Application of megakaryocyte in hematopoietic stem cell transplantation

Abstract

The invention relates to medical immunology, in particular to application of megakaryocytes in hematopoietic stem cell transplantation. According to the embodiment of the invention, the megakaryocyte can improve the efficiency of hematopoietic stem cell transplantation, specifically, the chimeric rate of exogenous leukocytes (such as myeloid cells, B cells, T cells, NK cells and neutrophils) and exogenous hematopoietic stem cells in a subject subjected to hematopoietic stem cell transplantation is improved.

Description

Technical Field

[0001] The present invention relates to medical immunology, and in particular to the use of megakaryocytes in hematopoietic stem cell transplantation. Background Art

[0002] Hematopoietic stem cell transplantation (HSCT) is a method of pre-treatment with high-dose radiotherapy and chemotherapy to remove tumors or abnormal cells in the recipient's body, and then transplant autologous or allogeneic hematopoietic stem cells to the recipient to reconstruct normal hematopoiesis and immune system. It is currently widely used in the treatment of malignant blood diseases, non-malignant refractory blood diseases, genetic diseases and certain solid tumors, and has achieved good results. According to the source of hematopoietic stem cells collected, it is divided into: bone marrow transplantation, umbilical cord blood hematopoietic stem cell transplantation, peripheral blood hematopoietic stem cell transplantation, etc. Peripheral blood hematopoietic stem cell transplantation is more commonly used now, which is less harmful to the donor than bone marrow transplantation. For patients who cannot match HLA fully matched, haploid hematopoietic stem cell transplantation and umbilical cord blood hematopoietic stem cell transplantation can be selected. Compared with umbilical cord blood transplantation, peripheral blood hematopoietic stem cell transplantation can achieve faster hematopoiesis and immune reconstruction, but it may induce severe graft-versus-host reaction GvHD. Umbilical cord blood hematopoietic stem cell transplantation has the advantages of significantly lower incidence of GvHD than peripheral blood hematopoietic stem cell transplantation, relatively lower requirements for HLA matching between unrelated donors and recipients, and convenient and abundant sources of umbilical cord blood collection. However, in clinical practice, it faces the problem of delayed engraftment of granulocytes and platelets. In summary, both peripheral blood hematopoietic stem cell transplantation and umbilical cord blood hematopoietic stem cell transplantation have the phenomenon of slow colonization. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention aims to provide a method for improving the effect of hematopoietic stem cell implantation.

[0004] The first aspect of the present application provides a use of megakaryocytes in hematopoietic stem cell transplantation.

[0005] In some embodiments, the use includes one or more of the following: use of megakaryocytes in improving the efficiency of hematopoietic stem cell transplantation; use of megakaryocytes in reducing complications after hematopoietic stem cell transplantation.

[0006] In some embodiments, the use of the megakaryocytes in improving the efficiency of hematopoietic stem cell transplantation includes one or more of the following: improving the chimerism rate of exogenous leukocytes in a subject undergoing hematopoietic stem cell transplantation; improving the chimerism rate of exogenous hematopoietic stem cells in a subject undergoing hematopoietic stem cell transplantation.

[0007] In some embodiments, the exogenous leukocytes include one or more of the following: exogenous myeloid cells, exogenous B cells, exogenous T cells, exogenous natural killer cells (NK cells), and exogenous neutrophils.

[0008] In some embodiments, the hematopoietic stem cells are derived from one or more of:

[0009] (a) Umbilical cord blood;

[0010] (b) bone marrow;

[0011] (c) peripheral blood;

[0012] (d) induced pluripotent stem cells;

[0013] (e) Transdifferentiation from differentiated cells of non-hematopoietic stem cell origin;

[0014] (f) embryonic stem cells;

[0015] (g) genetically modified hematopoietic stem cells;

[0016] (h) Other sources of multipotent or pluripotent cells.

[0017] In some embodiments, the hematopoietic stem cells are CD34 positive cells.

[0018] In some embodiments, the megakaryocytes include CD41 / CD61 positive cells, and CD41 / CD61 and CD42b positive cells.

[0019] In some embodiments, the megakaryocytes include megakaryotic progenitor cells, immature megakaryocytes, and mature megakaryocytes.

[0020] In some embodiments, the megakaryocytes include multinucleated megakaryocytes.

[0021] The second aspect of the present application provides a method for treating blood diseases or solid tumors by using megakaryocytes according to any one of the embodiments of the first aspect, wherein the blood diseases include: aplastic anemia, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, iron deficiency anemia, chronic kidney disease-related anemia, thalassemia, hemophilia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia; wherein solid tumors include embryonal tumors, neuroblastomas, lymphomas, and multiple myeloma.

[0022] The third aspect of the present application provides the use of megakaryocytes and hematopoietic stem cells in the preparation of a kit and / or a drug for treating blood diseases and / or solid tumors, wherein the blood diseases include: aplastic anemia, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, iron deficiency anemia, chronic kidney disease-related anemia, thalassemia, hemophilia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia; wherein solid tumors include embryonal tumors, neuroblastomas, lymphomas, and multiple myeloma.

[0023] The fourth aspect of the present application provides the use of megakaryocytes and hematopoietic stem cells in preparing a kit for hematopoietic stem cell transplantation.

[0024] In some embodiments, the hematopoietic stem cells are derived from one or more of:

[0025] (a) Umbilical cord blood;

[0026] (b) bone marrow;

[0027] (c) peripheral blood;

[0028] (d) induced pluripotent stem cells;

[0029] (e) Transdifferentiation from differentiated cells of non-hematopoietic stem cell origin;

[0030] (f) embryonic stem cells;

[0031] (g) genetically modified hematopoietic stem cells;

[0032] (h) Other sources of multipotent or pluripotent cells.

[0033] In some embodiments, the megakaryocytes include CD41 / CD61 positive cells, and CD41 / CD61 and CD42b positive cells.

[0034] In some embodiments, the megakaryocytes include megakaryotic progenitor cells, immature megakaryocytes, and mature megakaryocytes.

[0035] In some embodiments, the megakaryocytes include multinucleated megakaryocytes.

[0036] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0038] Figure 1 The figure shows the purity of megakaryocytes and the proportion of mature megakaryocytes detected by flow cytometry using CD41 / CD61 and CD42b antibody staining according to an embodiment of the present invention.

[0039] Figure 2 Shown is the ploidy analysis of megakaryocytes detected by flow cytometry using PI staining according to an embodiment of the present invention.

[0040] Figure 3The graph shows the percentage of human CD45 positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 at week 8 after CD34+ cell transplantation according to an embodiment of the present invention.

[0041] Figure 4 The graph shows the percentage of human CD33-positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 at week 8 after CD34+ cell transplantation according to an embodiment of the present invention.

[0042] Figure 5 The graph shows the percentage of human CD19 positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 at week 8 after CD34+ cell transplantation according to an embodiment of the present invention.

[0043] Figure 6 The graph shows the percentage of human CD3 positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 according to an embodiment of the present invention at week 8 after CD34+ cell transplantation.

[0044] Figure 7 The graph shows the percentage of human CD56-positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 at week 8 after CD34+ cell transplantation according to an embodiment of the present invention.

[0045] Figure 8 The graph shows the percentage of human CD66b-positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 at week 8 after CD34+ cell transplantation according to an embodiment of the present invention.

[0046] Fig. 9 The graph shows the percentage of human CD34 positive cells in the bone marrow and peripheral blood of mice in the experimental group, control group 1 and control group 2 at week 8 after CD34+ cell transplantation according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0048] Those skilled in the art understand that the process of hematopoietic stem cell differentiation into erythroid lineage under physiological conditions goes through the following process: Under physiological conditions, the process of hematopoietic stem cell differentiation into erythroid lineage goes through the following process: Hematopoietic stem cells (HSCs) → pluripotent stem cells (PSCs) → committed stem cells (CSCs) → granulocyte, erythrocyte, monocyte and megakaryocyte colony forming units (CFU-GEMMs) (also called pluripotent hematopoietic progenitor cells) → burst forming unit-megakaryocytes (BFU-MKs) (also called pre-megakaryocytic progenitor cells) → megakaryocyte colony forming units (CFU-GEMMs). unit-megakaryocytes, CFU-MKs) (also known as megakaryocyte progenitor cells) → promegakaryoblasts (also known as pre-megakaryoblasts) → primitive megakaryoblasts (also known as megakaryoblasts) → immature megakaryocytes (promegakaryocyte) → megakaryocytes (megakaryocyte) → platelets.

[0049] The first aspect of the present invention provides a method for using megakaryocytes in hematopoietic stem cell transplantation.

[0050] It is understood that the cells used in hematopoietic stem cell transplantation (HSCT) may include but are not limited to cells with CD34 positivity higher than, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. In some embodiments, the use includes one or more of the following: the use of megakaryocytes in improving the efficiency of hematopoietic stem cell transplantation; the use of megakaryocytes in reducing complications after hematopoietic stem cell transplantation.

[0051] In some embodiments, the use of megakaryocytes in improving the efficiency of hematopoietic stem cell transplantation includes one or more of the following:

[0052] Improving the chimerism rate of exogenous leukocytes in subjects who have undergone hematopoietic stem cell transplantation;

[0053] Improve the chimerism rate of exogenous hematopoietic stem cells in subjects undergoing hematopoietic stem cell transplantation.

[0054] In some embodiments, the exogenous leukocytes include one or more of the following: exogenous myeloid cells, exogenous B cells, exogenous T cells, exogenous natural killer cells (NK cells), and exogenous neutrophils.

[0055] It can be understood that the chimerism rate refers to the proportion of donor cells (exogenous cells) in the total cells of the recipient in the bone marrow or peripheral blood of the subject after hematopoietic stem cell transplantation (for example, the chimerism rate of exogenous B cells = the number of exogenous B cells in the sample to be tested / the total number of cells in the sample to be tested). The chimerism rate can be used to evaluate the efficiency of the transplantation and the survival and function of the exogenous cells after transplantation. The higher the chimerism rate, the higher the efficiency of hematopoietic stem cell transplantation. In some embodiments, human leukocyte antigen (HLA) sites can be used to distinguish donor cells from recipient cells.

[0056] It can be understood that CD45+ is used to identify white blood cells, CD33+ is used to identify myeloid cells, CD19+ is used to identify B cells, CD3+ is used to identify T cells, CD56+ is used to identify NK cells, CD66b+ is used to identify neutrophils, and CD34+ is used to identify hematopoietic stem cells.

[0057] In some embodiments, the hematopoietic stem cells are derived from one or more of:

[0058] (a) Umbilical cord blood;

[0059] (b) bone marrow;

[0060] (c) peripheral blood;

[0061] (d) induced pluripotent stem cells;

[0062] (e) Transdifferentiation from differentiated cells of non-hematopoietic stem cell origin;

[0063] (f) embryonic stem cells;

[0064] (g) genetically modified hematopoietic stem cells;

[0065] (h) Other sources of multipotent or pluripotent cells.

[0066] In some embodiments, the hematopoietic stem cells are CD34 positive cells (also referred to as CD34+ and CD34 in this specification). + ).

[0067] In some embodiments, the megakaryocytes include CD41 / CD61 positive cells, and CD41 / CD61 and CD42b positive cells.

[0068] In some embodiments, the megakaryocytes include megakaryotic progenitor cells, immature megakaryocytes, and mature megakaryocytes.

[0069] In some embodiments, the megakaryocytes include multinucleated megakaryocytes.

[0070] In some embodiments, the megakaryocytes comprise greater than 70% of total mature megakaryocytes.

[0071] The second aspect of the present application provides a method for treating blood diseases or solid tumors by using megakaryocytes according to any one of the embodiments of the first aspect, wherein the blood diseases include: aplastic anemia, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, iron deficiency anemia, chronic kidney disease-related anemia, thalassemia, hemophilia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia; wherein solid tumors include embryonal tumors, neuroblastomas, lymphomas, and multiple myeloma.

[0072] The third aspect of the present application provides the use of megakaryocytes and hematopoietic stem cells in the preparation of a kit and / or a drug for treating blood diseases and / or solid tumors, wherein the blood diseases include: aplastic anemia, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, iron deficiency anemia, chronic kidney disease-related anemia, thalassemia, hemophilia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia; wherein solid tumors include embryonal tumors, neuroblastomas, lymphomas, and multiple myeloma.

[0073] The fourth aspect of the present application provides the use of megakaryocytes and hematopoietic stem cells in preparing a kit for hematopoietic stem cell transplantation.

[0074] It is understandable that the present application embodiment provides the use of a combination of megakaryocytes and hematopoietic stem cells in preparing a kit for hematopoietic stem cell transplantation. As an example, in the kit, megakaryocytes and hematopoietic stem cells can exist independently or in combination.

[0075] In some embodiments, the hematopoietic stem cells are derived from one or more of:

[0076] (a) Umbilical cord blood;

[0077] (b) bone marrow;

[0078] (c) peripheral blood;

[0079] (d) induced pluripotent stem cells;

[0080] (e) Transdifferentiation from differentiated cells of non-hematopoietic stem cell origin;

[0081] (f) embryonic stem cells;

[0082] (g) genetically modified hematopoietic stem cells;

[0083] (h) Other sources of multipotent or pluripotent cells.

[0084] In some embodiments, the megakaryocytes include CD41 / CD61 positive cells, and CD41 / CD61 and CD42b positive cells.

[0085] In some embodiments, the megakaryocytes include megakaryotic progenitor cells, immature megakaryocytes, and mature megakaryocytes.

[0086] In some embodiments, the megakaryocytes include multinucleated megakaryocytes.

[0087] In some embodiments, the megakaryocytes comprise greater than 70% of total mature megakaryocytes.

[0088] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications are used. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0089] Example 1: Isolation of CD34+ cells

[0090] 1.1 Sedimentation of red blood cells

[0091] 1.11 Gently shake and mix 6% hydroxyethyl starch by mass / volume concentration, take 10 ml and add it to a 50 ml EP tube; then add 30 ml of anticoagulated human umbilical cord blood (blood:HES=3:1) to each tube, gently shake and mix, let stand at room temperature for 30 minutes, and then centrifuge at 20°C and 50g for 10 minutes to sediment the red blood cells.

[0092] 1.12 Collect the supernatant obtained after centrifugation in 1.11 into a new 50ml EP tube as much as possible (avoid aspirating red blood cells).

[0093] 1.13 Centrifuge the supernatant collected in 1.12 at 400 g for 10 min at 4°C to collect the total nucleated cells for later use.

[0094] 1.2 Mononuclear cell isolation

[0095] 1.21 Dilute the total nucleated cells collected in step 1.1 with an equal volume of PBS, take the diluted supernatant and gently and slowly add it to the surface of Ficoll human lymphocyte separation fluid along the closed tube (without disturbing or destroying the interface) (diluted supernatant: Ficoll = 2:1), and then centrifuge at 400g at 20°C for 30 minutes.

[0096] 1.22 After centrifugation, gently pipette the buffy coat into a new tube (try to pipette above the interface to avoid absorbing too much Ficoll in the lower layer) to obtain the buffy coat containing mononuclear cells.

[0097] 1.23 Gently shake the buffy coat to mix well and dilute with an equal volume of PBS, gently shake to mix well, centrifuge at 400g for 10 min at 4°C to obtain mononuclear cells for use.

[0098] 1.3 CD34+ cell isolation

[0099] 1.31 Resuspend the mononuclear cells collected in step 1.2 in PBS buffer containing 0.5% BSA and use the second generation EasySep TM The EasySepTM Human CD34 Positive Selection Kit II was used to separate and purify CD34+ cells according to the instructions provided by the manufacturer.

[0100] 1.22Finally, the isolated and purified CD34+ cells were resuspended in an appropriate volume of freezing solution and frozen.

[0101] 1.4 CD34+ cell purity identification

[0102] 1.41 Separate a portion of the CD34+ cells collected in step 1.3 for CD34 antibody staining, resuspend in a CD34 antibody mixture (PE-conjugated anti-human CD34, purchased from Biolegend) prepared in PBS buffer containing 0.5% BSA, pipette to mix, and incubate at 4°C in the dark for 30 min.

[0103] 1.42 After the incubation, add 1 mL of PBS buffer containing 0.5% BSA to each tube to wash the cells, and centrifuge at 400g for 10 min at 4°C. After centrifugation, discard the supernatant, resuspend the cell pellet in 200 μL PBS buffer, and then perform DAPI staining. Perform flow cytometric analysis on the stained samples using BD LSRFortessa SORP.

[0104] 1.43 Cells with CD34 positivity of not less than 95% are considered qualified "seed" cells for transplantation in subsequent experiments.

[0105] Example 2: Inducing differentiation of CD34+ cells into megakaryocytes

[0106] CD34+ cells were cultured in megakaryocyte differentiation induction medium (SFEMII+20ng / ml hSCF+100ng / ml hTPO) for 13 or 14 days and then harvested.

[0107] 2.1 Detection of megakaryocyte surface markers

[0108] 2.11 Take out a portion of the harvested megakaryocytes and centrifuge to remove the supernatant, resuspend in PBS buffer containing 0.5% BSA to prepare antibody mixture (PE-Cy7 conjugated anti-human CD41 / CD61, BV650 conjugated anti-human CD42b, both purchased from Biolegend), mix by pipetting, and incubate at 4°C in the dark for 30 minutes.

[0109] 2.12 After incubation, add 1 mL of PBS buffer containing 0.5% BSA to each tube to wash the cells, and centrifuge at 400g for 10 min at 4°C. After centrifugation, discard the supernatant, resuspend the cell pellet with 200 μL PBS buffer, and then perform DAPI staining. Perform flow cytometric analysis on the stained samples using BD LSRFortessa SORP.

[0110] 2.13 As Figure 1 As shown, the proportion of megakaryocytes indicated by CD41_CD61+ was 85.6%, of which the proportion of mature megakaryocytes indicated by CD41_CD61+ and CD42b+ was 76.2%.

[0111] 2.2 Megakaryocyte ploidy analysis

[0112] 2.21 Take out a portion of the harvested megakaryocytes and centrifuge to remove the supernatant, resuspend them in a PBS buffer containing 0.5% BSA to prepare an antibody mixture (FITC-conjugated anti-human CD41 / CD61, purchased from Biolegend), mix them by pipetting, and incubate them at 4°C in the dark for 30 minutes.

[0113] 2.22 After the incubation, add 1 mL of PBS buffer containing 0.5% BSA to each tube to wash the cells, and centrifuge at 400g for 10 min at 4°C. After centrifugation, discard the supernatant and immediately resuspend the cell pellet with 70% pre-cooled ethanol, blow it evenly, and place it at -20°C overnight for fixation.

[0114] 2.23 Take out the fixed cells and centrifuge them, remove the fixative and wash them with PBS.

[0115] 2.24 After centrifugation, remove the supernatant and resuspend the cells in PBS containing 0.01% Triton-X100, add RNase and PI dye, and incubate at room temperature in the dark for 20-30 minutes;

[0116] 2.25 After the incubation, wash with PBS and resuspend the stained cells in 200 μL PBS buffer. Analyze the samples by flow cytometry using BDLSFortessa SORP.

[0117] 2.26 As Figure 2 As shown, diploid megakaryocytes are the majority, but some are polyploid.

[0118] Example 3: Detection of megakaryocytes to assist in improving the effect of hematopoietic stem cell transplantation

[0119] 3.1CD34+ cell and megakaryocyte transplantation

[0120] 3.11 The overall experimental process is as follows Figure 3 As shown. On day 0, NCG immunodeficient mice (purchased from Jicui Pharmaceutical) were irradiated with X-rays at a dose of 200 cGy. After irradiation, an appropriate amount of Bainoxin was added to the drinking water of the mice to prevent Gram-positive, Gram-negative bacteria and mycoplasma infection. 4-6 hours after the end of irradiation, 3×10 4 CD34+ hematopoietic stem cells or PBS, experimental group (CD34 + cells+megakaryocytes group) and control group 1 (CD34 + Cells + PBS group) 3×10 4 The control group 2 (PBS+megakaryocyte group) was infused with an equal volume of PBS.

[0121] 3.12 On the 7th day after transplantation of CD34+ cells, a second transplantation was performed with megakaryocytes or PBS. + Each mouse was transplanted with 2×10 6 Megakaryocytes, control group 1 was infused with PBS (CD34 + cells+PBS group).

[0122] 3.2 Analysis of the proportion of human cells

[0123] 3.21 At 8 weeks after CD34+ cell transplantation, 100 μl of peripheral blood was collected from mice in the experimental group, control group 1, and control group 2 by tail snipation for subsequent flow cytometry analysis.

[0124] 3.22 After peripheral blood was collected, the mice were killed by cervical dislocation and their leg bones were removed for subsequent bone marrow cell isolation and flow cytometric analysis.

[0125] 3.23 The peripheral blood was lysed and bone marrow cells were separated and enriched by centrifugation. 2×10 6Peripheral blood mononuclear cells and bone marrow cells were used for staining. After centrifugation, the cell pellet was resuspended in PBS buffer containing 0.5% BSA and containing the following antibody mixtures (BV605-conjugated anti-mouse CD45, APC-conjugated anti-human CD45, APC-Cy7-conjugated anti-human CD33, PerCP-Cy5.5-conjugated anti-human CD19, BV711-conjugated anti-human CD56, PB-conjugated anti-human CD3, PE-conjugated anti-human CD66b, FITC-conjugated anti-human CD34, all antibodies were purchased from Biolegend), mixed by pipetting, and incubated at 4°C in the dark for 30 min.

[0126] 3.24 Centrifuge at 400g for 10 min at 4°C. After centrifugation, discard the supernatant, resuspend the cell pellet in 200 μL PBS buffer, and then stain with DAPI. The stained samples were analyzed by flow cytometry using BD LSRFortessa SORP.

[0127] 3.25 Figure 3-Figure 9 The results showed that megakaryocyte infusion can enhance the overall reconstruction effect of hematopoietic stem cell transplantation and accelerate the engraftment of myeloid and lymphoid lines. hCD45+ is used to identify human leukocytes (used to evaluate the overall reconstruction effect of hematopoietic stem cell transplantation), hCD33+ is used to identify human myeloid cells, hCD19+ is used to identify human B cells, hCD3+ is used to identify human T cells, hCD56+ is used to identify human NK cells, hCD66b+ is used to identify human neutrophils, and hCD34+ is used to identify human hematopoietic stem cells.

[0128] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0129] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. The use of megakaryocytes in hematopoietic stem cell transplantation.

2. The use according to claim 1, characterized in that The uses include one or more of the following: The use of megakaryocytes in improving the efficiency of hematopoietic stem cell transplantation; Use of megakaryocytes in reducing complications after hematopoietic stem cell transplantation.

3. The use according to claim 1 or 2, characterized in that: The use of the megakaryocytes in improving the efficiency of hematopoietic stem cell transplantation includes one or more of the following: Improving the chimerism rate of exogenous leukocytes in subjects who have undergone hematopoietic stem cell transplantation; Improve the chimerism rate of exogenous hematopoietic stem cells in subjects who have undergone hematopoietic stem cell transplantation, Optionally, the exogenous leukocytes include one or more of the following: exogenous myeloid cells, exogenous B cells, exogenous T cells, exogenous natural killer cells (NK cells) and exogenous neutrophils.

4. The use according to any one of claims 1 to 3, characterized in that The hematopoietic stem cells are derived from one or more of the following: (a) Umbilical cord blood; (b) bone marrow; (c) peripheral blood; (d) induced pluripotent stem cells; (e) Transdifferentiation from differentiated cells of non-hematopoietic stem cell origin; (f) embryonic stem cells; (g) genetically modified hematopoietic stem cells; (h) Other sources of multipotent or pluripotent cells.

5. The use according to claims 1 to 4, characterized in that The hematopoietic stem cells are CD34 positive cells.

6. The use according to any one of claims 1 to 5, characterized in that The megakaryocytes include CD41 / CD61 positive cells, and CD41 / CD61 and CD42b positive cells; Optionally, the megakaryocytes include megakaryocyte progenitor cells, immature megakaryocytes and mature megakaryocytes; Optionally, the megakaryocytes comprise multinucleated megakaryocytes.

7. A method for treating blood diseases or solid tumors by using megakaryocytes according to any one of claims 1 to 6, wherein the blood diseases include: Aplastic anemia, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, iron deficiency anemia, anemia associated with chronic kidney disease, thalassemia, hemophilia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia; solid tumors include embryonal tumor, neuroblastoma, lymphoma, and multiple myeloma.

8. Use of megakaryocytes and hematopoietic stem cells in the preparation of a kit and / or a drug for treating blood diseases and / or solid tumors, wherein the blood diseases include: Aplastic anemia, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, iron deficiency anemia, anemia associated with chronic kidney disease, thalassemia, hemophilia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia; solid tumors include embryonal tumor, neuroblastoma, lymphoma, and multiple myeloma.

9. Use of megakaryocytes and hematopoietic stem cells in the preparation of a kit for hematopoietic stem cell transplantation.

10. The use according to claim 8 or 9, characterized in that The hematopoietic stem cells are derived from one or more of the following: (a) Umbilical cord blood; (b) bone marrow; (c) peripheral blood; (d) induced pluripotent stem cells; (e) Transdifferentiation from differentiated cells of non-hematopoietic stem cell origin; (f) embryonic stem cells; (g) genetically modified hematopoietic stem cells; (h) other sources of multipotent or pluripotent cells; Optionally, the megakaryocytes include CD41 / CD61-positive cells, and CD41 / CD61- and CD42b-positive cells; Optionally, the megakaryocytes include megakaryocyte progenitor cells, immature megakaryocytes and mature megakaryocytes; Optionally, the megakaryocytes comprise multinucleated megakaryocytes.

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