Molecular markers of human megakaryocyte-biased hematopoietic stem cells and their detection kits

Abstract

The present invention provides a human megakaryocyte-biased hematopoietic stem cell molecular marker and a detection kit thereof. The detection marker of the present invention fills the gap in the prior art for the identification of surface molecular markers of human megakaryocyte-biased hematopoietic stem cells, creatively discovers that megakaryocyte hematopoietic stem cells are enriched in CD34 + CD38 ‑ CD45RA ‑ CD90 + CD49f low Cell populations, using this marker to identify cell subpopulations or screen for the preparation of cell therapy preparations has broad application prospects and great improvements in scientific research and clinical practice.

Description

Technical Field

[0001] The present invention belongs to the field of molecules, and in particular relates to a human megakaryocyte-biased hematopoietic stem cell molecular detection marker and a detection kit thereof. Background Art

[0002] Hematopoietic stem cells possess complex heterogeneity, primarily characterized by their lineage-biased differentiation and lineage restriction. One hallmark of hematopoietic stem cell heterogeneity is the presence of megakaryocyte / platelet-biased hematopoietic stem cells (Mk-biased HSCs). In various bone marrow failure diseases (e.g., aplastic anemia, AA), megakaryocyte hematopoiesis is the most sensitive and the most difficult to restore during treatment. In human hematopoietic stem cell transplantation, megakaryocyte hematopoiesis reconstruction is also relatively difficult. These clinical phenomena may be related to the characteristics of megakaryocyte-biased hematopoietic stem cells.

[0003] Recent studies have shown that megakaryocyte-biased hematopoietic stem cells are located at the top level of the hematopoietic stem cell differentiation spectrum, and a subpopulation of mouse megakaryocyte-biased hematopoietic stem cells with immunophenotypic markers has been identified. At the same time, studies of human myeloproliferative neoplasms have found that regulatory disorders at the hematopoietic stem cell level are closely associated with the development and progression of such diseases. Other studies have shown that in adult bone marrow hematopoiesis, human megakaryocyte differentiation can directly originate from multipotent hematopoietic progenitor cells (MPPs) without passing through the intermediate megakaryocyte-erythroid progenitor (MEP), suggesting that megakaryocyte-biased hematopoietic stem cells also exist in the human hematopoietic stem cell pool. However, their phenotypic characteristics remain unclear. Summary of the Invention

[0004] In view of this, the present invention aims to propose a molecular marker of human megakaryocyte-biased hematopoietic stem cells and a detection kit thereof, so as to overcome the defects of the existing technology of difficulty in megakaryocyte reconstruction and detection. The immunophenotypic marker discovered by the present invention can be used to analyze cell subpopulations enriched in human megakaryocyte-biased hematopoietic stem cells, and then sort out functional human megakaryocyte-biased hematopoietic stem cells.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] The first aspect of the present invention provides a functional hematopoietic stem cell population, wherein the functional hematopoietic stem cell population is a human megakaryocyte-biased hematopoietic stem cell population, wherein the human megakaryocyte-biased hematopoietic stem cell population is enriched in CD34 + CD38 - CD45RA - CD90 + CD49f low Among the cell subsets, the immunophenotype is: CD34 + CD38 - CD45RA - CD90 + CD49f lowCD62L - CD133 + .

[0007] A second aspect of the present invention provides a kit for identifying the functional hematopoietic stem cells according to claim 1, wherein the kit comprises a reagent for detecting the immunophenotype of the functional hematopoietic stem cells.

[0008] Preferably, the reagents in the kit are detection reagents for CD34, CD38, CD45RA, CD90, CD49f, CD62 and CD133.

[0009] Preferably, the detection reagent is an immunoassay reagent.

[0010] Preferably, the detection reagent is a detection reagent used in flow cytometry, and preferably the detection reagent includes: anti-CD34 antibody, anti-CD38 antibody, anti-CD45RA antibody, anti-CD90 antibody, anti-CD49flow antibody, anti-CD62 antibody and anti-CD133 antibody.

[0011] A third aspect of the present invention provides a method for identifying functional hematopoietic stem cells according to claim 1, the method comprising using a reagent to detect the immunophenotype of a cell population.

[0012] Preferably, the method is an immunological detection method.

[0013] Preferably, the immunological detection method is flow cytometry.

[0014] Preferably, CD34, CD38, CD45RA, CD90, CD49f, CD62 and CD133 are detected.

[0015] The fourth aspect of the present invention provides a pharmaceutical composition for cell therapy, which comprises functional hematopoietic stem cells and pharmaceutically acceptable excipients, wherein the functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells, and the immunophenotype of the human megakaryocyte-biased hematopoietic stem cells is: CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + .

[0016] The fifth aspect of the present invention provides an application of functional hematopoietic stem cells in the preparation of a preparation or medicament for treating blood diseases, wherein the functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells, and the human megakaryocyte-biased hematopoietic stem cells have an immunophenotype of CD34 + CD38- CD45RA - CD90 + CD49f low CD62L - CD133 + .

[0017] Preferably, the blood disease is a bone marrow failure disease.

[0018] Preferably, the bone marrow failure disease includes bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, Fanconi anemia FA, primary immune thrombocytopenia (ITP), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0020] Figure 1 a is a schematic diagram of the limiting dilution transplantation experiment flow in immunodeficient mice in Example 1, b is a typical flow cytometry plot of megakaryocytes in mouse bone marrow, and c is a statistical graph of the frequency of megakaryocyte hematopoietic stem cells;

[0021] Figure 2 .Umbilical cord blood CD34 in Example 2 + CD34 + CD38 - CD45RA - CD90 + Typical flow cytometry plots of CD49f expression distribution in subpopulation cells;

[0022] Figure 3 .Analysis of BD Rhapsody single-cell RNA-seq sequencing results in Example 2, a is the four cell populations identified by k-means clustering analysis. Each dot represents a cell, and the color represents a different cell population (C1-C4 population). The pie chart shows the percentage of each cell population in the blank control group and the microcarrier group. b is the expression of CD49f in the four cell populations, c is the pseudo-time analysis, d is the expression of specific genes in the four cell populations, the bubble color represents the Log2 average relative expression, and the bubble size represents the Log2 relative expression percentage. e is an expression heatmap of the top 10 most differentially expressed genes (DEGs) in the C2 cell population compared to the other populations, including the surface markers SELL (CD62L) and PROM1 (CD133);

[0023] Figure 4a is a typical flow cytometric graph of specific cell subsets of umbilical cord blood CD34+ cells before and after in vitro culture in Example 2; b is a typical flow cytometric graph of specific cell subsets of bone marrow mononuclear cells before and after in vitro culture;

[0024] Figure 5 a is the platelet count in the peripheral blood of immunodeficient mice after xenotransplantation in Example 3, and b is a typical flow cytometric graph of megakaryocytes in the bone marrow;

[0025] Figure 6 .a is a schematic diagram of the xenotransplantation process in immunodeficient mice in Example 6, b is the engraftment level of megakaryocytes in the mouse bone marrow after transplantation, the statistics of the reconstruction success rate, a typical flow cytometer, and the statistical graph of the frequency of megakaryocyte hematopoietic stem cells, and c is the engraftment level of human CD45+ cells in the mouse bone marrow after transplantation, the statistics of the reconstruction success rate, a typical flow cytometer, and the statistical graph of the frequency of hematopoietic stem cells. DETAILED DESCRIPTION

[0026] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0027] The present invention will be described in detail below with reference to the embodiments.

[0028] Example 1 In vitro expansion of functional human megakaryocyte-biased hematopoietic stem cells

[0029] 1. Cultivation of Functional Human Megakaryotic Hematopoietic Stem Cells

[0030] 1) Fresh human umbilical cord blood was mixed in a high-temperature and high-pressure sterilized glass plasma bottle at a ratio of umbilical cord blood to HESpan = 4:1, and then allowed to settle at room temperature for 45 minutes to fully crosslink and settle the red blood cells in the blood.

[0031] 2) Use a 10ml disposable sterile pipette and a 1ml pipette to carefully aspirate the pale yellow supernatant from the plasma bottle into two 50ml centrifuge tubes. Centrifuge at 1600r / min for 10min and discard the supernatant.

[0032] 3) Add a small amount of ACK red blood cell lysis buffer to the centrifuge tube and mix thoroughly by pipetting. Then add 40 ml of ACK and mix thoroughly by inversion. Incubate in a 37°C water bath for about 25 minutes. Centrifuge at 1500 rpm for 10 minutes and discard the supernatant.

[0033] 4) Wash the cells with an appropriate amount of sterile PBS buffer, centrifuge at 1500 r / min for 10 min, and discard the supernatant. In this step, human umbilical cord blood mononuclear cells are obtained, resuspended in an appropriate amount of PBS buffer, and 10 μl is taken for staining and counting using 0.4% Trypan Blue Solution.

[0034] 5) Resuspend the cells in PBS buffer according to the number of monocytes. Then, add appropriate amounts of FcRBlocking Reagent (to block nonspecific binding sites) and CD34 magnetic beads, mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 minutes. Invert the tube several times every 10 minutes to mix the cells.

[0035] 6) Wash the cells with an appropriate amount of sterile PBS buffer and pass them through LS column to collect CD34 + The cells were centrifuged at 1500 r / min for 10 min and resuspended in an appropriate amount of PBS buffer. 10 μl of the cells were stained and counted using 0.4% Trypan Blue Solution.

[0036] 7) Freshly prepare SFEMⅡ culture medium containing 1% PS, 100 ng / ml hTPO, and 10 ng / ml hSCF. Set up A. Uncultured group (Fresh) B. Blank control group (Control) C. 3D microcarrier group (Microniche):

[0037] A. For the uncultured group, umbilical cord blood CD34 + Cells are not cultured

[0038] B. For the blank control group, freshly isolated CD34 + cells to 2×10 6 / ml, add 1.9ml culture medium / well into a 24-well plate, then add 100μl cell suspension / well to a final system of 2ml / well, gently pipette to mix, and the initial cell concentration is 2×10 5 2 ml of PBS buffer was added to the culture wells at the edge of the plate.

[0039] C. For the three-dimensional microcarrier group, commercial three-dimensional microcarriers (3D F01, catalog number: F01-100, Beijing Huakan Biotechnology Co., Ltd.) were plated into a 24-well plate and irradiated with UV light for 20-30 minutes. 100 μl of cell suspension was pipetted into the wells to allow the cells and carriers to fully absorb the cell suspension. 2 ml of PBS buffer was added to the culture wells at the edge of the plate. After 2 hours in an incubator, 1.9 ml of culture medium was added to a final volume of 2 ml. The microcarriers were gently stirred with a pipette tip to disperse them and evenly distribute them in the culture medium. The initial cell concentration for the plate was 2 × 10 5 / hole.

[0040] 8) For Groups B and C, arrange 4-6 replicate wells per group and culture in a 37°C, 5% CO2 incubator for 7 days. After 7 days, harvest the cells in a clean bench. Mix the cells or the cell-material complex by repeated pipetting. Filter through a 30μm sterile nylon membrane and transfer from the plate to a flow cytometer. Wash the plate and nylon membrane once with an appropriate amount of PBS buffer. Wash the cells twice with sterile PBS buffer, centrifuge at 1500 rpm for 10 min at 4°C, and discard the supernatant.

[0041] 2. Based on the immunodeficient mouse transplantation experiment, the reconstruction of the megakaryocytes by each group of cells after culture was verified

[0042] 1) On the day of transplantation, NOG immunodeficient mice were irradiated with 120 cGy of X-rays. Transplantation was performed 4 hours after the end of irradiation.

[0043] 2) The cells were diluted to an appropriate concentration using sterile PBS buffer according to the gradient cell number and injected into the tail vein of NOG immunodeficient mice at 200 μL per mouse.

[0044] 3) Transplanted cells were derived from cells collected from the culture of functional human megakaryocyte-biased hematopoietic stem cells in 1. The number of transplanted cells in each group was set as follows:

[0045] A. Uncultured group: 100,000, 50,000, 10,000, 1,000, 100, 10 CD34 + cell;

[0046] B. Blank control group 50000, 10000, 2000, 400, 80, 16 corresponding to the initial CD34 + cell;

[0047] C. The three-dimensional microcarrier groups of 100,000, 20,000, 4,000, 800, 160, and 32 correspond to the initial CD34 + cell.

[0048] 4) Take 10 μL tail blood at the 4th, 8th, 12th and 16th week after transplantation to detect human CD45 + Implantation rate.

[0049] 5) At week 16, mice were sacrificed by CO2 inhalation and soaked in 75% alcohol for 10 minutes. The hind limbs, including the bilateral femurs, tibias, and iliac bones, were removed in a clean bench. The muscle tissue was stripped clean with sterile gauze. PBS buffer was drawn up with a 1 ml syringe to flush out the bone marrow cells into a flow cytometer, which was then temporarily stored on ice.

[0050] 6) Filter the collected mouse bone marrow cells through a 30 μm sterile nylon membrane to prepare a single-cell suspension. Centrifuge at 1500 × 10 min at 4°C. Discard the supernatant and resuspend in an appropriate amount of PBS buffer. For each mouse, remove 50% of the bone marrow cells for secondary transplantation. Remove an appropriate amount of cells and transfer them to a flow cytometer for flow cytometry analysis. Centrifuge the remaining cells and freeze them in cryopreservation buffer at -80°C.

[0051] 7) Use PBS buffer containing 2% FBS to prepare an antibody mixture (APC-Cy7-conjugated anti-Mouse CD45, APC-conjugated anti-Mouse CD41, Percp-Cy5.5-conjugated anti-MouseCD42d, BV786-conjugated anti-Mouse CD61, PE-Cy7-conjugated anti-Mouse Ter119, BV605-conjugated anti-Mouse CD71, FITC-conjugated anti-Human CD45, BV510-conjugated anti-Human CD41a, PE-conjugated anti-Human CD42b, BV650-conjugatedanti-Human CD61, Alexa Fluor700-conjugated anti-Human CD235a, BV711-conjugatedanti-Human CD71 flow cytometry antibody mixture) was added to the flow cytometry tube to label the cells. After pipetting and mixing, the cells were incubated at 4°C in the dark for 30-60 minutes.

[0052] 8) After incubation, add 1 mL of PBS buffer to each tube to wash the cells. Centrifuge at 1500 rpm for 10 min at 4°C, discard the supernatant, and resuspend in 100-200 μL of PBS buffer. Analyze on a BD LSRII / LSRFortessa / CantoII flow cytometer and analyze the expression of human CD45 in mouse bone marrow using FlowJo 10 software. + The cell ratio was determined to be a successful transplant if there was an obvious positive group. The transplantation success rate of each cell dose transplantation group was calculated to calculate the long-term stem cell frequency; the mouse CD45-CD41-CD42d-CD61-human CD45-human CD41a in the mouse bone marrow was detected. + 、CD41a + CD61 + CD42b-, CD41a + CD61 + CD42b+ Cell ratio, mouse CD45-human CD45 + CD34 + CD38 + Human CD41a + 、CD71 + CD110 + 、CD71-CD110 + The cell ratio was used to determine the reconstruction of the transplanted megakaryocyte lineage, and the reconstruction success rate of each megakaryocyte phenotype in each cell dose transplantation group was calculated to calculate the frequency of long-term megakaryocyte-biased stem cells.

[0053] 3. Analysis of experimental results:

[0054] Table 1 Statistics on the success rate of reconstruction of each megakaryocyte subpopulation,

[0055] Reconstruction of Mk lineage

[0056]

[0057] Table 1 shows the statistical success rate of reconstruction of each megakaryocyte subpopulation, which shows the difference in megakaryocyte reconstruction in different culture groups. The representative flow cytometry diagrams of each megakaryocyte subpopulation are shown in Figure 1 .

[0058] Umbilical cord blood CD34 cultured in vitro on three-dimensional microcarriers + Compared with the blank control group, the cells showed a significant decrease in CD45 - CD41 - CD42d - CD61 - human CD45 - Megakaryocyte CD41a represents three phenotypes of cells with different maturation levels + 、CD41a + CD61 + CD42b - 、CD41a + CD61 + CD42b + The flow cytometry positive groups were clearly divided and the reconstruction success rate was improved, with no significant difference compared with the uncultured group. According to the ELDA calculation of the three phenotypic megakaryocyte groups, it was found that the frequency of functional megakaryocytes in the three-dimensional microcarrier group that were biased towards long-term hematopoietic stem cells was increased to varying degrees compared with the uncultured group. + 、CD41a + CD61 + CD42b - 、CD41a + CD61 + CD42b+ The calculated frequencies were 3.36 (p<0.05), 2.85 and 2.72 times that of the uncultured group, respectively.

[0059] Mouse CD45 in xenograft mice - human CD45 + CD34 + CD38 + CD41a in different differentiation stages of progenitor cell subsets + 、CD71 + CD110 + 、CD71 - CD110 + Subpopulations were clearly divided in the uncultured group and microcarrier group, but no significant positive subpopulations were observed in the blank control group. + There was no significant difference in the success rate of subpopulation reconstruction between the uncultured group and the microcarrier group. + CD110 + and CD71 - CD110 + The success rate of subpopulation reconstruction in the microcarrier group was approximately 1.8 times and 3.7 times that of the uncultured group.

[0060] The above experimental identification shows that the inventors have successfully obtained a functional hematopoietic stem cell population by using three-dimensional microcarriers for expansion in vitro, and verified that the cell population has the ability of human megakaryocyte-biased hematopoietic stem cells through megakaryocyte reconstruction in animals.

[0061] Example 2 Proposing human megakaryocyte-biased differentiation surface markers based on single-cell sequencing

[0062] 1. Analysis object:

[0063] Umbilical cord blood hCD34 + The cell culture and cell harvesting methods of the cell microcarrier plate were the same as those in steps 1) to 8) of Example 1, and the grouping was the same as that in Example 1. B. blank control group and C. three-dimensional microcarrier group (3D F01, Catalog No.: F01-01, Beijing Huakan Biotechnology Co., Ltd.). Additionally, a new small molecule compound group (SR1 and UM171, small molecule compounds known in the art for in vitro stem cell culture) was added. Culture conditions were as follows: Both compound stock solutions were diluted to the appropriate concentrations using culture medium. StemRegenin (SR1) was used at a final concentration of 1 μM, as reported in the literature, and UM171 was used at a final concentration of 40 nM, as reported in the literature. Care was taken to protect the wells from light during the preparation process. 1.8 ml of culture medium was added to each well, followed by 100 μl of cell suspension, followed by 100 μl of compound dilution, and repeated pipetting to mix thoroughly.

[0064] BD Rhapsody single-cell sequencing targets CD34 cells obtained by sorting cells from three groups of cultured cells using FACS ArialIII flow cytometer. + CD38 - CD45RA - CD90 + cell.

[0065] 2. Flow cytometry analysis method:

[0066] Umbilical cord blood hCD34 + After the cells were cultured and harvested, the cell concentration per flow cytometry tube was adjusted to 2×10 5 -1×10 6 Between 1 and 2 days, add 100 μl of the prepared antibody mixture (FITC-conjugated anti-Human CD34, PE-Cy7-conjugated anti-Human CD38, APC-H7-conjugated anti-Human CD45RA, Percp-Cy5.5-conjugated anti-Human CD90, BV510-conjugated anti-Human CD49f, PE-conjugated anti-Human CD62L, APC-conjugated anti-Human CD133 flow cytometry antibody mixture prepared in PBS buffer containing 2% FBS), mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 minutes. On day 0, perform the same antibody labeling treatment on the uncultured CD34+ cells.

[0067] 2) After incubation, add 1 ml of PBS buffer to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min, discard the supernatant, resuspend the cells in an appropriate amount of PBS buffer, and stain with DAPI before loading.

[0068] 3) BD FACS CANTOⅡ / AriaⅢ flow cytometer was used for detection and FlowJo 10 software was used to gate and analyze the cell ratio and absolute number.

[0069] 3. BD Rhapsody single-cell sequencing:

[0070] (1) BD FACS AriaⅢ flow cytometry sorter was used to sort 2×10 4 CD34 + CD38 - CD45RA - CD90 + cell.

[0071] (2) Add appropriate amount of AbSeq antibody against CD34, CD38, CD45RA, CD90, and CD49f, incubate on ice for 30 minutes, and add appropriate amount of BD Pharmingen TM Wash the cells 2-3 times with Stain Buffer (FBS) by centrifugation.

[0072] (3) Use a low-adsorption pipette tip to draw up 620 μL of pre-cooled Sample Buffer (Cat. No. 650000062) and gently blow the cells to mix them evenly. Add 3.1 μL of 2 mM Calcein AM (BD Biosciences, Cat. No. 564061) and 3.1 μL of 0.3 mM Draq7 (BD Biosciences, Cat. No. 564904), and gently blow the mixture up and down about 10 times. Incubate at 37°C in the dark for 5 minutes, draw up 10 μL of the mixture for viable cell counting, and place the remaining cells on ice for later use.

[0073] (4) Set the P1200M pipette to Prime / Treat mode and process the cartridge as follows:

[0074] ①100% ethanol, 700 μL

[0075] ②Air

[0076] ③Cartridge Wash Buffer 1, 700μL, room temperature 1min

[0077] ④Air

[0078] ⑤Cartridge Wash Buffer 1, 700μL, room temperature 10min

[0079] ⑥Air

[0080] ⑦Cartridge Wash Buffer 2, 700 μL, room temperature ≤ 4 hours

[0081] (5) Loading cells and magnetic beads: Set the P1200M pipette to Prime / Treat mode, aspirate 700 μL of air into the cartridge, and then set the P1200M pipette to Cell Load mode to load the cells. Carefully remove the cartridge after loading the cells and immediately place it in the BD Scanner for counting.

[0082] (6) Loading magnetic beads: Set the P1200M pipette to Prime / Treat mode, aspirate 700 μL of air into the cartridge, and immediately set the P1200M pipette to Bead Load mode to load the magnetic beads. Let it stand for 3 minutes before scanning.

[0083] (7) Washing magnetic beads: Set the P1200M pipette to the Wash mode, treat the cartridge twice with 720 μL pre-cooled sample buffer, and then scan.

[0084] (8) Lyse cells, recover captured and wash magnetic beads: Set the P1200M pipette to Lysis mode, aspirate 550 μL of Lysis Buffer containing DTT, slide the front slider to the "BEADS" position after 2 minutes, slide the left slider to the "RETRIEVAL" position, set the P5000M pipette to Retrieval mode, slide the front slider to the "0" position after 30 seconds, and add 4950 μL of Lysis Buffer containing DTT. Slide the front slider to the "OPEN" position, take out the 5ml low adsorption tube, immediately place it on the 15mL magnetic stand for 1 minute, and then immediately wash the magnetic beads. Scan the cartridge again.

[0085] (9) Reverse transcription: Prepare 200 μL (1 library) of system cDNA Mix (including 40 μL RT Buffer, 20 μL dNTP, 10 μL RT 0.1 M DTT, 12 μL Bead RT / PCR Enhancer, 10 μL RNase Inhibitor, 10 μL Reverse Transcriptase, 98 μL nuclease-free water) and place on a metal shaker at 1200 rpm, 37°C, for 45 min.

[0086] (10) Exonuclease I treatment: Prepare 200 μL (1 library) of Exonuclease I Mix (containing 20 μL Exonuclease I Buffer, 10 μL Exonuclease I, and 170 μL nuclease-free water) and place on a metal shaker at 1200 rpm, 37°C, for 30 min. Incubate on a metal block at 80°C for 20 min without shaking. Incubate on ice for 1 min, then discard the supernatant on a magnetic stand. Resuspend in 200 μL of pre-chilled Bead Resuspension Buffer (Cat. No. 650000066) and store at 4°C.

[0087] (11) Construction of RPE for WTA library:

[0088] ① Prepare Random Primer Mix: For 1 library: 20 μL WTA Extension Buffer, 20 μL WTA Extension Primers, 134 μL nuclease-free water.

[0089] ② Place the beads on a magnetic rack and discard the supernatant. Resuspend in 75 μL Elution Buffer, incubate at 95°C for 5 minutes (without shaking), and remove the supernatant from the magnetic rack. Wash the beads with 200 μL Elution Buffer and discard the supernatant from the magnetic rack. Resuspend in Random Primer Mix and follow the steps below:

[0090] A. Incubate at 95°C for 5 minutes (without shaking);

[0091] B. Incubate at 37°C for 5 minutes (shaking at 1200 rpm);

[0092] C. Incubate at 25°C for 15 minutes (shaking at 1200 rpm).

[0093] ③ Prepare Primer Extension Enzyme Mix. For 1 library: 8 μL 10 mM dNTPs, 12 μL Bead RT / PCR Enhancer, 6 μL WTA Extension Enzyme. Follow the steps below:

[0094] A. Incubate at 25°C for 10 minutes (shaking at 1200 rpm);

[0095] B. Incubate at 37°C for 15 minutes (shaking at 1200 rpm);

[0096] C. Incubate at 45°C for 10 minutes (shaking at 1200 rpm).

[0097] D. Incubate at 55°C for 10 minutes (shaking at 1200 rpm).

[0098] ④ Discard the supernatant from the magnetic rack. Resuspend in Elution Buffer and incubate at 95°C for 5 minutes (without shaking). Shake at 1200 rpm for 10 seconds. Remove the supernatant from the magnetic rack and resuspend the beads in 200 μL of pre-chilled Bead Resuspension Buffer. Store at 4°C.

[0099] (12) PCR1 for WTA+AbSeq+SMK library construction:

[0100] Component For 1library(μL)

[0101] PCR Master Mix(Cat.No.91-1118)100

[0102] Universal Oligo(Cat.No.650000074)20

[0103] Bead RT / PCR Enhancer(Cat.No.91-1082)12

[0104] Sample Tag PCR1 Primer(Cat.No.91-1088)1.2

[0105] AbSeq PCR1 Primer(Cat.No.91-1086)12

[0106] Total 145.2

[0107] Step Temperature Time Circle

[0108] Hot start 95℃180s 1

[0109] Denaturation 95℃30s 11-15

[0110] Annealing 60℃30s

[0111] Extension 72℃60s

[0112] Final Extension 72℃300s 1

[0113] Hold 4℃1

[0114] (13) RPE product purification: Prepare 50 mL of 80% alcohol and AMPure XP magnetic beads at room temperature and vortex thoroughly for 1 min. Pipette 360 μl of AMPure XP magnetic beads into 200 μl of RPE product, pipette to mix, incubate at room temperature for 10 min, discard the supernatant on the magnetic rack, keep it on the magnetic rack, carefully add 1 mL of 80% anhydrous ethanol, and discard the supernatant after 30 seconds. Repeat once, dry at room temperature for 5 min, resuspend in 40 μl of Elution Buffer, incubate at room temperature for 2 min, and remove the supernatant on the magnetic rack.

[0115] (14) Prepare RPE PCR Mix: For 1library: 60μL PCR MasterMix, 10μL UniversalOligo, 10μL WTA Amplification Primer.

[0116] Step Temperature Time Circle

[0117] Hot start 95℃180s 1

[0118] Denaturation 95℃30s 12-13

[0119] Annealing 60℃60s

[0120] Extension 72℃60s

[0121] Final Extension 72℃120s 1

[0122] Hold 4℃1

[0123] (15) RPE PCR products and AbSeq+SMK PCR1 products were purified, quantified using Qubit, and the library fragment size was measured using 2100.

[0124] (16) Perform WTA Index PCR: For 1 library: 60 μL PCR MasterMix, 5 μL LibraryForward Primer, 5 μL nuclease-free water.

[0125] Step Temperature Time Circle

[0126] Hot start 95℃180s 1

[0127] Denaturation 95℃30s 8-9

[0128] Annealing 60℃30s

[0129] Extension 72℃30s

[0130] Final Extension 72℃120s 1

[0131] Hold 4℃1

[0132] (17) Purify the WTA Index PCR product, quantify it using Qubit, and measure the library fragment size using 2100.

[0133] (18) Perform AbSeq Index PCR: For 1 library: 25 μL PCR MasterMix, 2 μL LibraryForward Primer, 18 μL nuclease-free water.

[0134] Step Temperature Time Circle

[0135] Hot start 95℃300s 1

[0136] Denaturation 95℃30s 6-8

[0137] Annealing 60℃30s

[0138] Extension 72℃30s

[0139] Final Extension 72℃60s 1

[0140] Hold 4℃1

[0141] (19) Purify the AbSeq Index PCR product, quantify it using Qubit, and measure the library fragment size using 2100.

[0142] (20) Perform Sample Tag PCR2: For 1 library: 25 μL PCR Master Mix, 2 μL Universal Oligo, 3 μL Sample Tag PCR2 Primer, 15 μL nuclease-free water.

[0143] Step Temperature Time Circle

[0144] Hot start 95℃180s 1

[0145] Denaturation 95℃30s 10

[0146] Annealing 60℃180s

[0147] Extension 72℃60s

[0148] Final Extension 72℃300s 1

[0149] Hold 4℃1

[0150] (21) Purify the Sample Tag PCR2 product and quantify it using Qubit.

[0151] (22) Perform Sample Tag Index PCR: For 1 library: 25 μL PCR MasterMix, 2 μL Library Forward Primer, 18 μL nuclease-free water.

[0152] Step Temperature Time Circle

[0153] Hot start 95℃180s 1

[0154] Denaturation 95℃30s 6-8

[0155] Annealing 60℃30s

[0156] Extension 72℃30s

[0157] Final Extension 72℃60s 1

[0158] Hold 4℃1

[0159] (23) Purify the Sample Tag Index PCR product, quantify it using Qubit, and measure the library fragment size using 2100.

[0160] (24) BD SeqGeq software was used to analyze the single-cell sequencing results.

[0161] 4. Analysis of experimental results:

[0162] Generally speaking, CD49f expression is divided into CD49f negative (CD49f-), CD49f weak positive (CD49f low ) and CD49f strong positive (CD49f high ) three subpopulations, CD49f positive (CD49f + ) including CD49f low and CD49f high .Depend on Figure 2 and Figure 4 It is known that in human umbilical cord blood CD34 + cells or bone marrow mononuclear cells, CD34 + CD38 - CD45RA - CD90 +The distribution of CD49f expression in the phenotype was different in the uncultured group, control group and 3D microcarrier group. In the uncultured group and 3D microcarrier group, CD49f+ was mainly expressed as CD49f low subpopulation, while in the control group, CD49f high This phenomenon is more obvious in bone marrow mononuclear cells. However, the CD34+ cells of umbilical cord blood or bone marrow mononuclear cells cultured in vitro with three-dimensional microcarriers have + CD38 - CD45RA - CD90 + CD49f low The proportion and absolute number of subpopulation cells were increased compared with the uncultured group, blank control group and small molecule compound group.

[0163] The distribution of CD49f is known in the prior art as CD49f - (C1), CD49f low (C2) and CD49f high (C3 and C4), and based on the known literature records on stem cell and megakaryocyte marker genes, it was found that primitive stem cell marker genes tend to be enriched in the C2 subpopulation; multipotent progenitor cell marker genes that are biased towards the megakaryocyte are enriched in the C3 subpopulation; megakaryocyte progenitor cell marker genes tend to be enriched in the C3 and C4 subpopulations. Figure 3 The results of single-cell sequencing showed that k-means clustering analysis showed that CD34 + CD38 - CD45RA - CD90 + The cells were divided into four subpopulations, which clearly matched the known distribution of CD49f: CD49f - (C1), CD49f low The 3D microcarrier culture group contained nearly all C2 cells and a subset of C3 cells, while the blank control group contained mostly C1 and C4 cell subsets. Pseudo-time analysis revealed that the C2 subpopulation resides at the apex of the hematopoietic hierarchy. Therefore, C2 is considered an important subpopulation for the maintenance of HSC function mediated by 3D microcarriers. Among the marker genes of the C2 subpopulation, two surface markers were identified: SELL (CD62L) and PROM1 (CD133).

[0164] Depend on Figure 4 By using umbilical cord blood CD34 + By flow cytometry analysis of cells and bone marrow mononuclear cells, we identified a specific CD34 + CD38- CD45RA - CD90 + CD49f low CD62L - CD133 + The cell population was only contained in the uncultured group and the three-dimensional microcarrier group, but was almost lost in the blank control group cells. This phenomenon was more obvious in bone marrow mononuclear cells.

[0165] In addition, SR1, UM171, PVA and other small molecules and polymers with the existing in vitro expansion function of human hematopoietic stem cells have been reported to culture umbilical cord blood CD34 + cells can also retain CD62L - CD133 + Cell populations were detected (although not as significantly as with 3D microcarriers), whereas this characteristic subset was not retained when culturing bone marrow mononuclear cells.

[0166] The above results indicate that the three-dimensional microcarrier culture in vitro maintains the natural subpopulation of human megakaryocyte-biased hematopoietic stem cells that is easily lost in blank control culture (also present in uncultured cells); CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + It can be used as a surface marker for human megakaryocyte-biased hematopoietic stem cells.

[0167] Example 5 Physiological state and in vitro culture harvested human megakaryopoietic stem cell population transplanted into the medullary cavity of immunodeficient mice

[0168] 1. Experimental Methods

[0169] On the day of transplantation, NOG immunodeficient mice were irradiated with X-rays at a dose of 120 cGy, and transplantation was performed 4 hours after the end of irradiation.

[0170] 2) Umbilical cord blood hCD34 + The method for harvesting cells cultured on microcarrier well plates was the same as steps 1) to 8) in Example 1, and the grouping was the same as in Example 1. A. Fresh group C. Three-dimensional microcarrier group. + Wash cells twice with sterile PBS buffer in a clean bench, centrifuge at 1500 rpm for 10 min at 4°C, and discard the supernatant.

[0171] 1) Resuspend cells in an appropriate amount of PBS buffer and add 100 μL of the prepared antibody mixture to a flow cytometry tube to label the cells. Mix thoroughly by pipetting and incubate at 4°C in the dark for 30-60 minutes. (For flow cytometry, prepare the FITC-conjugated anti-HumanCD34, PE-Cy7-conjugated anti-Human CD38, APC-H7-conjugated anti-Human CD45RA, Percp-Cy5.5-conjugated anti-Human CD90, and PE-conjugated anti-Human CD49f antibody mixture in PBS buffer containing 2% FBS.)

[0172] 2) After incubation, 1 mL of PBS buffer was added to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min at 4°C, discard the supernatant, resuspend in an appropriate amount of PBS buffer, and stain with DAPI before loading. The cells were loaded onto a BD Aria III flow cytometer, using an appropriate amount of sterile PBS buffer as the receiving fluid. Two-way sorting of DAPI was performed on the two groups of cells. - CD34 + CD38 - CD45RA - CD90 + CD49f low (CD49f low ) or DAPI - CD34 + CD38 - CD45RA - CD90 + CD49f neg&high (nonCD49f low )cell.

[0173] 3) Adjust the concentration to an appropriate level according to the number of cells obtained using sterile PBS buffer (200 cells / 25 μL / mouse), and inject the solution into the medullary cavity of anesthetized NOG immunodeficient mice.

[0174] 4) At the 4th, 8th, 12th, and 16th week after transplantation, 10 μL of tail blood was collected for flow cytometry testing of human CD45+ engraftment rate. At the same time, 15 μL of tail blood was added to 105 μL of CBC diluent (1:7) for detection by an automatic hematology analyzer.

[0175] 5) At week 16, mice were sacrificed by CO2 inhalation and soaked in 75% alcohol for 10 minutes. The hind limbs, including the bilateral femurs, tibias, and iliac bones, were removed in a clean bench. The muscle tissue was stripped clean with sterile gauze. PBS buffer was drawn up with a 1 ml syringe to flush out the bone marrow cells into a flow cytometer, which was then temporarily stored on ice.

[0176] 6) Filter the collected mouse bone marrow cells through a 30 μm sterile nylon membrane to prepare a single-cell suspension. Centrifuge at 1500 × 10 min at 4°C. Discard the supernatant and resuspend in an appropriate amount of PBS buffer. Remove an appropriate amount of cells and transfer them to a flow cytometer for flow cytometry analysis. Centrifuge the remaining cells and freeze them in freezing medium at -80°C.

[0177] 7) Use PBS buffer containing 2% FBS to prepare an antibody mixture (APC-Cy7-conjugated anti-Mouse CD45, APC-conjugated anti-Mouse CD41, Percp-Cy5.5-conjugated anti-MouseCD42d, BV786-conjugated anti-Mouse CD61, PE-Cy7-conjugated anti-Mouse Ter119, BV605-conjugated anti-Mouse CD71, FITC-conjugated anti-Human CD45, BV510-conjugated anti-Human CD41a, PE-conjugated anti-Human CD42b, BV650-conjugatedanti-Human CD61, Alexa Fluor700-conjugated anti-Human CD235a, BV711-conjugatedanti-Human CD71 Add appropriate amount of antibody mixture to each flow cytometry tube to label cells, mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 min.

[0178] 8) After incubation, add 1 mL of PBS buffer to each tube to wash the cells, centrifuge at 1500 r for 10 min at 4°C, discard the supernatant, resuspend in 100-200 μL of PBS buffer, and stain with DAPI before loading. BD Aria III flow cytometer was used to detect mouse CD45 in mouse bone marrow using FlowJo10 software. - CD41 - CD42d - CD61 - human CD45 - Human CD41a + The proportion of cells was used to determine the reconstruction of the transplanted megakaryocyte lineage, and the success rate of megakaryocyte phenotype reconstruction in each group was calculated.

[0179] 2. Result analysis:

[0180] Table 2. Statistics of the success rate of megakaryocyte reconstruction

[0181] hCD41a Positive rate in recipients

[0182]

[0183] Depend on Figure 5 a The results of blood routine tests showed that at 16 weeks after transplantation, the CD49f low The number of platelets in the peripheral blood of recipient mice was significantly higher than that of nonCD49f low There was no significant difference in platelet level between the uncultured group and the 3D microcarrier group.

[0184] Depend on Figure 5 b Flow cytometry and the above Table 2. Statistical table of the success rate of megakaryocyte reconstruction show that compared with nonCD49f low Recipient mice, uncultured group and microcarrier group had more CD49f low Reconstitution of mCD45 in recipient mice - mCD41 - mCD42d - mCD61 - hCD45 - hCD41a + Megakaryocytes (with obvious megakaryocyte-positive population), while microcarriers CD49f low Group (7 / 9) and uncultured CD49f low The success rate of megakaryocyte reconstruction was improved compared with the group (4 / 7), and the microcarrier nonCD49f low Group (4 / 7) and uncultured CD49f low The success rate of megakaryocyte reconstruction was similar in both groups (4 / 7).

[0185] The results showed that megakaryocyte hematopoietic stem cells were enriched in CD34 in both physiological state and three-dimensional microcarrier culture system. + CD38 - CD45RA - CD90 + CD49f low cell population, and microcarrier three-dimensional culture expanded this functional human megakaryocyte-biased hematopoietic stem cell subset in vitro.

[0186] Example 6 Functional Verification of Human Megakaryotic Hematopoietic Stem Cells Obtained from Single Umbilical Cord Blood Culture

[0187] 1. Experimental Methods

[0188] 1) Collect a bag of 60-200mL of newborn umbilical cord blood 10 8 -10 9The mononuclear cells were placed in a clean and sterile 200ml plasma bottle, and HES was added according to the volume ratio of hydroxyethyl starch (HES): umbilical cord blood = 1:4. After thorough shaking, the bottle was left to stand at room temperature for 45 minutes to 1 hour to fully settle the red blood cells in the blood.

[0189] 2) Use a 10ml disposable sterile pipette to gently aspirate the supernatant of the umbilical cord blood after sedimentation, place it in a 50ml centrifuge tube, and centrifuge at 1600 rpm for 10 minutes.

[0190] 3) Discard the supernatant and resuspend the cells in culture medium (SFEM + 10 ng / mL SCF + 100 ng / mL TPO + 1% PS). Count the cells and add four equal portions of the cell suspension to four pre-cleaned, autoclaved 125 mL to 1 L bioreactors, each containing 60 mL of culture medium. Add the above culture medium to 60 mL.

[0191] 4) 100 mg (10 mg / tablet) of three-dimensional microcarrier material was added to the experimental group and mixed evenly. The bioreactor culture bottles of the experimental group were placed in a 5% CO2, 37°C constant temperature incubator and dynamically cultured at 40 rpm.

[0192] 5) On day 3 after culture, mix the cells in the experimental group by pipetting, filter through a 500-mesh yellow membrane to remove any material, and centrifuge the filtrate (1500 rpm, 10 min, 4°C) to lyse the cells. Wash the cells, resuspend and count the cells, and centrifuge (1500 rpm, 10 min, 4°C) to discard the supernatant. Resuspend the cells in an appropriate amount of PBS and add 100 μL of the prepared antibody mixture to a flow cytometry tube to label the cells. Mix by pipetting, and incubate at 4°C in the dark for 30-60 min. (FITC-conjugated anti-Human CD34, PE-Cy7-conjugated anti-Human CD38, APC-H7-conjugated anti-Human CD45RA, Percp-Cy5.5-conjugated anti-Human CD90, PE-conjugated anti-Human CD49f, Alexa Fluor 700-conjugated anti-Human CD62L, and APC-conjugated anti-Human CD133 flow cytometry antibody mixture was prepared in PBS buffer containing 2% FBS.)

[0193] 6) After incubation, add 1 mL of PBS buffer to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min at 4°C, discard the supernatant, resuspend in an appropriate amount of PBS buffer, and stain with DAPI before loading. Load the BD Aria III flow cytometer with an appropriate amount of sterile PBS buffer as the receiving fluid. Perform two-way DAPI sorting on the two groups of cells.- CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + (CD62L - CD133 + ) or DAPI - CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 - &CD62L + CD133 -&+

[0194] (nonCD62L-CD133+) cells.

[0195] 7) On the day of transplantation, NOG immunodeficient mice were irradiated with X-rays at a dose of 120 cGy. Transplantation was performed 4 hours after the end of irradiation.

[0196] 8) Adjust the concentration of the suspension to the following concentration gradient using sterile PBS buffer according to the number of cells obtained: 1000, 500, 100, and 50 cells / 25 μL cell suspension / mouse, and inject the suspension into the medullary cavity of anesthetized NOG immunodeficient mice.

[0197] 9) At the 4th, 8th, 12th, and 16th week after transplantation, 10 μL of tail blood was collected for flow cytometry testing of human CD45+ engraftment rate. At the same time, 15 μL of tail blood was added to 105 μL of CBC diluent (1:7) for detection by an automatic hematology analyzer.

[0198] 10) At week 16, mice were sacrificed by CO2 inhalation and soaked in 75% alcohol for 10 minutes. The hind limbs, including the bilateral femurs, tibias, and iliac bones, were removed in a clean bench. The muscle tissue was stripped clean with sterile gauze. PBS buffer was drawn up with a 1 ml syringe to flush out the bone marrow cells into a flow cytometer, which was then temporarily stored on ice.

[0199] 11) Filter the collected mouse bone marrow cells through a 30 μm sterile nylon membrane to prepare a single-cell suspension. Centrifuge at 1500 × 10 min at 4°C. Discard the supernatant and resuspend in an appropriate amount of PBS buffer. Remove an appropriate amount of cells and transfer them to a flow cytometer for flow cytometry analysis. Centrifuge the remaining cells and freeze them in freezing medium at -80°C.

[0200] 12) Use PBS buffer containing 2% FBS to prepare an antibody mixture (APC-Cy7-conjugated anti-Mouse CD45, APC-conjugated anti-Mouse CD41, Percp-Cy5.5-conjugated anti-MouseCD42d, BV786-conjugated anti-Mouse CD61, PE-Cy7-conjugated anti-Mouse Ter119, BV605-conjugated anti-Mouse CD71, FITC-conjugated anti-Human CD45, BV510-conjugated anti-Human CD41a, PE-conjugated anti-Human CD42b, BV650-conjugatedanti-Human CD61, Alexa Fluor700-conjugated anti-Human CD235a, BV711-conjugatedanti-Human CD71 Add appropriate amount of antibody mixture to each flow cytometry tube to label cells, mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 min.

[0201] 13) After incubation, 1 mL of PBS buffer was added to each tube to wash the cells. The cells were centrifuged at 1500 r / min for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 100-200 μL of PBS buffer and stained with DAPI before loading. The cells were loaded onto a BD Aria III flow cytometer and gated using FlowJo10 software to detect mouse CD45 in mouse bone marrow. - CD41 - CD42d - CD61 - human CD45 - Human CD41a + The cell ratio was used to determine the reconstruction of the transplanted megakaryocyte lineage and the success rate of megakaryocyte phenotype reconstruction in each group was calculated. - human CD45 + Cell ratio to determine the level of engraftment.

[0202] 2. Experimental conclusions:

[0203] mCD45 was detected in the bone marrow cells of the recipient mice 16 weeks after transplantation. - hCD45 - mCD41 - mCD42d - mCD62 - hCD41a+ The proportion of subpopulation cells is not less than 0.01% as the standard for successful reconstruction of megakaryocytes, mCD45 - hCD45 + The proportion of subpopulation cells should be no less than 0.01% as the standard for successful engraftment of human leukocytes.

[0204] Depend on Figure 6 It can be seen that at different cell transplantation doses, the mouse bone marrow successfully implanted human CD45 + cells and can reconstitute megakaryocyte CD41a + Among them, transplantation of 1000, 500, and 100 cell doses of CD62L - CD133 + The success rate of mouse megakaryocyte reconstruction by subpopulation cells was higher than that by transplanting nonCD62L - CD133 + Mouse subpopulation cells; based on megakaryocyte mCD45 - hCD45 - mCD41 - mCD42d - mCD62 - hCD41a + Phenotype ELDA calculation, we know CD62L - CD133 + The frequency of functional megakaryocytes in the group is biased towards long-term hematopoietic stem cells compared with nonCD62L - CD133 + The group with nonCD62L transplantation increased by 3.19 times (p<0.05). - CD133 + The success rate of myeloid and lymphoid reconstitution (CD45+ engraftment) in mice with subpopulation cells is higher than that of transplantation of CD62L - CD133 + The frequency of long-term hematopoietic stem cells was calculated by ELDA based on the hCD45+ phenotype of mice, nonCD62L - CD133 + Compared with CD62L - CD133 + The serum creatinine level in the control group increased by 11.85 times (p<0.0001).

[0205] The results of Example 5 further show that megakaryocyte hematopoietic stem cells are enriched in CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 +Cell populations, in vitro sorting of cell lines with the above-mentioned immunophenotypes for cell transplantation can improve the effect of megakaryocyte reconstruction.

[0206] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A functional hematopoietic stem cell population, characterized in that: The functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells, and the human megakaryocyte-biased hematopoietic stem cells are enriched in CD34 + CD38 - CD45RA - CD90 + CD49f low Among the cell subsets, the immunophenotype is: CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + ; The functional hematopoietic stem cells include adding three-dimensional microcarriers to the expansion culture medium to culture human non-adherent cells, and the non-adherent cells are human monocytes; the three-dimensional microcarrier is 3D TableTrix® Microslide® F01 of Huakan Biotechnology Co., Ltd.

2. A method for identifying functional hematopoietic stem cells according to claim 1, characterized in that The method includes detecting the immunophenotype of a cell population using a reagent; The reagent is a reagent for detecting CD34, CD38, CD45RA, CD90, CD49f, CD62 and CD133; The judgment criteria of the method are: CD34 positive, CD38 negative, CD45RA negative, CD90 positive, low expression of CD49f, CD62L negative, and CD133 positive.

3. The method according to claim 2, characterized in that The method is an immunological detection method.

4. The method according to claim 3, characterized in that The immunological detection method is flow cytometry.

5. Use of the functional hematopoietic stem cell population according to claim 1 in preparing a preparation for treating blood diseases, characterized in that: The blood disease is a bone marrow failure disease.

6. Use of the functional hematopoietic stem cell population according to claim 1 in preparing a preparation for treating blood diseases, characterized in that: The blood disease is immune thrombocytopenia (ITP).

7. Use according to claim 5, characterized in that The bone marrow failure disease is selected from the group consisting of: bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, and Fanconi anemia FA.

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