Method for in-vitro preparation and expansion of hematopoietic stem cells

Abstract

The present application relates to a method for in-vitro preparation and expansion of hematopoietic stem cells. Specifically, the method comprises: 1) obtaining isolated umbilical cord blood mononuclear cells; 2) mixing the isolated umbilical cord blood mononuclear cells, a fibrinogen solution, and a thrombin solution to obtain a culture; and 3) enabling the culture to be in contact with a dispase and followed by passaging to obtain hematopoietic stem cells. The present application also relates to a kit for in-vitro preparation and expansion of hematopoietic stem cells, a use of fibrinogen and thrombin in preparation of the kit for in-vitro preparation and expansion of hematopoietic stem cells, and hematopoietic stem cells obtained by means of the method or kit in the present application.

Description

Methods for preparing and expanding hematopoietic stem cells in vitro Technical Field

[0001] This application relates to the fields of biomedicine and immunotherapy. Specifically, it relates to methods for the in vitro preparation and expansion of hematopoietic stem cells. Background Technology

[0002] Hematopoietic stem cells (HSCs) are a type of adult tissue stem cell that can differentiate into lymphoid and myeloid hematopoietic progenitor cells through directed differentiation. These cells then proliferate and differentiate into various mature, functional blood cells to maintain hematopoietic homeostasis throughout the body. HSCs possess biological characteristics such as self-renewal, multilineage differentiation, apoptosis, migration, and quiescent states, and can be used to treat a variety of diseases, including benign and malignant hematologic disorders, autoimmune diseases (such as mucopolysaccharidosis type II), and solid tumors (such as neuroblastoma).

[0003] In recent years, with the continuous development of immunological technology and the characteristics of HSCs in supporting hematopoiesis and reconstructing the entire blood system after transplantation, stem cell gene therapy, including haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and umbilical cord blood stem cell transplantation (UCBT), has become a mainstream research hotspot and is gradually becoming one of the main treatment methods for hematological malignancies in clinical practice. However, the insufficient number of stem cells limits the widespread application of stem cell transplantation in clinical practice. Therefore, it is urgent to solve the fundamental problem of stable culture and expansion of HSCs in vitro.

[0004] Currently, in vitro HSC cell expansion methods mainly rely on cytokine induction. This involves combining and adding multiple cytokines, including SCF, Flt3L, G-CSF, GM-CSF, IL-3, IL-6, TPO, and TNFSF15, to activate various signaling pathways, such as JAK-STAT, Notch, and Wnt, thereby maintaining the in vivo HSC cell phenotype while inducing large-scale in vitro expansion of HSCs. However, this method yields a relatively small number of HSCs, which is insufficient for clinical applications. Summary of the Invention

[0005] Human fibrinogen is a glycoprotein synthesized and secreted by hepatocytes. It is one of the most abundant clotting factors in blood plasma and can be converted into fibrin by thrombin, forming a stable, porous fibrin gel upon coagulation. As an insoluble protein, fibrin can be digested and degraded by dispersants. Utilizing a fibrin gel culture system can provide a 3D culture environment for cells, better mimicking the in vivo environment and thus offering experimental conditions closer to physiological states. This is of great significance for studying cell behavior, cell-cell interactions, and cell-microenvironment interactions.

[0006] Without being limited to specific theories, the technical solution of this application is at least partly based on the following discovery: applying the aforementioned 3D culture system to HSC cell culture helps to achieve large-scale expansion of HSCs, especially long-term hematopoietic stem cells (LT-HSCs) with high self-renewal capacity and multi-directional differentiation potential, and to develop more effective HSC expansion strategies.

[0007] This application describes a method for large-scale in vitro expansion of HSC cells by establishing a 3D fibrin gel culture system using fibrinogen and thrombin. HSC cells prepared using this method not only possess the ability to expand on a large scale in vitro, self-renew, and have longer survival, but also exhibit hematopoietic reconstruction capacity and the potential for targeted differentiation into various lineages after in vivo infusion.

[0008] This application is submitted to address the challenge of the inability to prepare and expand large quantities of HSC cells in vitro.

[0009] In a first aspect, this application provides a method for preparing and expanding hematopoietic stem cells in vitro, which includes or consists of the following steps:

[0010] 1) Obtain isolated umbilical cord blood-derived mononuclear cells (UBMCs);

[0011] 2) The isolated UBMC cells, fibrinogen solution, and thrombin solution were mixed to obtain a culture;

[0012] 3) Contact the culture with the dispersing enzyme and passage it to obtain hematopoietic stem cells.

[0013] In some embodiments, in step 1) of the method of this application, the umbilical cord blood mononuclear cells are isolated from the umbilical cord blood of a mammal, which is a non-human mammal or a human, preferably a human.

[0014] In some embodiments, in step 2) of the method of this application, the isolated UBMC cells are mixed with a fibrinogen solution, seeded into a culture plate pre-incubated with thrombin solution, incubated for 30 minutes to allow the fibrinogen to be completely converted into fibrin gel and encapsulate the umbilical cord blood mononuclear cells, and then culture medium is added, wherein the fibrinogen is human fibrinogen and the thrombin is human thrombin.

[0015] In some embodiments, the final concentration of fibrinogen is 1-20 mg / ml, such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml.

[0016] In some embodiments, the concentration of the thrombin is 0.01-1 U / μl, such as, but not limited to, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl.

[0017] In some implementations, the culture medium added after 30 minutes of incubation is a hematopoietic stem cell culture medium containing recombinant human FLT3L protein, recombinant human TPO protein, and recombinant human SCF protein.

[0018] In some embodiments, in step 3) of the method of this application, after culturing for 4-6 days, preferably 5 days, the culture obtained in step 2) is contacted with a dispersing enzyme and passaged to obtain hematopoietic stem cells; preferably, the concentration of the dispersing enzyme is 1-10 mg / ml, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml; preferably, the passage is performed at least 3 times.

[0019] In a second aspect, this application provides a kit for the in vitro preparation and expansion of hematopoietic stem cells, comprising:

[0020] Isolated umbilical cord blood mononuclear cells;

[0021] Fibrinogen;

[0022] thrombin; and

[0023] Optionally, a dispersant enzyme.

[0024] In some embodiments, the fibrinogen is human fibrinogen, and the thrombin is human thrombin.

[0025] In a further embodiment, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml; the concentration of thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl.

[0026] In a further embodiment, the concentration of the dispersing enzyme is 1-10 mg / ml, preferably 2-5 mg / ml, and more preferably 4 mg / ml.

[0027] In a third aspect, this application provides the use of fibrinogen and thrombin in the preparation of kits for the in vitro preparation and expansion of hematopoietic stem cells.

[0028] In some embodiments, the fibrinogen is human fibrinogen, and the thrombin is human thrombin.

[0029] In a further embodiment, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml; the concentration of thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl.

[0030] In a fourth aspect, this application provides hematopoietic stem cells obtained by the method or kit of this application.

[0031] In some embodiments, the hematopoietic stem cells have one or more of the following characteristics:

[0032] 1) Increased Lin - CD45RA - CD38 - CD34 + CD90 + CD49F + Long-term hematopoietic stem cell ratio;

[0033] 2) Significantly upregulated expression levels of HSC-related genes RUNX1, CD93, THY1, CD34, EPCR, ITGA3, and TAL1;

[0034] 3) In vitro multi-lineage clone formation capability;

[0035] 4) The body's self-renewal capacity; and

[0036] 5) Highly efficient in vivo lineage reconstruction capability.

[0037] Through extensive experimental research, the applicant has discovered that HSC cells cultured using 3D fibrin gel can maintain a long-term hematopoietic stem cell phenotype (Lin). - CD45RA - CD38 - CD34 + CD90 + CD49F + The cells exhibit a significantly increased stem phenotype with each passage; they highly express HSC-related genes (RUNX1, CD93, THY1, CD34, EPCR, ITGA3, and TAL1); they possess a large-scale amplification capacity in vitro; and they exhibit self-renewal and efficient lineage reconstruction capabilities in vivo. This application provides a technical solution for the large-scale production and amplification of functional HSC cells.

[0038] In this application, when numerical values ​​are mentioned, they should be understood to include an appropriate error range, such as ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±8%, ±9%, and ±10%. This is mainly due to reasonable errors introduced by measurement methods, personnel operation, raw materials themselves, operating environment, and instruments and equipment. For example, since the accuracy of the measurement method for concentration and enzyme activity, as well as the skill level of the personnel and sampling, are related to various factors, a concentration of 100 IU / ml can refer to 100 IU / ml within the error range, and a technician can determine the specific error range based on the context. When there is no explicit limitation, the error range is ±10%, that is, "100 IU / ml" should be understood as 90-110 IU / ml.

[0039] When referring to time, it should not be understood as being accurate to the second; errors are allowed, such as ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±8%, ±9%, and ±10%. For example, due to the influence of human operation and timer errors, there may be an error of ±10% within 5 days.

[0040] The method described in this application does not involve the diagnosis or treatment of disease. Attached Figure Description

[0041] Figures 1A to 1G show that UBMC cells can be cultured in 3D fibrin gel and expanded into HSC cells in large quantities.

[0042] Figure 2 shows that UBMC cells amplified by 3D fibrin gel have higher levels of stem molecule expression.

[0043] Figure 3 shows that UBMC cells amplified by 3D fibrin gel have the ability to form multiple lineage clones in vitro.

[0044] Figures 4A to 4H show that UBMC cells expanded by 3D fibrin gel have self-renewal capacity and efficient lineage reconstruction capacity in vivo. Detailed Implementation

[0045] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0046] Various cell lines, animals, etc. used in the examples:

[0047] Human umbilical cord blood was obtained from volunteers; immunodeficient NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0048] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0049] Example 1: UBMC cells can be cultured in 3D fibrin gel and HSC cells can be expanded in large quantities.

[0050] 1. Experimental Procedure

[0051] 3D fibrin gel HSC cell culture system:

[0052] 1) Collect human umbilical cord blood, separate UBMC cells using Ficoll reagent, and lyse red blood cells using red blood cell lysis buffer;

[0053] 2) Collect the isolated UBMC cells and arrange them at an initial density of 5 × 10⁶ cells / year. 5 Fla-UBMC cells were seeded per well into 24-well plates to prepare a control rigid plate culture. 3.5 μL of thrombin solution (0.1 U / μL, Sea Run Holdings, Cat: SEA-135) was pre-added to the 24-well plates, followed by seeding with 5 × 10⁶ cells per well. 5The collected UBMC cells were mixed with human fibrinogen solution (RiaSTAP, Cat: NDC 63833-891-90) to a final fibrinogen concentration of 4 mg / ml (450 Pa). 250 μL of the mixture was immediately seeded into each well of a 24-well cell culture plate and incubated at 37°C with 5% CO2 for 30 minutes to allow fibrinogen to completely transform into fibrin gel and encapsulate the UBMC cells to form a network structure. 1 ml of StemSpan containing recombinant human Flt-3 ligand (FLT3L) protein (PeproTech, Cat: 300-19-50UG), recombinant human thrombopoietin (TPO) protein (PeproTech, Cat: 300-18-50UG), and recombinant human stem cell factor (SCF) protein (PeproTech, Cat: 300-07-50UG) was then added. TM SFEMII (STEMCELL, Cat: 09655) medium was added to each well for continuous amplification culture to prepare 3D-UBMC cells;

[0054] 3) Continue to culture Fla-UBMC cells and 3D-UBMC cells in the culture medium described in 2) above, and passage them on day 5 and day 10 respectively according to cell growth, and change the medium every 2 days.

[0055] 4) After 15 days, discard the supernatant from each well and add 400 μL of dispersin II (5 mg / mL, ROCHE, REF: 04942078001). Incubate at 37°C in a 5% CO2 incubator until the fibrin is completely degraded and the encapsulated 3D-UBMC cells are released. Simultaneously, harvest the Fla-UBMC cells. Count the total number of Fla-UBMC and 3D-UBMC cells under a microscope using a cell counting chamber.

[0056] 5) All samples were stained with Live / Dead dye at room temperature for 10 minutes, and then Fla-UBMC cells and 3D-UBMC cells were stained with HSC-related antibody CD34, respectively. The fold increase and number of CD34-positive cells were counted. Long-term hematopoietic stem cell (LT-HSC) phenotype-related antibodies (Lin, CD45RA, CD38, CD34, CD90, CD49F) were used to count LT-HSC (Lin... - CD45RA - CD38 - CD34 + CD90 + CD49F +The amplification fold and number of positive cells were determined. Data were collected using Invitrogen Attune NxT software and analyzed using FlowJo software.

[0057] 2. Experimental Results

[0058] 2.1 Umbilical cord blood-derived UBMC cells in a 3D fibrin gel culture system (3D-UBMC) exhibited a faster proliferation rate than those in a rigid plate (Fla-UBMC), and the cumulative increase in proliferation rate was more significant with increasing passage number (Figure 1A, Figure 1B): After 15 days, the proliferation rate of UBMC cells derived from umbilical cord blood in a rigid plate (Fla-UBMC) increased from the initial density of 5 × 10⁻⁶ cells / year. 5 Amplification to 2.3 × 10⁻⁶ cells / well 6 The amplification rate was 3.6 × 10⁶ cells / well in 3D fibrin gel (3D-UBMC) and 3.6 × 10⁶ cells / well. 6 The amplification folds were 4.6x and 7.2x, respectively, per well.

[0059] 2.2 Umbilical cord blood-derived UBMC cells exhibited a faster CD34-positive cell proliferation rate in the 3D fibrin gel culture system (3D-UBMC) than in the rigid plate (Fla-UBMC), and the cumulative increase in the CD34-positive cell proliferation rate was more significant with increasing passage number (Figures 1C to 1E): After 15 days, the proportion of CD34-positive cells in the rigid plate (Fla-UBMC) increased from the initial 6% to 18%, while in the 3D fibrin gel (3D-UBMC), it increased to 45%, and the number of CD34-positive cells increased from the initial 3 × 10⁻⁶ cells. 4 Each of the 4.14 × 10⁴ cells was amplified into the Fla-UBMC group. 5 1.62 × 10 in the 3D-UBMC group 6 The amplification folds were 13.7 times and 54 times, respectively.

[0060] 2.3 Umbilical cord blood-derived UBMC cells in the 3D fibrin gel culture system (3D-UBMC) had a higher proportion of LT-HSC positive cells than those in rigid plates (Fla-UBMC), and the cumulative increase in the proportion of LT-HSC positive cells was more significant with increasing passage number (Figure 1F, Figure 1G): the proportion of LT-HSC in freshly isolated UBMC cells was 0.005% on day 0, and the proportion in 3D-UBMC rose to 1% on day 15, while the proportion in Fla-UBMC was 0.09%, that is, the proportion of LT-HSC cells increased by 222 times and 21 times in the 3D-UBMC and Fla-UBMC groups, respectively.

[0061] Example 2: 3D-UBMC cells have higher HSC molecule expression levels

[0062] 1. Experimental Procedure

[0063] qPCR: 3D-UBMC cells and Fla-UBMC cells were collected, lysed with Trizol, and RNA was extracted and reverse transcribed into cDNA. qPCR was performed using primers corresponding to HSC-related genes (RUNX1, CD93, THY1, CD34, EPCR, ITGA3, and TAL1).

[0064] 2. Experimental Results

[0065] qPCR results showed that, compared with Fla-UBMC cells, the mRNA levels of CD93, THY1, CD34, and EPCR were significantly upregulated in 3D-UBMC cells, while the mRNA levels of RUNX1, ITGA3, and TAL1 were slightly increased (Figure 2).

[0066] Example 3: 3D-UBMC cells exhibit multi-lineage clone-forming ability in vitro

[0067] 1. Experimental Procedure

[0068] 3D-UBMC or Fla-UBMC cells cultured for five days were used at a total number of 2×10⁻⁶. 3 The MethodCult in the CFU kit (Stemcell, catalog number: 04435) TM The culture medium was mixed to 1.1 mL and inoculated into 6-well plates, which were then incubated in a humidified incubator at 37°C and 5% CO2. After 14 days, the number and type of colonies were counted and analyzed.

[0069] 2. Experimental Results

[0070] Counting and analysis of colony numbers and types revealed a significant increase in the number of CFUs in 3D-UBMC cells, including burst-forming unit-erythroid lineage (BFU-E), CFU-granulocyte / macrophage (CFU-GM), and CFU-granulocyte / erythroid / macrophage / megakaryocytic cell (CFU-GEMM), with numbers of 53, 31, and 10, respectively. In contrast, the Fla-UBMC group showed a weaker ability to generate CFUs, with BFU-E, CFU-GM, and CFU-GEMM numbers of 42, 17, and 2, respectively (Figure 3).

[0071] Example 4: 3D-UBMC cells possess self-renewal capacity and efficient lineage reconstitution ability in vivo.

[0072] 1. Experimental Procedure

[0073] 1.1 Initial Transplantation Assay: Immunodeficient NCG mice were irradiated with a dose of 1.5 Gray beforehand. Human 3D-UBMC and Fla-UBMC cells, cultured on 3D fibrin gels and rigid plates for 14 days, were then collected. The cells were then divided into two groups of 5 × 10⁶ cells per mouse. 5 The dose of cells was administered via tail vein grafting to irradiated mice. Mice were sacrificed after 16 weeks, and peripheral blood, bone marrow, and spleen were collected from each mouse. Single-cell suspensions were prepared, and the percentage of human HSCs in each group was analyzed using hCD45 and CD34 antibodies; the percentage of human myeloid cells in each group was analyzed using hCD45 and CD15 antibodies; the percentage of human B cells in each group was analyzed using hCD45 and CD19 antibodies; and the percentage of human T cells in each group was analyzed using hCD45 and CD3 antibodies. Data were collected using Invitrogen Attune NxT software and analyzed using FlowJo software.

[0074] 1.2 Secondary transplantation assay: Bone marrow was isolated from mice in the 3D-UBMC group and Fla-UBMC group 16 weeks after the initial transplantation, and 1×10⁻⁶ marrow samples were collected from each mouse. 6 The dose of cells was transferred via tail vein to a new batch of NCG mice irradiated with a dose of 1.5 Gray for secondary transplantation assays. Mice were sacrificed 16 weeks later, and peripheral blood, bone marrow, and spleen were collected from each mouse. Single-cell suspensions were prepared, and the percentage of human HSCs in each group was analyzed using hCD45 and CD34 antibodies; the percentage of human myeloid cells in each group was analyzed using hCD45 and CD15 antibodies; the percentage of human B cells in each group was analyzed using hCD45 and CD19 antibodies; and the percentage of human T cells in each group was analyzed using hCD45 and CD3 antibodies. Data were collected using Invitrogen Attune NxT software and analyzed using FlowJo software.

[0075] 2. Experimental Results

[0076] Flow cytometry analysis was performed on the proportion of each cell type in peripheral blood, bone marrow, and spleen of mice after adoptive 3D-UBMC and Fla-UBMC transplantation. After the initial transplantation (Fig. 4A-Fig. 4D) and the second transplantation (Fig. 4E-Fig. 4H), the proportion of human HSC cells, myeloid cells, and lymphocytes (T cells and B cells) in the adoptive 3D-UBMC cell group was significantly higher than that in the adoptive Fla-UBMC group. Furthermore, the proportion of each cell type after the second transplantation was significantly higher than that after the initial transplantation in all groups.

[0077] In summary, the solution proposed in this application has the following effects:

[0078] 1. This application utilizes a 3D fibrin gel culture system formed by fibrinogen and thrombin to prepare and expand HSC cells on a large scale, which features high expansion efficiency, low differentiation degree, and self-renewal capability.

[0079] 2. HSC cells prepared using the method and kit of this application have high levels of stem gene expression and expression levels of transcription factors related to maintaining HSC self-renewal.

[0080] 3. Using the method and kit of this application, a large number of LT-HSC cell populations can be generated, which can be directed to differentiate into various lineages of cells in vivo and reconstitute hematopoietic stem cells. Moreover, the mature cells generated have extremely strong biodynamics, which is expected to overcome the limitations of existing hematopoietic stem cell transplantation and has great clinical application prospects.

Claims

1. A method for preparing and expanding hematopoietic stem cells in vitro, comprising: 1) Obtain isolated umbilical cord blood mononuclear cells; 2) The isolated umbilical cord blood mononuclear cells, fibrinogen solution, and thrombin solution were mixed to obtain a culture; 3) Contact the culture with the dispersing enzyme and passage it to obtain hematopoietic stem cells.

2. The method according to claim 1, wherein: In step 1), the umbilical cord blood mononuclear cells are isolated from the umbilical cord blood of a mammal, which is either a non-human mammal or a human, preferably a human.

3. The method according to claim 1, wherein: In step 2), the isolated cord blood mononuclear cells are mixed with fibrinogen solution and inoculated into a culture plate pre-incubated with thrombin solution. After incubation for 30 minutes to allow fibrinogen to be completely converted into fibrin gel and encapsulate the cord blood mononuclear cells, culture medium is added.

4. The method according to claim 3, wherein: The fibrinogen is human fibrinogen, and the thrombin is human thrombin; Preferably, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml ± 10%. Preferably, the final concentration of the thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl ± 10%. Preferably, the culture medium added after 30 minutes of incubation is a hematopoietic stem cell culture medium containing recombinant human Flt-3 ligand protein, recombinant human thrombopoietin protein, and recombinant human stem cell factor protein.

5. The method according to claim 1, wherein: In step 3), after culturing for 4-6 days, preferably 5 days, the culture obtained in step 2) is contacted with the dispersing enzyme and passaged. Preferably, the final concentration of the dispersing enzyme is 1-10 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml ± 10%. Preferably, the passage is performed at least three times.

6. A kit for in vitro preparation and expansion of hematopoietic stem cells, comprising: Isolated umbilical cord blood mononuclear cells; Fibrinogen; thrombin; and Optionally, a dispersant enzyme.

7. The kit according to claim 6, wherein: The fibrinogen is human fibrinogen, and the thrombin is human thrombin; Preferably, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml ± 10%. Preferably, the concentration of the thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl ± 10%. Preferably, the final concentration of the dispersing enzyme is 1-10 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml ± 10%.

8. The uses of fibrinogen and thrombin in the preparation of kits for the in vitro preparation and expansion of hematopoietic stem cells, wherein: The fibrinogen is human fibrinogen, and the thrombin is human thrombin; Preferably, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 2-5 mg / ml, and even more preferably 4 mg / ml ± 10%. Preferably, the concentration of the thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl ± 10%.

9. A hematopoietic stem cell, obtained by the method according to any one of claims 1 to 5 or the kit according to claim 6 or 7, and having one or more of the following characteristics: 1) Increased Lin - CD45RA - CD38 - CD34 + CD90 + CD49F + Long-term hematopoietic stem cell ratio; 2) Significantly upregulated expression levels of HSC-related genes RUNX1, CD93, THY1, CD34, EPCR, ITGA3, and TAL1; 3) In vitro multi-lineage clone formation capability; 4) The body's self-renewal capacity; and 5) Highly efficient in vivo lineage reconstruction capability.

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