, as adult stem cells, possess robust self-renewal and multipotential differentiation abilities, capable of directed proliferation and differentiation into various blood cell types, including myeloid and lymphoid lineages, thereby completing the entire hematopoietic system. Since the concept of bone marrow transplantation was first proposed in the 1950s, hematopoietic stem cell transplantation (HSCT) has become an effective treatment for a variety of hematologic disorders. According to statistics from the China Clinical Trial Registry (ChiCTR), over 190 clinical trials involving hematopoietic stem cell transplantation have been registered in my country since 2008. Beyond its traditional application in the treatment of hematologic malignancies, HSCT can also be used to treat a variety of solid tumors, including breast cancer, ovarian cancer, neuroblastoma, and small cell lung cancer. HSCT also has demonstrated therapeutic efficacy in immune system disorders, such as severe combined immunodeficiency syndrome (SCID).

[0003] At present, the sources of HSCs are mainly divided into the following four types: bone marrow (BM), mobilized peripheral blood (mPB), placenta and umbilical cord blood (UCB). Among them, UCB-HSCs have many advantages. First, compared with bone marrow and mobilized peripheral blood, the content of hematopoietic stem and progenitor cells (HSPCs) in umbilical cord blood is higher and has better biological characteristics. For example, studies have found that when inoculated at the same time, UCB-HSCs divide 24 hours earlier than BM-HSCs, and at the same in vitro culture time, the expansion generation of HSCs derived from umbilical cord blood is significantly higher than that of HSCs in bone marrow, which means that the cycle required for hematopoietic system reconstruction by UCB-HSCs is shorter. However, due to the limited absolute number of hematopoietic stem cells in a single umbilical cord blood, its application range is greatly limited. Usually, the volume of a single umbilical cord blood is about 50-150 ml, which contains nucleated cells and CD34 + The number of cells is generally 0.5-1×10 9 and 0.6-10.6×106 The cell dose required for clinical treatment is based on nucleated cells or CD34 + The cell count should be at least 2.5 × 10 7 / kg or 2×10 5 / kg patient weight. If the patient weighs 75kg, then at least 2×10 9 nucleated cells or 1.5 × 10 7 CD34 + cell.

[0004] It can be seen that the number of hematopoietic stem cells contained in a single cord blood unit is generally insufficient to meet the number of cells required for the treatment of an adult patient and can only be used to treat children and low-weight patients (<30 kg). In order to overcome the bottleneck problem of the absolute scarcity of hematopoietic stem cells in cord blood, many solutions have been proposed, such as improving the hematopoietic stem cell collection method from a single cord blood unit to increase cell yield, improving cell freezing and thawing methods to avoid the loss of hematopoietic stem cells during the process, simultaneously transfusing two cord blood units, and using cell culture technology to expand hematopoietic stem cells in vitro. Although the first two methods can maximize the acquisition and preservation of hematopoietic stem cells in a single cord blood unit, they still cannot significantly increase the total number of hematopoietic stem cells.

[0005] On the other hand, while the simultaneous transfusion of two cord blood units increases the number of hematopoietic stem cells transplanted, studies have shown that transfusing two cord blood units does not improve the delayed recovery of neutrophil and platelet counts seen in patients receiving cord blood hematopoietic stem cell transplants, but instead increases the risk of GVHD. It is particularly important to note that only one of the two cord blood units can be engrafted into the patient and achieve long-term hematopoietic and immune reconstitution. Furthermore, the simultaneous transplantation of two cord blood units increases the cost of clinical treatment. Clearly, none of these methods can fundamentally address the bottleneck of limited hematopoietic stem cell numbers. Therefore, how to effectively expand cord blood hematopoietic stem cells in vitro and increase their number has become an important topic and key technology in the current research on the clinical application of cord blood. Summary of the Invention

[0006] In some embodiments, the present invention provides a composition comprising DJ001 and UM171.

[0007] In some embodiments, the composition further comprises one or more of STR3025, ALA, and Trolox.

[0008] In some embodiments, the composition further comprises IL-3, G-CSF, and GM-CSF.

[0009] In some embodiments, the composition further comprises STR3025, ALA and Trolox simultaneously.

[0010] In some embodiments, the concentrations of DJ001 and UM171 are 50-200 ng / mL and 10-50 nM, respectively.

[0011] In some embodiments, the concentrations of DJ001 and UM171 are 50-150 ng / mL and 20-40 nM, respectively.

[0012] In some embodiments, the concentrations of DJ001 and UM171 are 50-120 ng / mL and 25-40 nM, respectively.

[0013] In some embodiments, the concentrations of STR3025, ALA and Trolox are 0.5-3 uM, 5-150 ug / ml and 50-600 nM, respectively.

[0014] In some embodiments, the concentrations of STR3025, ALA and Trolox are 0.5-2.5 uM, 10-150 ug / ml and 100-500 nM, respectively.

[0015] In some embodiments, the concentrations of STR3025, ALA and Trolox are 0.5-2 uM, 20-100 ug / ml and 200-500 nM, respectively.

[0016] In some embodiments, the present application provides use of the composition in the preparation of a medium for hematopoietic stem cell culture.

[0017] In some embodiments, the present application provides a medium comprising the composition.

[0018] In some embodiments, the medium is a serum-free medium.

[0019] In some embodiments, the medium further comprises a basal medium, including but not limited to one or a combination of DMEM / F12, DMEM, IMDM, knockout DMEM and a-MEM.

[0020] In some embodiments, the medium further comprises one or more of putrescine, selenium, insulin, transferrin, polyvinyl alcohol, B27, SCF, Flt3L and TPO.

[0021] In some embodiments, the culture medium further contains putrescine 50-150 μM, selenium 1-10 ng / mL, insulin 0.5-10 μg / mL, transferrin 20-100 μg / mL, polyvinyl alcohol 0.5-5 w / v%, B27 supplement 0.5-3% v / v, SCF 100-300 ng / mL, Flt3L 100-300 ng / mL and TPO 10-40 ng / mL.

[0022] In some embodiments, the culture medium further contains putrescine 80-120 μM, selenium 2-10 ng / mL, insulin 0.5-5 μg / mL, transferrin 30-60 μg / mL, polyvinyl alcohol 0.5-2 w / v%, B27 supplement 1-3% v / v, SCF 100-150 ng / mL, Flt3L 100-150 ng / mL, and TPO 10-30 ng / mL.

[0023] In some embodiments, the culture medium is a hematopoietic stem cell culture medium.

[0024] In some embodiments, the source of hematopoietic stem cells is selected from bone marrow, mobilized peripheral blood, placenta, or umbilical cord blood.

[0025] In some embodiments, the hematopoietic stem cells include CD133, CD34 + cells or LT-HSCs.

[0026] In some embodiments, the present invention provides use of the culture medium in expanding hematopoietic stem cells.

[0027] In some embodiments, the source of hematopoietic stem cells is selected from bone marrow, mobilized peripheral blood, placenta, or umbilical cord blood.

[0028] In some embodiments, the hematopoietic stem cells include CD133, CD34 + cells or LT-HSCs.

[0029] In some embodiments, the present invention provides a method for culturing hematopoietic stem cells, comprising culturing using a culture medium comprising the above-mentioned culture medium. In some embodiments, the hematopoietic stem cells are derived from bone marrow, mobilized peripheral blood, placenta, or umbilical cord blood.

[0030] Although the infusion of allogeneic hematopoietic stem cells can reconstruct the hematopoietic system, increasing evidence shows that HSC is a cell population with heterogeneous characteristics from the perspectives of cell proliferation, differentiation, self-renewal and lifespan, including multipotent progenitor cells (MPP), short-term HSC (ST-HSC) and long-term HSC (LT-HSC).

[0031] In the hematopoietic system, quiescent, long-term repopulating hematopoietic stem cells (LT-HSCs) reside at the apex of all mature blood cells. Their potential for self-renewal and asymmetric cell division maintains a naive multipotent pool throughout life. Under steady-state conditions, progeny derived from these cells rapidly proliferate in response to peripheral stimulation, replacing activated, exhausted, or aged blood cells. ST-HSCs and LT-HSCs are the most classically recognized HSC classifications. ST-HSCs and LT-HSCs are distinguished not only by phenotype but also by the duration of their ability to maintain repopulation. ST-HSCs and their progenitors have the ability to maintain normal hematopoiesis for 6–8 weeks, while LT-HSCs are HSCs that persist for more than 16 weeks. However, LT-HSCs are present at extremely low levels in hematopoietic tissues, with approximately 1 in 100,000 cells in the bone marrow. Umbilical cord blood is rich in a variety of hematopoietic stem / progenitor cells, containing approximately 10 times the number of different hematopoietic progenitor cell types observed in adult blood. Therefore, increasing the abundance of LT-HSCs is key to improving the long-term engraftment success of hematopoietic stem cells. This study invented a culture medium that specifically expands LT-HSC cells, which can expand LT-HSC cells more than 500 times within 14 days and has great clinical therapeutic value.

[0032] In some embodiments, the present invention provides the use of hematopoietic stem cells obtained by the method described in preparing drugs for treating diseases, including blood diseases, breast cancer, ovarian cancer, neuroblastoma or small cell lung cancer.

[0033] In some embodiments, the hematological disorder includes but is not limited to leukemia, aplastic anemia, or myelodysplastic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The effect of culture medium of different concentration groups on the expansion of HSCs. Figure 1 A: Flow cytometry analysis of the effects of different culture media on CD34 + The influence of the proportion of LT-HSCs cells; Figure 1B&C: Analysis of the proportion of the two cell types; Figure 1 D: Cell counting results after the culture was completed.

[0035] Figure 2 The effects of different culture methods on the expansion of HSCs. Figure 2 A: Schematic diagram of the culture method; Figure 2 B: Flow cytometry analysis of the effect of culture medium on CD34 + The influence of the proportion of LT-HSCs cells; Figure 2 C&D: Comparative analysis of the proportions of the two cell types in different groups; Figure 2 E: Cell counting results after culture.

[0036] Figure 3 To verify the amplification effect of different samples on the culture system. Figure 3 A: Flow cytometry analysis of CD34 expression between samples in the amplification culture system + The proportion of LT-HSCs cells; Figure 3 B&C: Analysis of the proportion of the two cell types among different samples; Figure 3 D: CD34 in two culture modes + Statistics of the proportion of LT-HSCs; Figure 3 E: Cell counting results during the culture process of the two culture modes.

[0037] Figure 4 The effect of the culture system on the expansion of CD34+ cells in peripheral blood and bone marrow after mobilization. Figure 4 A: Flow cytometry analysis of CD34 in peripheral blood and bone marrow after mobilization in the expansion culture system + The proportion of LT-HSCs cells; Figure 4 B&C: Analysis of the proportion of the two cell types between samples.

[0038] Figure 5 Comparison of trilineage differentiation abilities in different culture modes. Figure 5 A: Schematic diagram of cell colonies 10 days after inoculation (BFU-E: erythroid; CFU-E: erythroid; CFU-M: megakaryocyte; CFU-GM: granulocyte; CFU-GEMM: granulocyte-macrophage-megakaryocyte); Figure 5 B. Comparison of colony-forming ability. (*P<0.05, **P<0.01, ***P<0.001)

[0039] Figure 6 The effects of different culture media on cell proliferation. Figure 6 A: Flow cytometry analysis of the effects of different small molecule compounds on CD34 in the culture system + The influence of cell ratio; Figure 6B: Flow cytometry analysis of the effects of different small molecule compounds on the proportion of LT-HSCs in the culture system; Figure 6 C&D: Analysis of the proportion of the two cell types; E: Effects of different small molecule compounds on CD34 + The influence of cell expansion multiples.

[0040] Figure 7 The effects of different culture media on the expansion of HSCs. Figure 7 A: Flow cytometry analysis of the effects of different small molecule compounds on CD34 in the culture system + The influence of the proportion of LT-HSCs cells; Figure 7 B&C: Analysis of the proportion of the two cell types; Figure 7 D&E: Effect of insulin concentration on cell proliferation; Figure 7 F: Cell counting analysis during the culture process of each group. DETAILED DESCRIPTION

[0041] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0042] In the following examples herein, the formula of the basal medium (i.e., Basic) is as follows:

[0043] Table 1

[0044]

[0045]

[0046] Preparation method: Components 1-10 were prepared into 100X solutions, and then added to DMEM / F12 medium. The final concentrations of each component in the basal medium are shown in Table 1, thereby obtaining the basal medium (Basic).

[0047] The experimental materials and methods used in the examples herein are as follows:

[0048] 1. Experimental Materials

[0049] 1.1 Selection of research subjects: The hematopoietic stem cells selected in this study were umbilical cord blood CD34 + cells, mobilized peripheral blood CD34 + cells, bone marrow CD34 + cell.

[0050] 1.2 Drugs and Reagents

[0051] The main reagents used in this study are shown in Table 1.

[0052] Table 1 Reagent Description

[0053]

[0054]

[0055] 1.3 Solution preparation

[0056] Before the experiment begins, dissolve cytokines and small molecule compounds according to the reagent instructions to prepare a 1000× storage solution, and then dilute them in the corresponding proportion according to the requirements of each round of experiments for later use.

[0057] 1.4 Instruments and Equipment

[0058] The same instruments used in this part of the experiment are shown in Table 2.

[0059] Table 2 Instruments and equipment

[0060]

[0061]

[0062] 2. Experimental methods

[0063] 2.1 Isolation and Counting of UCB-HSCs

[0064] Remove the anticoagulated cord blood from the blood bag and transfer it to a clean 50ml centrifuge tube. Count the sample volume and add approximately half the volume of TBD lymphocyte separation medium to the 50ml centrifuge tube. Tilt the tube to a 45° angle and, using a pipette, slowly add the blood along the tube wall to the surface of the separation medium until a clear separation interface forms. Wash the centrifuge tube with sterile PBS and slowly drip the rinse solution into the centrifuge tube containing the separation medium. Transfer the centrifuge tube to a swing-out centrifuge and centrifuge at 1000 rpm for 15 minutes at room temperature. You will see distinct bands in the tube. Carefully transfer the buffy coat layer to a fresh tube and resuspend the cells in five times the volume of PBS. Centrifuge at 1000 rpm for 10 minutes at room temperature. Discard the supernatant to obtain the cell pellet. Repeat this washing process three times.

[0065] 2.2 Conventional culture of UCB-HSCs

[0066] Count the viable cells of the isolated cells and then inoculate equal amounts into the culture medium of the different groups. Place the inoculated culture plates in a 37°C, 5% CO2 incubator for conventional cell culture. Day 0 is designated as the day after inoculation. Thereafter, fresh culture medium should be added to the culture system every 36 hours, depending on the cell population, to ensure adequate growth factors. Strict sterility should be maintained throughout the entire operation.

[0067] 2.3 Colony formation assay

[0068] Return the methylcellulose culture medium to room temperature and add 1% blue chain double antibody to prevent contamination during the culture process. Take 3 ml of the prepared methylcellulose culture medium and transfer it to a 6 cm culture dish for use. Then wash the hematopoietic stem cells to be tested once with 1× PBS, remove the residual culture medium, resuspend the cells, count the viable cells, and dilute them to an appropriate concentration. Take 800-1000 hematopoietic stem cells and inoculate them into the prepared methylcellulose culture medium. Gently pipette and mix. Note that this step should avoid the generation of bubbles. Transfer the inoculated culture dish to a 37 degree Celsius CO2 constant temperature incubator, and on the 10th day after inoculation, count the BFU-E, CFU-E, CFU-G, CFU-M, CFU-GM, and CFU-GEMM under a microscope, and statistically analyze the differences between the samples in each group.

[0069] 2.4 Flow cytometry phenotyping

[0070] The hematopoietic stem cells to be tested were collected using Graphpad Prism and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 350 g for 5 min at room temperature to remove excess culture medium. 5 mL of 1× PBS was added to the cell pellet to resuspend the cells. The cells were gently pipetted to mix and centrifuged again to wash away any culture medium that may remain on the cell surface. The viable cells were counted and the number of cells was calculated based on the number of cells per 5-10 × 10 6 Resuspend the cells at a concentration of 100 μl / mL. Pipette 100 μl of the diluted cell suspension and add CD34 antibody, CD38 antibody, CD90 antibody, CD49f, and CD45RA antibody in sequence at a dosage of 5 μl / 10 μl. 5 Cells were surface labeled and incubated at 4°C in the dark for 20 minutes. The incubated cells were removed and centrifuged at 350g for 5 minutes. The supernatant was removed. An appropriate amount of 1× sterile PBS was then added to the cell pellet. Gently pipette and mix the cells. Centrifuge at 350g for 5 minutes. Repeat the wash cycle to remove excess unlabeled antibody. Resuspend the cell pellet in 500 μL of PBS and prepare for analysis.

[0071] 2.5 Statistical analysis

[0072] Graphpad prism7 statistical analysis software was used to perform statistical analysis on the data. The two groups were compared using the t-test, with *P < 0.05 indicating that the difference was statistically significant. Multiple groups were compared using ANOVA analysis, with *P < 0.05 also indicating that the difference was statistically significant.

[0073] Example 1 Culture medium formulation and its application

[0074] Culture medium formulations AB are divided into the following groups:

[0075] Group A: Basic+UM171 (35 nM)+DJ001 (100 ng / ml)+SRT3025 (1.5 uM);

[0076] Group B: Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+ALA(10ug / ml);

[0077] Group C: Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+Trolox(100nM);

[0078] Group D: Basic+UM171 (35 nM)+DJ001 (100 ng / ml)+SRT3025 (1.5 uM)+ALA (10 ug / ml)+Trolox (100 nM);

[0079] Group E: Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+ALA(50ug / ml)+Trolox(300nM);

[0080] Group F: Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+ALA(100ug / ml)+Trolox(500nM).

[0081] The culture medium is prepared as follows:

[0082] Preparation method: UM171, DJ001, SRT3025, ALA, and Trolox were dissolved in DMSO to prepare 1000X solutions, and then added to basic culture medium. The specific concentrations of the components in each group are shown in Groups A to F of this example.

[0083] According to the above “experimental method”, 1×10 5CD34 + The cells were inoculated into the culture medium and cultured in a fed-batch manner. Cell counts were performed on the 4th and 9th days of culture, and CD34 expression was analyzed by flow cytometry on the 14th day of expansion culture. + cells and LT-HSCs (CD34 + CD38-CD90 + CD45RA-CD49f + ) in the total cell population, the results are as follows Figure 1 shown.

[0084] Figure 1 Description of grouping in: Figure 1 The Basic group in the table refers to group A culture medium; Figure 1 ALA-Low in the table refers to Group B culture medium; Figure 1 Trolox-low in refers to Group C culture medium; Figure 1 Mix-Low in the table refers to the culture medium of group D; Figure 1 Mix-medium in the text refers to group E culture medium; Figure 1 Mix-high in the text refers to the F group culture medium.

[0085] The experimental results showed that different formulations and different small molecule compounds can affect LT-HSCs and CD34 + Taking into account the proportion of the two cell types and the number of cell expansion, the culture medium formula E group had the best effect.

[0086] Example 2 Selection of culture scheme

[0087] This round of culture will focus on two aspects: simulating the bone marrow niche and reducing the O2 content in the environment. The culture medium used is Formula E in Example 1.

[0088] Specific steps of the experiment:

[0089] Basic group or basic medium group: take 1×10 5 CD34 + The cells were added to the expansion medium (Formula E in Example 1) and cultured according to the "Conventional Culture of UCB-HSCs" method described in Example 1.

[0090] Basic+oil group (Mineral oil group): take 1×10 5 CD34 +The cells were added to the expansion medium (Formula E in Example 1), and 2 mL of sterile mineral oil was gently added to the surface of the culture medium. The cells were cultured according to the "Conventional Culture of UCB-HSCs" method described in Example 1.

[0091] Cultispher group: 1×10 5 CD34 + The cells were added with expansion medium (Formula E in Example 1) and 1% of the volume of the culture medium as a cultispher carrier, and cultured according to the "conventional culture of UCB-HSCs" method described in Example 1.

[0092] Cultispher+oil group: take 1×10 5 CD34 + The cells were added to expansion medium (Formulation E in Example 1), 1% of the culture volume of cultispher carrier, and 2 mL of sterile mineral oil was gently added to the surface of the culture medium.

[0093] The experimental diagram is as follows Figure 2 As shown in A. Each group was analyzed by flow cytometry and cell counting (cultured to day 14). The results are shown in Figure 2 The results show that both the addition of mineral oil and microcarriers to the culture medium are beneficial to increasing the proportion of LT-HSCs in the culture system, and the addition of macroporous gelatin microcarriers is more conducive to increasing the number of cells in terms of cell proliferation.

[0094] Note: The CD34 + The cells refer to the CD34 cells isolated and cultured according to the above-mentioned "experimental method" and obtained after magnetic bead sorting. + cell.

[0095] Example 3 Multiple Sample Repeated Verification

[0096] After confirming that the two culture methods significantly increased the proportion of LT-HSCs, the experiments in this example began to use multiple samples to further verify the stability of the expansion culture. At the same time, this round of experiments also verified the expansion culture medium of the present invention on the CD34 + Comparison of cell expansion effects and the three-component differentiation ability of cells after expansion and culture. The specific results are shown below:

[0097] 1. Repeatability of amplification culture medium between samples

[0098] The amplification culture system of Example 1 medium formula E has good reproducibility between samples, such as Figure 3As shown in B&C, the CD34 + There was no significant difference in the proportion of cells and LT-HSCs. In addition, the number of cell proliferation ( Figure 3 E), the amplified number of macroporous gelatin microcarriers was significantly higher than that of the oil-sealed group.

[0099] Remark: Figure 3 The cell seeding density of each source in A-3E was the same, with an initial seeding density of 2×10 5 / mL, cultured for 14 days and then flow cytometry was performed. The results were as follows Figure 3 As shown in A-3E.

[0100] The Cultispher group and the Mineral oil group in this embodiment are the same as those in Example 2.

[0101] 2. Comparative experiment on the expansion culture of HSCs from different sources using expansion medium

[0102] Combined with the culture medium formula E of Example 1, the results are as follows Figure 4 As shown in A-4C, CD34 + The cell content shows that within the same culture period, this expansion culture system can be used to culture CD34 cells from mobilized peripheral blood. + The cells showed significant proliferation and were higher than those in umbilical cord blood CD34 + However, from the perspective of the proportion of LT-HSCs, the proportion of LT-HSCs obtained by culturing cells derived from umbilical cord blood using medium formula E in Example 1 was significantly higher than that of cells derived from peripheral blood and bone marrow after mobilization. + The invention further illustrates that the expansion culture medium of the present invention has a specific expansion effect on LT-HSCs derived from umbilical cord blood, and is particularly suitable for the expansion culture of LT-HSCs derived from umbilical cord blood.

[0103] Remark: Figure 4 The cell seeding density of each source in A-4C was the same, with an initial seeding density of 2×10 5 / mL, cultured for 14 days and then flow cytometry was performed. The results were as follows Figure 4 As shown in A-4C.

[0104] in Figure 4 UCB-HSCs-1 and UCB-HSCs-2 shown in B represent cells derived from two different individuals.

[0105] 3. Detection of the three-dimensional differentiation ability of cells

[0106] After 14 days of expansion culture, cells were collected and counted, and 10 3 The cells were inoculated into hematopoietic stem cell differentiation medium (Stemcell, Cat. No. 04434) and cultured for 14 days. Figure 5 As shown, significant differences in trilineage differentiation capacity were observed between HSCs cultured in the two culture methods and those isolated from fresh cord blood. Both cultures exhibited higher lineage differentiation capacity than the unexpanded group, demonstrating that expanded HSCs possessed enhanced hematopoietic differentiation capacity. Furthermore, no significant differences in colony morphology were observed after expansion, demonstrating the stability of the expansion system.

[0107] in, Figure 5 ( Figure 5 A-5B):

[0108] (1) Un-expanded group (also known as “fresh” group): refers to HSCs isolated from fresh umbilical cord blood directly added to the trilineage differentiation medium;

[0109] (2) Cultispher group: HSCs isolated from fresh umbilical cord blood were cultured using the medium E of Example 1 and 1% of the volume of the culture medium was added to the microcarriers for 14 days (the cell expansion culture stage was the same as that of the Cultispher group in Example 2) and then inoculated into the trilineage differentiation medium;

[0110] (3) Mineral oil group: HSCs isolated from fresh umbilical cord blood were cultured using the medium E of Example 1, and 2 mL of mineral oil was added to isolate the air and cultured for 14 days (the cell expansion culture stage was the same as the Basic+oil group in Example 2) and then inoculated into the tri-lineage differentiation medium.

[0111] Example 4 Culture medium formulation and HSCs culture

[0112] Media formulations are divided into the following groups:

[0113] (1)Basic

[0114] (2) Basic+SR1 (750nM)

[0115] (3) Basic+UM171 (35nM)

[0116] (4) Basic+CHIR99021(1uM)

[0117] (5) Basic+GW9662(1uM)

[0118] (6) Basic+ALA (100ug / ml)

[0119] (7) Basic + NAM (4ug / ml)

[0120] (8) Basic + DJ001 (100ng / ml)

[0121] (9) Basic + Trolox (0.5uM)

[0122] (10) Basic + SRT3025 (1.5uM)

[0123] The medium preparation method of the (1)-(10) groups is as follows:

[0124] The additives SR1, UM171, CHIR99021, GW9662, ALA, NAM, DJ001, Trolox, SRT3025 of each group are respectively dissolved with DMSO to configure 1000X solution, and then added to the Basic medium to the final concentration.

[0125] According to the above "experimental method", the separation and culture of UCB-HSCs are carried out, and 1x10 5 CD34 + cells obtained by magnetic bead sorting are inoculated into the medium, and the fed-batch culture is used, and the cell counting is carried out on the 4th day and the 9th day of culture, and the flow cytometry analysis is carried out on the 14th day of expansion culture CD34 + cells and LT-HSCs (CD34 + CD38-CD90 + CD45RA-CD49f + ) in the total cell population, and the results of this round of expansion are shown in Figure 6 .

[0126] As can be seen from Figure 6 ( Figure 6 A-6E), the sensitivity of UCB-HSCs (umbilical cord blood-derived HSCs) to different small molecule compounds is different. After being treated with different small molecule compounds, part of the cell population in the expansion group is obviously different.

[0127] As shown in Figure 6 A, in the CD34 + cell population, SR1 has obvious four cell groups, while the cell groups of UM171, CHIR99021, NAM and SRT3025 are relatively single, indicating that the cell differentiation is weak.

[0128] As shown in Figure 6As shown in B, from the perspective of the cell proportion of LT-HSCs, the percentage of LT-HSCs in the UM171, NAM, DJ001 and SRT3025 groups was higher, and after adding the small molecule compounds in each group, both CD34 + The proportion of cells and LT-HSCs in the culture medium was significantly higher than that in the single-component basal medium. In terms of the number of cells expanded, on the fourth day of culture, there was little difference in the number of cells expanded among the different groups, but they were all higher than those in the basal medium group.

[0129] Example 5: Formulation of culture medium and culture of HSCs

[0130] Media formulations are divided into the following groups:

[0131] a)Basic+UM171(35nM)+DJ001(100ng / ml);

[0132] b)Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM);

[0133] c)Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+ALA(100ug / ml);

[0134] d)Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+ALA(100ug / ml)+Trolox(0.5uM);

[0135] e)Basic+UM171(35nM)+DJ001(100ng / ml)+SRT3025(1.5uM)+ALA(100ug / ml)+Trolox(0.5uM)+IL-3(15ng / ml)+G-CSF(10ng / ml)+GM-CSF(10ng / ml);

[0136] f) The culture medium of group e) was changed to a low concentration of insulin (25 ng / ml);

[0137] g) Based on the culture medium of group e), the insulin concentration was changed to a medium concentration (1 μg / ml);

[0138] h) Based on the culture medium of group e), the insulin concentration was changed to a high concentration (25ug / ml).

[0139] According to the above “experimental method”, 1×10 5 CD34+ The cells were inoculated into the culture medium and cultured in a fed-batch manner. Cell counts were performed on the 4th and 9th days of culture, and CD34 expression was analyzed by flow cytometry on the 14th day of expansion culture. + cells and LT-HSCs (CD34 + CD38-CD90 + CD45RA-CD49f + ) in the total cell population, the results of this round of expansion are as follows Figure 7 A-7F shown.

[0140] After this round of expansion, Figure 7 A It can be clearly seen that when a variety of small molecule compounds are added to the culture medium, CD34 + The cell clustering phenomenon is significantly weakened, indicating that the cell types in the expansion system tend to be single, and the proportion of LT-HSCs also increases with the increase in the number of compound types. On the other hand, it is worth noting that with the increase in the number of small molecule compounds, the total number of cell expansion in the culture system shows an inverse trend. This indirectly reflects that too many small molecule compounds may have inhibited cell expansion to a certain extent, causing the cells to be in a relatively static state to a large extent. Figure 7 B and Figure 7 In C, the culture medium of group E is supplemented with three cytokines: IL-3, G-CSF, and GM-CSF. It can be seen that although the addition of these three cytokines promoted cell proliferation to a certain extent, it was weak in maintaining stemness ( Figure 7 The proportion of LT-HSCs in C decreased significantly, indicating a decrease in the proportion of stem cells), and there was no increase in the proportion, especially the proportion of LT-HSCs decreased significantly. In addition, when the insulin concentration was changed, there was indeed a change in the cell proportion ( Figure 7 D and Figure 7 E). Therefore, in subsequent experiments, an intermediate concentration of insulin, i.e., 1 ug / mL, was selected for subsequent culture.

Claims

1. A hematopoietic stem cell culture medium, characterized in that The culture medium contains DJ001, UM171, SRT3025, ALA and Trolox.

2. The culture medium according to claim 1, wherein The culture medium also contains IL-3, G-CSF and GM-CSF.

3. The culture medium according to claim 1, wherein The concentrations of DJ001 and UM171 are 50-200 ng / mL and 10-50 nM, respectively.

4. The culture medium according to claim 1, wherein The concentrations of DJ001 and UM171 are 50-150 ng / mL and 20-40 nM, respectively.

5. The culture medium according to claim 1, wherein The concentrations of DJ001 and UM171 are 50-120 ng / mL and 25-40 nM, respectively.

6. The culture medium according to claim 1, wherein The concentrations of SRT3025, ALA and Trolox are 0.5~3 uM, 5~150 ug / ml and 50~600 nM, respectively.

7. The culture medium according to claim 1, wherein The concentrations of SRT3025, ALA and Trolox are 0.5-2.5 uM, 10-150 ug / ml and 100-500 nM, respectively.

8. The culture medium according to claim 1, wherein The concentrations of SRT3025, ALA and Trolox are 0.5~2 uM, 20~100 ug / ml and 200~500 nM, respectively.

9. The culture medium according to any one of claims 1 to 8, wherein The culture medium is a serum-free culture medium.

10. The culture medium according to any one of claims 1 to 8, wherein The culture medium further contains a basal culture medium, including one or a combination of DMEM / F12, DMEM, IMDM, knockout DMEM and a-MEM.

11. The culture medium according to any one of claims 1 to 8, wherein The culture medium further contains one or more of putrescine, selenium, insulin, transferrin, polyvinyl alcohol, B27, SCF, Flt3L and TPO.

12. The culture medium according to any one of claims 1 to 8, wherein The culture medium also contains putrescine 50-150 μM, selenium 1-10 ng / mL, insulin 0.5-10 μg / mL, transferrin 20-100 μg / mL, polyvinyl alcohol 0.5-5 w / v%, B27 supplement 0.5-3% v / v, SCF 100-300 ng / mL, Flt3L 100-300 ng / mL and TPO 10-40 ng / mL.

13. The culture medium according to any one of claims 1 to 8, wherein The culture medium also contains putrescine 80-120 μM, selenium 2-10 ng / mL, insulin 0.5-5 μg / mL, transferrin 30-60 μg / mL, polyvinyl alcohol 0.5-2 w / v%, B27 supplement 1-3% v / v, SCF 100-150 ng / mL, Flt3L 100-150 ng / mL, and TPO 10-30 ng / mL.

14. Use of the culture medium according to any one of claims 1 to 8 for expanding hematopoietic stem cells.

15. The use according to claim 14, characterized in that The hematopoietic stem cell source is selected from bone marrow, mobilized peripheral blood, placenta or umbilical cord blood.

16. The use according to claim 14, characterized in that The hematopoietic stem cells include CD34 + cells or LT-HSCs.

17. A method for culturing hematopoietic stem cells, characterized in that: The culture medium according to any one of claims 1 to 8 is used for culturing.

18. The method according to claim 17, wherein The hematopoietic stem cell source is selected from bone marrow, mobilized peripheral blood, placenta or umbilical cord blood.

19. The method according to claim 17, wherein The hematopoietic stem cells include CD34 + cells or LT-HSCs.

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